Molecules

25 pages, 3895 KiB

Open AccessReview

Mitochondria: Central Organelles for Melatonin′s Antioxidant and Anti-Aging Actions

by Russel J. Reiter, Dun Xian Tan, Sergio Rosales-Corral, Annia Galano, Xin Jia Zhou and Bing Xu

Molecules 2018, 23(2), 509; https://doi.org/10.3390/molecules23020509 - 24 Feb 2018

Cited by 290 | Viewed by 25517

Abstract

Melatonin, along with its metabolites, have long been known to significantly reduce the oxidative stress burden of aging cells or cells exposed to toxins. Oxidative damage is a result of free radicals produced in cells, especially in mitochondria. When measured, melatonin, a potent [...] Read more.

Melatonin, along with its metabolites, have long been known to significantly reduce the oxidative stress burden of aging cells or cells exposed to toxins. Oxidative damage is a result of free radicals produced in cells, especially in mitochondria. When measured, melatonin, a potent antioxidant, was found to be in higher concentrations in mitochondria than in other organelles or subcellular locations. Recent evidence indicates that mitochondrial membranes possess transporters that aid in the rapid uptake of melatonin by these organelles against a gradient. Moreover, we predicted several years ago that, because of their origin from melatonin-producing bacteria, mitochondria likely also synthesize melatonin. Data accumulated within the last year supports this prediction. A high content of melatonin in mitochondria would be fortuitous, since these organelles produce an abundance of free radicals. Thus, melatonin is optimally positioned to scavenge the radicals and reduce the degree of oxidative damage. In light of the “free radical theory of aging”, including all of its iterations, high melatonin levels in mitochondria would be expected to protect against age-related organismal decline. Also, there are many age-associated diseases that have, as a contributing factor, free radical damage. These multiple diseases may likely be deferred in their onset or progression if mitochondrial levels of melatonin can be maintained into advanced age. Full article

(This article belongs to the Special Issue Melatonin as an Antioxidant and a Functionally Pleiotropic Molecule: Synthesis, Metabolism and Activities in Organisms)

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Figure 1
The chemical reduction or the addition of energy to ground state oxygen generates products referred to as reactive oxygen species (ROS). The most reactive of these derivatives are peroxynitrite and the hydroxyl radical. The conversion of hydrogen peroxide to the hydroxyl radical requires a transition metal with the conversion usually being referred to as the Fenton reaction. The red asterisk (*) identifies products that have been reported to be directly scavenged by melatonin and its metabolites. The evidence of these scavenging reactions is much stronger for some ROS than for others. Melatonin also stimulates antioxidant enzymes, e.g., superoxide dismutases (SOD), glutathione peroxidases (GPx), and glutathione reductase (GRd) to indirectly remove toxic ROS. The most toxic species, i.e., peroxynitrite and the hydroxyl radical, are not enzymatically degraded; they can only be removed by a direct scavenger. CAT = catalase. Full article ">Figure 2
This figure illustrates the structure of a mitochondrion and the location of the complexes (CI-CV) that constitute the electron transport chain that engages in oxidative phosphorylation, which results in the generation of energy in the form of ATP. Free radicals are formed when electrons leak and reduce nearby oxygen (O2) molecules to form the superoxide anion radical (O2•−). CI releases electrons into the mitochondrial matrix, while CIII releases them into both the matrix and the intramembrane space. Once formed, the O2•− can be dismutated by superoxide dismutase (SOD) to hydrogen peroxide (H2O2) with its eventual conversion to the hydroxyl radical (•OH). O2•− can also couple with nitric oxide (NO•) to produce the peroxynitrite anion (ONOO−). Since melatonin is both taken up and synthesized in mitochondria, it is in an optimal position to scavenge these toxic species. Full article ">Figure 3
The structure of melatonin and some of its metabolites that have been shown to detoxify reactive oxygen and reactive nitrogen species. Additionally, some of these have other actions that enhance their ability to reduce oxidative stress, e.g., chelation of transition metal ions, promotion of antioxidant enzymes, inhibition of pro-oxidant enzymes, reducing electron leaking from the respiratory chain complexes, etc. Also shown is the sequence by which these metabolites are formed. This sequential formation of metabolites from melatonin, along with their ability to scavenge radicals, is referred to as melatonin′s antioxidant cascade. Full article ">Figure 4
The oligopeptide transporters, PEPT1/2, have recently been reported to be present in mitochondrial membranes. These transporters are believed to move melatonin into mitochondria against a gradient. This may explain the much higher concentration of melatonin in mitochondria than in other subcellular compartments. Moreover, high melatonin levels in mitochondria would be consistent with the marked ability of this antioxidant to protect these organelles from free radical damage as it occurs during aging and as a result of diseases of aging that have a free radical component. Full article ">Figure 5
This is an illustration of what is referred to as the endosymbiotic theory for the origin of mitochondria (and chloroplasts) and why these organelles in present-day eukaryotes likely have the ability to produce melatonin. A couple of billion years ago, prokaryotes phagocytized proteobacteria, which are known to synthesize melatonin; these bacteria were initially digested and used as nutrition. During evolution, the ingested bacteria eventually developed a mutually-beneficial symbiotic relationship with the cells that ingested them and they evolved into mitochondria. When they did so, the evolved mitochondria retained the ability to produce melatonin (brown image). As a result, present-day eukaryotic cells synthesize melatonin as shown in several reports cited in the current review. Likewise, some of the same prokaryotes also engulfed photosynthetic, melatonin-producing bacteria which evolved into chloroplasts of plant cells (green image); chloroplasts also have been shown to be involved in melatonin synthesis. Since plant cells have both chloroplasts and mitochondria may explain why plants generally have higher cellular concentrations of melatonin than do animal cells, which only have mitochondria. Full article ">Figure 6
This figure is a flow diagram that links free radicals and the associated oxidative damage with the progression of the aging phenotype and the onset and development of age-related diseases. The cloud at the top lists many of the iterations of the free radical theory of aging that have been introduced over the last 60 years. In the current review, we discuss the evidence that melatonin could be relevant to the processes summarized. ROS = Reactive oxygen species; AD = Alzheimer disease; PD = Parkinson disease; HD = Huntington disease; MS = Multiple sclerosis; ALS = amyotrophic lateral sclerosis. Full article ">

17 pages, 7348 KiB

Open AccessArticle

Physicochemical Characterization and Functional Analysis of the Polysaccharide from the Edible Microalga Nostoc sphaeroides

by Haifeng Li, Linnan Su, Sheng Chen, Libin Zhao, Hongyu Wang, Fei Ding, Hong Chen, Ruona Shi, Yulan Wang and Zebo Huang

Molecules 2018, 23(2), 508; https://doi.org/10.3390/molecules23020508 - 24 Feb 2018

Cited by 45 | Viewed by 7535

Abstract

Nostoc colonies have been used as food and medicine for centuries, and their main supporting matrix is polysaccharides, which help Nostoc cells resist various environmental stresses including oxidative stress. Here we isolated a polysaccharide, nostoglycan, from cultured Nostoc sphaeroides colonies and determined its [...] Read more.

Nostoc colonies have been used as food and medicine for centuries, and their main supporting matrix is polysaccharides, which help Nostoc cells resist various environmental stresses including oxidative stress. Here we isolated a polysaccharide, nostoglycan, from cultured Nostoc sphaeroides colonies and determined its physicochemical properties, which revealed a characteristic infrared absorption spectrum typical of polysaccharides and an amorphous morphology with rough surfaces. We also show that nostoglycan has strong moisture absorption and retention capacities and a high relative viscosity. Using Caenorhabditis elegans models, we then demonstrate that nostoglycan is capable of improving overall survival rate of the animals under increased oxidative stress caused by paraquat. Nostoglycan also reduces reactive oxygen species level, inhibits protein carbonyl formation and lipid peroxidation, and increases activities of superoxide dismutase and catalase in paraquat-exposed nematodes. As oxidative stress may drive tumor progression, we further demonstrate that nostoglycan can suppress the proliferation of several types of tumor cells and induce apoptosis of human lung adenocarcinoma A549 cells via caspase-3 activation. Together, our results yield important information on the physicochemical characteristics and demonstrate the antioxidant and anti-proliferative functions of nostoglycan, and thus provide an insight into its potential in food and health industries. Full article

(This article belongs to the Special Issue Advances in Natural Polysaccharides Research)

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Spectroscopic and physicochemical characterization of nostoglycan. (A) FTIR spectrum of nostoglycan in the frequency range of 4000–400 cm−1; (B) Triple helical conformation analysis of polysaccharides in aqueous solution. The maximum absorption wavelength of Congo red in the absence or presence of an indicated polysaccharide was recorded by a spectrophotometer under increasing alkaline conditions (0–0.5 M NaOH); (C,D) Surface morphology of nostoglycan by scanning electron microscopy at 200× and 2000× magnification, respectively. Full article ">Figure 2
Moisture absorption and retention rates of nostoglycan. For moisture absorption analysis, the dried polysaccharides were placed at 43% (A) or 81% (B) RH for the indicated times. For moisture retention determination, the humidified polysaccharides were first placed at 43% RH for 12 h (C) and then in a silica gel chamber for the indicated times (D). Both moisture gain and loss were determined gravimetrically. Data are representative of three independent experiments and shown as mean ± SD. Full article ">Figure 3
Effect of nostoglycan on the survival rate and ROS level of C. elegans under oxidative stress. (A) Oxidative survival curves of nematodes with or without nostoglycan treatment. Young adult nematodes were first treated with nostoglycan or EGCG at the indicated concentrations and then exposed to 70 mM paraquat. Live nematodes were scored every 12 h until all dead. Representative Kaplan–Meier curves are presented from three independent experiments; (B) Representative micrographs of DCF fluorescence in paraquat-exposed nematodes. The nematodes were treated with or without 0.5 mg/mL nostoglycan prior to 10 mM paraquat exposure and then stained with the fluorescent probe DCFH-DA. The fluorescent images were captured by an ImageXpress Micro System. Scale bars, 200 μm; (C) DCF fluorescence intensity of paraquat-exposed nematodes. The nematodes were treated as in (B), and the DCF intensity was measured with a fluorescence microplate reader after DCFH-DA treatment. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 (as compared to paraquat-exposed nematodes). Full article ">Figure 4
Effect of nostoglycan on the proliferation of tumor cells. Human tumor cell lines A549 (A); HepG2 (B); HL-60 (C); PC3 (D); MCF-7 (E); and Jurkat (F) were used to evaluate the anti-proliferative activity of nostoglycan. The tumor cells were treated with 0.1–1.0 mg/mL nostoglycan or 100 μg/mL 5-FUDR for 48 h, and the cell viabilities were measured by MTT method. All data are normalized to the untreated cells and presented as mean ± SEM of three independent experiments. * p < 0.05. Full article ">Figure 5
Effect of nostoglycan on apoptosis and caspase-3 activation in tumor cells. Quantitative analysis of apoptosis was performed by flow cytometry using human lung adenocarcinoma cell line A549. The cells were treated with medium (control; A), 100 μg/mL 5-FUDR (B) or 1.0 mg/mL nostoglycan (C) for 48 h and then subjected to Annexin V-FITC/PI staining and flow cytometry analysis. Apoptosis rate was calculated as the percentage of Annexin V-positive cells in >10,000 total cells (D). The activity of caspase-3 in A549 cells was determined by colorimetric assay (E). Data are presented as mean ± SEM of three independent experiments. * p < 0.05. Full article ">

13 pages, 10518 KiB

Open AccessArticle

Resveratrol-Inspired Benzo[b]selenophenes Act as Anti-Oxidants in Yeast

by Dominika Mániková, Zuzana Šestáková, Jana Rendeková, Danuša Vlasáková, Patrícia Lukáčová, Edgars Paegle, Pavel Arsenyan and Miroslav Chovanec

Molecules 2018, 23(2), 507; https://doi.org/10.3390/molecules23020507 - 24 Feb 2018

Cited by 19 | Viewed by 5894

Abstract

Resveratrol is a natural (poly)phenol primarily found in plants protecting them against pathogens, as well as harmful effects of physical and chemical agents. In higher eukaryotic cells and organisms, this compound displays a remarkable range of biological activities, such as anti-oxidant, anti-inflammatory, anti-cancer, [...] Read more.

Resveratrol is a natural (poly)phenol primarily found in plants protecting them against pathogens, as well as harmful effects of physical and chemical agents. In higher eukaryotic cells and organisms, this compound displays a remarkable range of biological activities, such as anti-oxidant, anti-inflammatory, anti-cancer, anti-aging, cardio- and neuro-protective properties. Here, biological activities of synthetic selenium-containing derivatives of resveratrol—benzo[b]selenophenes—have been studied in lower eukaryotes Saccharomyces cerevisiae. Their toxicity, as well as DNA damaging and reactive oxygen species (ROS) inducing potencies, manifested through their ability to act as redox active anti-microbial agents, have been examined. We show that some benzo[b]selenophenes can kill yeast cells and that the killing effects are not mediated by DNA damage types that can be detected as DNA double-strand breaks. These benzo[b]selenophenes could potentially be used as anti-fungal agents, although their concentrations relevant to application in humans need to be further evaluated. In addition, most of the studied benzo[b]selenophenes display redox-modulating/anti-oxidant activity (comparable or even higher than that of resveratrol or Trolox) causing a decrease in the intracellular ROS levels in yeast cells. Therefore, after careful re-evaluation in other biological systems these observations might be transferred to humans, where resveratrol-inspired benzo[b]selenophenes could be used as supra-anti-oxidant supplements. Full article

(This article belongs to the Special Issue Small Molecule Catalysts with Therapeutic Potential)

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14 pages, 2265 KiB

Open AccessArticle

Triterpene Acids from Frankincense and Semi-Synthetic Derivatives That Inhibit 5-Lipoxygenase and Cathepsin G

by Andreas Koeberle, Arne Henkel, Moritz Verhoff, Lars Tausch, Stefanie König, Dagmar Fischer, Nicole Kather, Stefanie Seitz, Michael Paul, Johann Jauch and Oliver Werz

Molecules 2018, 23(2), 506; https://doi.org/10.3390/molecules23020506 - 24 Feb 2018

Cited by 19 | Viewed by 7479

Abstract

Age-related diseases, such as osteoarthritis, Alzheimer’s disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) [...] Read more.

Age-related diseases, such as osteoarthritis, Alzheimer’s disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging. Full article

(This article belongs to the Special Issue Plant Derived Natural Products and Age Related Diseases)

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10 pages, 1690 KiB

Open AccessArticle

Separation of Seven Polyphenols from the Rhizome of Smilax glabra by Offline Two Dimension Recycling HSCCC with Extrusion Mode

by Wei Guo, Hongjing Dong, Daijie Wang, Bin Yang, Xiao Wang and Luqi Huang

Molecules 2018, 23(2), 505; https://doi.org/10.3390/molecules23020505 - 24 Feb 2018

Cited by 13 | Viewed by 5065

Abstract

An offline two-dimensional recycling high-speed countercurrent chromatography (2D R-HSCCC) strategy with extrusion mode was developed for isolating polyphenols from the rhizome of Smilax glabra. Firstly, the ethyl acetate extract was divided into two fractions, Fr.1 and Fr.2, by silica gel column chromatography. [...] Read more.

An offline two-dimensional recycling high-speed countercurrent chromatography (2D R-HSCCC) strategy with extrusion mode was developed for isolating polyphenols from the rhizome of Smilax glabra. Firstly, the ethyl acetate extract was divided into two fractions, Fr.1 and Fr.2, by silica gel column chromatography. Then, HSCCC was applied to separate polyphenols from the two fractions using a solvent system consisting of petroleum ether–ethyl acetate–methanol–water (1:3:0.5:5, v/v). Fifty milligrams of Fr.1 was separated by conventional HSCCC, yielding 5-O-caffeoylshikimic acid (1, 15.8 mg) and taxifolin (2, 4.8 mg). Offline 2D R-HSCCC with extrusion mode was used to separate Fr.2, and astilbin (4, 37.3 mg), neoisoastilbin (5, 8.8 mg), engeletin (7, 7.9 mg), and a mixture of two polyphenols were obtained from 100 mg of Fr.2. The mixture of two polyphenols was further separated by pre-HPLC, yielding neoastilbin (3, 15.2 mg) and isoastilbin (6, 9.9 mg). The purities of these seven compounds were all over 96.0%. Their structures were identified by MS and NMR. The results demonstrated that the strategy based on offline 2D R-HSCCC with extrusion mode was a powerful tool to separate the main compounds from the rhizome of Smilax glabra and valued for the preparative separation compounds with broad K-values and similar structures. Full article

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Figure 1
Structures of the compounds from the rhizome of S. glabra. (R: Rectus, right-handed; S: Sinister, left-handed). Full article ">Figure 2
UPLC chromatograms of the EtOAc extract and the fractions (A: the EtOAc extract; B: Fr.1; C: Fr.2) Experimental conditions: an ACQUITY UPLC T3 column (100 mm × 2.1 mm i.d., 1.8 μm). Flow rate: 0.35 mL/min. Column temperature: 40 °C. Injection volume: 2 μL. Detection: 254 nm. UPLC conditions are as follows: acetonitrile (A) and water containing 0.1% (v/v) formic acid (B), the gradient elution mode was set as follows: 0–12 min, 5–23% A; 12–12.5 min, 23–31% A; 12.5–15.5 min, 31% A; 15.5–16 min, 31–43% A; 16–19 min, 43% A; 19–19.5 min, 43–100% A; 19.5–22.5 min, 100% A. Full article ">Figure 3
The switch sketch of three different patterns. Full article ">Figure 4
HSCCC chromatogram of Fr.1 and HPLC chromatogram of the two HSCCC fractions (Peak 1: 5-O-caffeoylshikimic acid; Peak 2: taxifolin). HSCCC condition: solvent system: PEMW (1:3:0.5:5, v/v); mobile phase: lower phase; stationary phase: upper phase; revolution speed: 850 rpm; sample loading: 50 mg of Fr.1 dissolved in 10 mL of lower phase; detection wavelength: 254 nm; flow rate: 2 mL/min; separation temperature: 25 °C; retention of the stationary phase: 62.1%. Full article ">Figure 5
1D HSCCC chromatogram of Fr.2 and HPLC chromatogram of the three HSCCC fractions (Peak 3 + 4 + 6: the mixture of neoastilbin, astilbin, and isoastilbin; Peak 5: neoisoastilbin; Peak 7: engeletin). HSCCC condition was the same as that of <a href="#molecules-23-00505-f004" class="html-fig">Figure 4</a>. Full article ">Figure 6
2D R-HSCCC chromatogram of Fr.2 and HPLC chromatogram of the two HSCCC fractions (Peak 3 + 6: the mixture of neoastilbin and isoastilbin; Peak 4: astilbin). HSCCC conditions were the same as it in <a href="#molecules-23-00505-f004" class="html-fig">Figure 4</a>. Full article ">

11 pages, 1211 KiB

Open AccessArticle

Discovery of Flavonoids from Scutellaria baicalensis with Inhibitory Activity Against PCSK 9 Expression: Isolation, Synthesis and Their Biological Evaluation

by Piseth Nhoek, Hee-Sung Chae, Jagadeesh Nagarajappa Masagalli, Karabasappa Mailar, Pisey Pel, Young-Mi Kim, Won Jun Choi and Young-Won Chin

Molecules 2018, 23(2), 504; https://doi.org/10.3390/molecules23020504 - 24 Feb 2018

Cited by 25 | Viewed by 4968

Abstract

Nine flavonoids were isolated and identified from a chloroform-soluble fraction of the roots of Scutellaria baicalensis through a bioactivity-guided fractionation using a proprotein convertase subtilisin/kexin type 9 (PCSK9) monitoring assay in HepG2 cells. All structures were established by interpreting the corresponding spectroscopic data [...] Read more.

Nine flavonoids were isolated and identified from a chloroform-soluble fraction of the roots of Scutellaria baicalensis through a bioactivity-guided fractionation using a proprotein convertase subtilisin/kexin type 9 (PCSK9) monitoring assay in HepG2 cells. All structures were established by interpreting the corresponding spectroscopic data and comparing measured values from those in the literature. All compounds were assessed for their ability to inhibit PCSK9 mRNA expression; compounds 1 (3,7,2′-trihydroxy-5-methoxy-flavanone) and 4 (skullcapflavone II) were found to suppress PCSK9 mRNA via SREBP-1. Furthermore, compound 1 was found to increase low-density lipoprotein receptor protein expression. Also, synthesis of compound 1 as a racemic mixture form (1a) was completed for the first time. Natural compound 1 and synthetic racemic 1a were evaluated for their inhibitory activities against PCSK9 mRNA expression and the results confirmed the stereochemistry of 1 was important. Full article

(This article belongs to the Section Natural Products Chemistry)

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The effects of (total) methanol extracts, hexane (Hex), chloroform (CHCl3), butyl alcohol (BuOH) and aqueous fractions of S. baicalensis on PCSK9 mRNA expression in HepG2 cells. (Atorva = Atorvastatin). Full article ">Figure 2
Chemical structures isolated from S. baicalensis. Full article ">Figure 3
Effects of compounds from S. baicalensis on Cell viability, PCSK9 and LDL-R expression in HepG2 cells by MTT, qRT-PCR and western blot analysis. (A) Cells grown were treated with 20 μM of compounds for 24 h, and cell viability was assessed by the MTT assay; (B) Expression of PCSK9 was assayed by qRT-PCR in cells treated with compounds 1–9 except for 7, and berberine (50 μM) for 24 h; (C) Expression of PCSK9 and LDL-R were assayed by western blots in cells treated with (1) and atorvastatin for 24 h. (Ber50, berberine 50 μM). Full article ">Figure 4
Expressions of PCSK9 (A) LDLR (B) and SREBF1 (C) were assayed by qRT-PCR in cells treated with compounds 1a, 1, 4 and Ber (berberine) at a indicated concentrations. Full article ">Scheme 1
Synthesis of racemic compound 1a. Full article ">

12 pages, 783 KiB

Open AccessCommunication

Bacillus Cellulase Molecular Cloning, Expression, and Surface Display on the Outer Membrane of Escherichia coli

by Daehwan Kim and Seockmo Ku

Molecules 2018, 23(2), 503; https://doi.org/10.3390/molecules23020503 - 24 Feb 2018

Cited by 27 | Viewed by 6993

Abstract

One of the main challenges of using recombinant enzymes is that they are derived from genetically-modified microorganisms commonly located in the intracellular region. The use of these recombinant enzymes for commercial purposes requires the additional processes of cell disruption and purification, which may [...] Read more.

One of the main challenges of using recombinant enzymes is that they are derived from genetically-modified microorganisms commonly located in the intracellular region. The use of these recombinant enzymes for commercial purposes requires the additional processes of cell disruption and purification, which may result in enzyme loss, denaturation, and increased total production cost. In this study, the cellulase gene of Bacillus licheniformis ATCC 14580 was cloned, over-expressed, and surface displayed in recombinant Escherichia coli using an ice-nucleation protein (INP). INP, an outer membrane-bound protein from Pseudomonas syringae, was utilized as an anchor linker, which was cloned with a foreign cellulase gene into the pET21a vector to develop a surface display system on the outer membrane of E. coli. The resulting strain successfully revealed cellulase on the host cell surface. The over-expressed INP-cellulase fusion protein was confirmed via staining assay for determining the extracellular cellulase and Western blotting method for the molecular weight (MW) of cellulase, which was estimated to be around 61.7 kDa. Cell fractionation and localization tests demonstrated that the INP-cellulase fusion protein was mostly present in the supernatant (47.5%) and outer membrane (19.4%), while the wild-type strain intracellularly retained enzymes within cytosol (>61%), indicating that the INP gene directed the cellulase expression on the bacteria cell surface. Further studies of the optimal enzyme activity were observed at 60 °C and pH 7.0, and at least 75% of maximal enzyme activity was preserved at 70 °C. Full article

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Figure 1
Targeted gene insertion and expression in E. coli. (A) The gel electrophoresis of amplified PCR cellulose products from B. licheniformis ATCC 14580 (lane 1) and INP from P. syringae KCTC 1832 (lane 2). M: 1 kb DNA marker; (B) SDS-PAGE analysis of the recombinant cells; M: standard protein size marker (molecular biomasses in kilodaltons), lane 1: the supernatant fraction of recombinant cell culture medium, lane 2: the total cell lysates of recombinant cell; (C) The purified fusion proteins following Ni-nitrilotriacetic acid (NTA)-sepharose resin treatment; M: standard protein size marker (kDa), lane 1: imidazole concentration of 20 mM in the binding buffer, lane 2: imidazole concentration of 50 mM in the binding buffer, lane 3: imidazole concentration of 100 mM in the binding buffer; (D) Western blot analysis of the purified fusion protein from SDS-PAGE results probed with anti-His-tag antibody, respectively. Full article ">Figure 2
Congo-red staining assay of wild-type B. licheniformis (A); non-cellulase producing E. coli (B); and recombinant E. coli harboring INP-cellulase genes (C); all of which were topped and cultivated on agar medium of 0.5% CMC at 30 °C for 18 h. The hollow clear zones around the colonies indicate the degradation of CMC as a result of the cellulolytic enzymes. Full article ">Figure 3
Relative cellulase activity analysis of various pH and temperature conditions. (A) Enzymatic hydrolysis of 0.5% CMC was carried out at 50 °C for 3 h with an agitation of 200 rpm; (B) Enzymatic hydrolysis of 0.5% CMC was carried out at pH 7.0 for 3 h with an agitation of 200 rpm. Error bars indicate the standard deviation of triplicated tests. Full article ">

9 pages, 4396 KiB

Open AccessArticle

Pilot Study of ⁶⁴CuCl₂ for PET Imaging of Inflammation

by Lei Jiang, Dongli Song, Hao Chen, Ao Zhang, Huoqiang Wang and Zhen Cheng

Molecules 2018, 23(2), 502; https://doi.org/10.3390/molecules23020502 - 24 Feb 2018

Cited by 9 | Viewed by 5331

Abstract

Copper(II) ion (Cu²⁺) is the essential element for numerous pathophysiological processes in vivo. Copper transporter 1 (CTR1) is mainly responsible for maintaining Cu²⁺ accumulation in cells, which has been found to be over-expressed in inflammatory tissues. Therefore, we explored the [...] Read more.

Copper(II) ion (Cu²⁺) is the essential element for numerous pathophysiological processes in vivo. Copper transporter 1 (CTR1) is mainly responsible for maintaining Cu²⁺ accumulation in cells, which has been found to be over-expressed in inflammatory tissues. Therefore, we explored the potential application of ⁶⁴CuCl₂ for PET imaging of inflammation through targeting CTR1. The animal models of H₂O₂ induced muscle inflammation and lipopolysaccaharide induced lung inflammation were successfully established, then imaged by small animal PET (PET/CT) post-injection of ⁶⁴CuCl₂, and PET images were quantitatively analyzed. H&E and immunohistochemical (IHC) staining and western blot experiments were performed for evaluating CTR1 levels in the inflammatory and control tissues. Both inflammatory muscle and lungs can be clearly imaged by PET. PET image quantitative analysis revealed that the inflammatory muscle and lungs showed significantly higher ⁶⁴Cu accumulation than the controls, respectively (p < 0.05). Furthermore, IHC staining and western blot analysis demonstrated that compared with the controls, CTR1 expression was increased in both the inflammatory muscle and lungs, which was consistent with the levels of ⁶⁴Cu²⁺ accumulation in these tissues. ⁶⁴CuCl₂ can be used as a novel, simple, and highly promising PET tracer for CTR1 targeted imaging of inflammation. Full article

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24 pages, 3796 KiB

Open AccessArticle

Kinetics and Optimization of Lipophilic Kojic Acid Derivative Synthesis in Polar Aprotic Solvent Using Lipozyme RMIM and Its Rheological Study

by Nurazwa Ishak, Ahmad Firdaus B. Lajis, Rosfarizan Mohamad, Arbakariya B. Ariff, Mohd Shamzi Mohamed, Murni Halim and Helmi Wasoh

Molecules 2018, 23(2), 501; https://doi.org/10.3390/molecules23020501 - 24 Feb 2018

Cited by 16 | Viewed by 6303

Abstract

The synthesis of kojic acid derivative (KAD) from kojic and palmitic acid (C16:0) in the presence of immobilized lipase from Rhizomucor miehei (commercially known as Lipozyme RMIM), was studied using a shake flask system. Kojic acid is a polyfunctional heterocycles that acts as [...] Read more.

The synthesis of kojic acid derivative (KAD) from kojic and palmitic acid (C16:0) in the presence of immobilized lipase from Rhizomucor miehei (commercially known as Lipozyme RMIM), was studied using a shake flask system. Kojic acid is a polyfunctional heterocycles that acts as a source of nucleophile in this reaction allowing the formation of a lipophilic KAD. In this study, the source of biocatalyst, Lipozyme RMIM, was derived from the lipase of Rhizomucor miehei immobilized on weak anion exchange macro-porous Duolite ES 562 by the adsorption technique. The effects of solvents, enzyme loading, reaction temperature, and substrate molar ratio on the reaction rate were investigated. In one-factor-at-a-time (OFAT) experiments, a high reaction rate (30.6 × 10⁻³ M·min⁻¹) of KAD synthesis was recorded using acetone, enzyme loading of 1.25% (w/v), reaction time of 12 h, temperature of 50 °C and substrate molar ratio of 5:1. Thereafter, a yield of KAD synthesis was optimized via the response surface methodology (RSM) whereby the optimized molar ratio (fatty acid: kojic acid), enzyme loading, reaction temperature and reaction time were 6.74, 1.97% (w/v), 45.9 °C, and 20 h respectively, giving a high yield of KAD (64.47%). This condition was reevaluated in a 0.5 L stirred tank reactor (STR) where the agitation effects of two impellers; Rushton turbine (RT) and pitch-blade turbine (PBT), were investigated. In the STR, a very high yield of KAD synthesis (84.12%) was achieved using RT at 250 rpm, which was higher than the shake flask, thus indicating better mixing quality in STR. In a rheological study, a pseudoplastic behavior of KAD mixture was proposed for potential application in lotion formulation. Full article

(This article belongs to the Special Issue Lipases and Lipases Modification)

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16 pages, 2808 KiB

Open AccessArticle

Graphene Oxide as a Nanocarrier for a Theranostics Delivery System of Protocatechuic Acid and Gadolinium/Gold Nanoparticles

by Muhammad Sani Usman, Mohd Zobir Hussein, Aminu Umar Kura, Sharida Fakurazi, Mas Jaffri Masarudin and Fathinul Fikri Ahmad Saad

Molecules 2018, 23(2), 500; https://doi.org/10.3390/molecules23020500 - 24 Feb 2018

Cited by 34 | Viewed by 5381

Abstract

We have synthesized a graphene oxide (GO)-based theranostic nanodelivery system (GOTS) for magnetic resonance imaging (MRI) using naturally occurring protocatechuic acid (PA) as an anticancer agent and gadolinium (III) nitrate hexahydrate (Gd) as the starting material for a contrast agent,. Gold nanoparticles (AuNPs) [...] Read more.

We have synthesized a graphene oxide (GO)-based theranostic nanodelivery system (GOTS) for magnetic resonance imaging (MRI) using naturally occurring protocatechuic acid (PA) as an anticancer agent and gadolinium (III) nitrate hexahydrate (Gd) as the starting material for a contrast agent,. Gold nanoparticles (AuNPs) were subsequently used as second diagnostic agent. The GO nanosheets were first prepared from graphite via the improved Hummer’s protocol. The conjugation of the GO and the PA was done via hydrogen bonding and π–π stacking interactions, followed by surface adsorption of the AuNPs through electrostatic interactions. GAGPA is the name given to the nanocomposite obtained from Gd and PA conjugation. However, after coating with AuNPs, the name was modified to GAGPAu. The physicochemical properties of the GAGPA and GAGPAu nanohybrids were studied using various characterization techniques. The results from the analyses confirmed the formation of the GOTS. The powder X-ray diffraction (PXRD) results showed the diffractive patterns for pure GO nanolayers, which changed after subsequent conjugation of the Gd and PA. The AuNPs patterns were also recorded after surface adsorption. Cytotoxicity and magnetic resonance imaging (MRI) contrast tests were also carried out on the developed GOTS. The GAGPAu was significantly cytotoxic to the human liver hepatocellular carcinoma cell line (HepG2) but nontoxic to the standard fibroblast cell line (3T3). The GAGPAu also appeared to possess higher T1 contrast compared to the pure Gd and water reference. The GOTS has good prospects of serving as future theranostic platform for cancer chemotherapy and diagnosis. Full article

(This article belongs to the Section Nanochemistry)

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18 pages, 3901 KiB

Open AccessArticle

Establishment of the Volatile Signature of Wine-Based Aromatic Vinegars Subjected to Maceration

by Rosa Perestrelo, Catarina L. Silva, Pedro Silva and José S. Câmara

Molecules 2018, 23(2), 499; https://doi.org/10.3390/molecules23020499 - 23 Feb 2018

Cited by 14 | Viewed by 5630

Abstract

The flavoring of vinegars with aromatic fruits and medicinal herbs is a practice with increasing trend mostly in countries with oenological tradition, resulting in a product of improved quality and consumer attractiveness. This study was directed towards the evaluation of the impact of [...] Read more.

The flavoring of vinegars with aromatic fruits and medicinal herbs is a practice with increasing trend mostly in countries with oenological tradition, resulting in a product of improved quality and consumer attractiveness. This study was directed towards the evaluation of the impact of the maceration process on the volatile signature of wine-based aromatic vinegars (WBAVs). The evaluation was performed using solid phase microextraction (SPME) combined with gas chromatography combined with mass spectrometry (GC-MS). Experimental parameters influencing headspace solid (HS)-SPME extraction efficiency, were optimized using an univariate experimental design. The best results were achieved using a polydimethylsiloxane (PDMS) fiber, 10 mL of vinegar sample, at 50 °C for 30 min of extraction. This way One hundred and three volatile organic compounds (VOCs), belonging to different chemical families including ethyl esters (37), higher alcohols (20), fatty acids (10), terpenoids (23), carbonyl compounds (six), lactones (five) and volatile phenols (two), were identified in wine vinegar (control) and WBAV. As far as we know, 34 of these VOCs are reported for the first time in macerated vinegars. Higher alcohols and lactones are the major chemical families in WBAV macerated with apple, whereas terpenoids are predominant in WBAV macerated with banana. The obtained data represent a suitable tool to guarantee the authenticity and genuineness of WBAV, as well as to promote the production of WBAV with improved sensorial and organoleptic properties. To the best of our knowledge, there are no reported studies dealing with the volatile signature of WBAV enriched with banana, passion fruit, apple and pennyroyal. Full article

(This article belongs to the Section Analytical Chemistry)

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Full article ">Figure 1
Flowchart showing the general production methods for vinegar [<a href="#B2-molecules-23-00499" class="html-bibr">2</a>,<a href="#B3-molecules-23-00499" class="html-bibr">3</a>,<a href="#B4-molecules-23-00499" class="html-bibr">4</a>]. Full article ">Figure 2
Effect of (a) fiber coating; (b) extraction time and extraction temperature; and (c) ionic strength, sample volume, desorption time on the volatile compounds extraction efficiency from wine vinegar. Error bars represent mean standard error (n = 3 for each data point). * Number of identified compounds. Full article ">Figure 3
Total GC-MS peak area of chemical families identified in wine (control) and WBAV (banana, passion fruit, apple, pennyroyal) vinegars. Full article ">Figure 4
(a) Principal components (PC) PC1 × PC2 of scores scatter plot of wine (control) and WBAV vinegars; (b) Loading plot of the main source of variability between volatile profile and wine vinegars—derived products by maceration with fruits and plant (attribution of the peak number shown in <a href="#molecules-23-00499-t001" class="html-table">Table 1</a>). Full article ">Figure 5
Dendrogram for the HCA results using Ward’s cluster algorithm of the volatile profile obtained from wine (control) and WBAV vinegars. The Square Euclidean distances are shown on the x-axis. Full article ">

13 pages, 3030 KiB

Open AccessArticle

Antioxidation and Cytoprotection of Acteoside and Its Derivatives: Comparison and Mechanistic Chemistry

by Xican Li, Yulu Xie, Ke Li, Aizhi Wu, Hong Xie, Qian Guo, Penghui Xue, Yerkingul Maleshibek, Wei Zhao, Jiasong Guo and Dongfeng Chen

Molecules 2018, 23(2), 498; https://doi.org/10.3390/molecules23020498 - 23 Feb 2018

Cited by 30 | Viewed by 5218

Abstract

The study tried to explore the role of sugar-residues and mechanisms of phenolic phenylpropanoid antioxidants. Acteoside, along with its apioside forsythoside B and rhamnoside poliumoside, were comparatively investigated using various antioxidant assays. In three electron-transfer (ET)-based assays (FRAP, CUPRAC, PTIO•-scavenging at pH 4.5), [...] Read more.

The study tried to explore the role of sugar-residues and mechanisms of phenolic phenylpropanoid antioxidants. Acteoside, along with its apioside forsythoside B and rhamnoside poliumoside, were comparatively investigated using various antioxidant assays. In three electron-transfer (ET)-based assays (FRAP, CUPRAC, PTIO•-scavenging at pH 4.5), the relative antioxidant levels roughly ruled as: acteoside >forsythoside B > poliumoside. Such order was also observed in H⁺-transfer-involved PTIO•-scavenging assay at pH 7.4, and in three multiple-pathway-involved radical-scavenging assays, i.e., ABTS⁺•-scavenging, DPPH•-scavenging, and •O₂⁻-scavenging. In UV-vis spectra, each of them displayed a red-shift at 335→364 nm and two weak peaks (480 and 719 nm), when mixed with Fe²⁺; however, acteoside gave the weakest absorption. In Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC−ESI−Q−TOF−MS/MS) analysis, no radical-adduct-formation (RAF) peak was found. MTT assay revealed that poliumoside exhibited the highest viability of oxidative-stressed bone marrow-derived mesenchymal stem cells. In conclusion, acteoside, forsythoside B, and poliumoside may be involved in multiple-pathways to exert the antioxidant action, including ET, H⁺-transfer, or Fe²⁺-chelating, but not RAF. The ET and H⁺-transfer may be hindered by rhamnosyl and apiosyl moieties; however, the Fe²⁺-chelating potential can be enhanced by two sugar-residues (especially rhamnosyl moiety). The general effect of rhamnosyl and apiosyl moieties is to improve the antioxidant or cytoprotective effects. Full article

(This article belongs to the Special Issue The Antioxidant Capacities of Natural Products)

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14 pages, 3343 KiB

Open AccessArticle

Characterization, Stability and Biological Activity In Vitro of Cathelicidin-BF-30 Loaded 4-Arm Star-Shaped PEG-PLGA Microspheres

by Yueli Bao, Shanrong Wang, Hongli Li, Yunjiao Wang, Haiyun Chen and Minglong Yuan

Molecules 2018, 23(2), 497; https://doi.org/10.3390/molecules23020497 - 23 Feb 2018

Cited by 15 | Viewed by 5107

Abstract

BF-30 is a single chain polypeptide of an N-segment with an α-helix from cathelicidin gene encoding, and it contains 30 amino acid residues, with a relative molecular mass and isoelectric point of 3637.54 and 11.79, respectively. Cathelicidin-BF-30 was entrapped in four-arm star-shaped poly(ethylene [...] Read more.

BF-30 is a single chain polypeptide of an N-segment with an α-helix from cathelicidin gene encoding, and it contains 30 amino acid residues, with a relative molecular mass and isoelectric point of 3637.54 and 11.79, respectively. Cathelicidin-BF-30 was entrapped in four-arm star-shaped poly(ethylene glycol-b-dl-lactic acid-co-glycolic acid) block copolymers (4-arm-PEG-PLGA) by a double-emulsion solvent-evaporation method. Three release phases of cathelicidin-BF-30loaded 4-arm-PEG-PLGA microspheres were observed, including an initial burst-release phase, followed by a lag phase with minimal drug release and finally a secondary zero-order release phase. The delivery system released BF-30 over more than 15 days in vitro. Furthermore, the material for preparing the microspheres has good biocompatibility and biodegradability. Additionally, based on the drug resistance of food pathogenic bacteria, the antibacterial effects of BF-30 on Shigella dysenteriae CMCC 51105 (Sh. dysenteriae CMCC 51105), Salmonella typhi (S. typhi) and Staphylococcus aureus (S. aureus) as well as the stability of the in vitro release of the BF-30-loded microspheres were studied. The α-helix secondary structure and antibacterial activity of released BF-30 were retained and compared with native peptide. These BF-30 loaded microspheres presented <10% hemolysis and no toxicity for HEK293T cells even at the highest tested concentration (150 μg/mL), indicating that they are hemocompatible and a promising delivery and protection system for BF-30 peptide. Full article

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1H nuclear magnetic resonance (NMR) spectrum of 4-arm-PEG-PLGA. Full article ">Figure 2
Scanning electron microscopy (SEM)images (a,b) and size distribution (c) of 4-arm-PEG-PLGA microspheres. Full article ">Figure 3
The degradation profiles of microspheres in phosphate-buffered saline (PBS) (pH = 7.40): the mass (a) pH (b) and Mn (c) changed with time. Full article ">Figure 4
Release of BF-30 loaded microspheres in vitro for two weeks (a). Circular dichroism spectra of native and released BF-30 in one dayof release (b). Full article ">Figure 5
Hemolytic activity (A) and anti-clotting activity (B) of BF-30 encapsulated in 4-arm-PEG-PLGA microspheres. *** p < 0.001 versus positive control. Full article ">Figure 6
The different concentrations of BF-30-loaded microspheres were used to determine the cytotoxicity. The cell growth of 4-arm-PEG-PLGA microspheres loaded with BF-30 using human HaCaT keratinocyte cells (a) and human HEK293T embryonic kidney cells (b). * p < 0.05, ** p < 0.01 and *** p < 0.001 versus microspheres without compound (negative control). Values are expressed as the mean ± SD of three independent experiments. Full article ">Figure 7
The antimicrobial activities of free BF-30 (b), extracted BF-30 (c), and released BF-30 (d). PBS (a) was used as a negative control in this investigation. Full article ">Figure 8
Preparation process of microspheres using the emulsification technique. Full article ">Scheme 1
Synthesis of 4-arm-PEG-PLGA. Full article ">

16 pages, 5502 KiB

Open AccessArticle

In Silico-Based Repositioning of Phosphinothricin as a Novel Technetium-99m Imaging Probe with Potential Anti-Cancer Activity

by Tamer M. Sakr, Mohammed A. Khedr, Hassan M. Rashed and Maged E. Mohamed

Molecules 2018, 23(2), 496; https://doi.org/10.3390/molecules23020496 - 23 Feb 2018

Cited by 26 | Viewed by 5618

Abstract

l-Phosphinothricin (glufosinate or 2-amino-4-((hydroxy(methyl) phosphinyl) butyric acid ammonium salt (AHPB)), which is a structural analog of glutamate, is a recognized herbicide that acts on weeds through inhibition of glutamine synthetase. Due to the structural similarity between phosphinothricin and some bisphosphonates (BPs), this [...] Read more.

l-Phosphinothricin (glufosinate or 2-amino-4-((hydroxy(methyl) phosphinyl) butyric acid ammonium salt (AHPB)), which is a structural analog of glutamate, is a recognized herbicide that acts on weeds through inhibition of glutamine synthetase. Due to the structural similarity between phosphinothricin and some bisphosphonates (BPs), this study focuses on investigating the possibility of repurposing phosphinothricin as a bisphosphonate analogue, particularly in two medicine-related activities: image probing and as an anti-cancer drug. As BP is a competitive inhibitor of human farnesyl pyrophosphate synthase (HFPPS), in silico molecular docking and dynamic simulations studies were established to evaluate the binding and stability of phosphinothricin with HFPPS, while the results showed good binding and stability in the active site of the enzyme in relation to alendronate. For the purpose of inspecting bone-tissue accumulation of phosphinothricin, a technetium (^99mTc)–phosphinothricin complex was developed and its stability and tissue distribution were scrutinized. The radioactive complex showed rapid, high and sustained uptake into bone tissues. Finally, the cytotoxic activity of phosphinothricin was tested against breast and lung cancer cells, with the results indicating cytotoxic activity in relation to alendronate. All the above results provide support for the use of phosphinothricin as a potential anti-cancer drug and of its technetium complex as an imaging probe. Full article

(This article belongs to the Special Issue Molecular Imaging and Treatment Monitoring of Cancer)

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13 pages, 3627 KiB

Open AccessArticle

Physicochemical Properties and Chemical Stability of β-Carotene Bilayer Emulsion Coated with Bovine Serum Albumin and Arabic Gum Compared to Monolayer Emulsions

by Bulei Sheng, Lin Li, Xia Zhang, Wenjuan Jiao, Di Zhao, Xue Wang, Liting Wan, Bing Li and Hui Rong

Molecules 2018, 23(2), 495; https://doi.org/10.3390/molecules23020495 - 23 Feb 2018

Cited by 29 | Viewed by 7814

Abstract

β-carotene is a lipophilic micronutrient that is considered beneficial to human health. However, there are some limitations in utilizing β-carotene in functional foods or dietary supplements currently because of its poor water dispersibility and chemical stability. A new type of β-carotene bilayer emulsion [...] Read more.

β-carotene is a lipophilic micronutrient that is considered beneficial to human health. However, there are some limitations in utilizing β-carotene in functional foods or dietary supplements currently because of its poor water dispersibility and chemical stability. A new type of β-carotene bilayer emulsion delivery system was prepared by a layer-by-layer electrostatic deposition technique, for which were chosen bovine serum albumin (BSA) as the inner emulsifier and Arabic gum (GA) as the outer emulsifier. The physicochemical properties of bilayer emulsions were mainly characterized by droplet size distribution, zeta potential, rheological behavior, Creaming Index (CI), and encapsulation ratio of β-carotene. Besides this, the effects of processing conditions (pH, thermal treatment, UV radiation, strong oxidant) and storage time on the chemical stability of bilayer emulsions were also evaluated. The bilayer emulsion had a small droplet size (221.27 ± 5.17 nm) and distribution (PDI = 0.23 ± 0.02), strong zeta potential (−30.37 ± 0.71 mV), good rheological behavior (with the highest viscosity that could reduce the possibility of flocculation) and physical stability (CI = 0), high β-carotene encapsulation ratio (94.35 ± 0.71%), and low interfacial tension (40.81 ± 0.86 mN/m). It also obtained better chemical stability under different environmental stresses when compared with monolayer emulsions studied, because it had a dense and thick bilayer structure. Full article

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14 pages, 3201 KiB

Open AccessArticle

Melatonin-Mediated Development of Ovine Cumulus Cells, Perhaps by Regulation of DNA Methylation

by Yi Fang, Shoulong Deng, Jinlong Zhang, Haijun Liu, Yihai Li, Xiaosheng Zhang and Yixun Liu

Molecules 2018, 23(2), 494; https://doi.org/10.3390/molecules23020494 - 23 Feb 2018

Cited by 17 | Viewed by 5054

Abstract

Cumulus cells of pre-pubertal domestic animals are dysfunctional, perhaps due to age-specific epigenetic events. This study was designed to determine effects of melatonin treatment of donors on methylation modification of pre-pubertal cumulus cells. Cumulus cells from germinal vesicle stage cumulus oocyte complexes (COCs) [...] Read more.

Cumulus cells of pre-pubertal domestic animals are dysfunctional, perhaps due to age-specific epigenetic events. This study was designed to determine effects of melatonin treatment of donors on methylation modification of pre-pubertal cumulus cells. Cumulus cells from germinal vesicle stage cumulus oocyte complexes (COCs) were collected from eighteen lambs which were randomly divided into control group (C) and melatonin group given an 18 mg melatonin implant subcutaneous (M). Compared to the C group, the M group had higher concentrations of melatonin in plasma and follicular fluid (p < 0.05), greater superovulation, a higher proportion of fully expanded COCs, and a lower proportion of apoptotic cumulus cells (p < 0.05). Real-time PCR results showed that melatonin up-regulated expression of genes MT1, Bcl2, DNMT1, DNMT3a and DNMT3b, but down-regulated expression of genes p53, Caspase 3 and Bax (p < 0.05). Furthermore, melatonin increased FI of FITC (global methylation level) on cumulus cells (p < 0.05). To understand the regulation mechanism, the DNMTs promoter methylation sequence were analyzed. Compared to the C group, although there was less methylation at two CpG sites of DNMT1 (p < 0.05) and higher methylation at two CpG sites of DNMT3a (p < 0.05), there were no significant differences in methylation of the detected DNMT1 and DNMT3a promoter regions. However, there were lower methylation levels at five CpG sites of DNMT3b, which decreased methylation of detected DNMT3b promoter region on M group (p < 0.05). In conclusion, alterations of methylation regulated by melatonin may mediate development of cumulus cells in lambs. Full article

(This article belongs to the Special Issue Melatonin as an Antioxidant and a Functionally Pleiotropic Molecule: Synthesis, Metabolism and Activities in Organisms)

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Melatonin concentrations in plasma and follicular fluid. (n = 9). * p < 0.05 in <a href="#molecules-23-00494-f001" class="html-fig">Figure 1</a>A; a,b in <a href="#molecules-23-00494-f001" class="html-fig">Figure 1</a>B, for columns, means without a common superscript differed (p < 0.05). (A) Melatonin concentrations of plasma, (B) melatonin concentrations of follicular fluid. Full article ">Figure 2
(A) Ovaries after superovulation, (B) HE histological appearance (bar = 500 μm) and (C) expansion of cumulus cell after IVM (×10). Full article ">Figure 3
Effects of melatonin on cumulus cells apoptosis. Flow cytometry (A: C group; B: M group); mean ± SEM cumulus cells apoptosis (C). The experiment was repeated three times; Data presented as mean ± SEM; a,b in <a href="#molecules-23-00494-f003" class="html-fig">Figure 3</a>C, for columns, means without a common superscript differed (p < 0.05). Full article ">Figure 4
Effects of melatonin on expression of related genes in cumulus cells. Expression of related apoptosis genes in cumulus cells (A); expression of melatonin synthetase and receptors genes (B). The experiment was repeated three times; data presented as mean ± SEM; a,b for adjacent columns, means without a common superscript differed (p < 0.05). Full article ">Figure 5
Abundance of 5-methylcytosine (5mC) and gene expression of DNMTs in cumulus cells collected from C and M groups; (A) Representative immunofluorescence images of 5mC (green), and propidium iodide-stained nuclei (red). Scale bars, 20 μm. (B) global 5mC DNA in cumulus cells. (C) relative mRNA levels of DNMTs. The experiments were repeated three times; data presented as mean ± SEM; a,b in <a href="#molecules-23-00494-f005" class="html-fig">Figure 5</a>B,C, for adjacent columns, means without a common superscript differed (p < 0.05). Full article ">Figure 6
Methylation sequencing in cumulus cells; the experiment was repeated three times; loci within a circle differed (p < 0.05). Full article ">Figure 7
Gene structures of detected promoter region of DNMT1, DNMT3a and DNMT3b. Locations of CpG sites in this study indicated in blue were analyzed, whereas those indicated in red were either not uniquely discriminated in the spectra or had low call rates. Full article ">Figure A1
Superovulation scheme (n = 9). Full article ">

19 pages, 1793 KiB

Open AccessReview

Natural Alkaloids and Heterocycles as G-Quadruplex Ligands and Potential Anticancer Agents

by Tong Che, Yu-Qing Wang, Zhou-Li Huang, Jia-Heng Tan, Zhi-Shu Huang and Shuo-Bin Chen

Molecules 2018, 23(2), 493; https://doi.org/10.3390/molecules23020493 - 23 Feb 2018

Cited by 46 | Viewed by 6918

Abstract

G-quadruplexes are four-stranded nucleic acid secondary structures that are formed in guanine-rich sequences. G-quadruplexes are widely distributed in functional regions of the human genome and transcriptome, such as human telomeres, oncogene promoter regions, replication initiation sites, and untranslated regions. Many G-quadruplex-forming sequences are [...] Read more.

G-quadruplexes are four-stranded nucleic acid secondary structures that are formed in guanine-rich sequences. G-quadruplexes are widely distributed in functional regions of the human genome and transcriptome, such as human telomeres, oncogene promoter regions, replication initiation sites, and untranslated regions. Many G-quadruplex-forming sequences are found to be associated with cancer, and thus, these non-canonical nucleic acid structures are considered to be attractive molecular targets for cancer therapeutics with novel mechanisms of action. In this mini review, we summarize recent advances made by our lab in the study of G-quadruplex-targeted natural alkaloids and their derivatives toward the development of potential anticancer agents. Full article

(This article belongs to the Special Issue G-Quadruplex Ligands and Cancer)

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18 pages, 3775 KiB

Open AccessFeature PaperReview

Enantiomeric Mixtures in Natural Product Chemistry: Separation and Absolute Configuration Assignment

by Andrea N. L. Batista, Fernando M. dos Santos, João M. Batista and Quezia B. Cass

Molecules 2018, 23(2), 492; https://doi.org/10.3390/molecules23020492 - 23 Feb 2018

Cited by 52 | Viewed by 10014

Abstract

Chiral natural product molecules are generally assumed to be biosynthesized in an enantiomerically pure or enriched fashion. Nevertheless, a significant amount of racemates or enantiomerically enriched mixtures has been reported from natural sources. This number is estimated to be even larger since the [...] Read more.

Chiral natural product molecules are generally assumed to be biosynthesized in an enantiomerically pure or enriched fashion. Nevertheless, a significant amount of racemates or enantiomerically enriched mixtures has been reported from natural sources. This number is estimated to be even larger since the enantiomeric purity of secondary metabolites is rarely checked in the natural product isolation pipeline. This latter fact may have drastic effects on the evaluation of the biological activity of chiral natural products. A second bottleneck is the determination of their absolute configurations. Despite the widespread use of optical rotation and electronic circular dichroism, most of the stereochemical assignments are based on empirical correlations with similar compounds reported in the literature. As an alternative, the combination of vibrational circular dichroism and quantum chemical calculations has emerged as a powerful and reliable tool for both conformational and configurational analysis of natural products, even for those lacking UV-Vis chromophores. In this review, we aim to provide the reader with a critical overview of the occurrence of enantiomeric mixtures of secondary metabolites in nature as well the best practices for their detection, enantioselective separation using liquid chromatography, and determination of absolute configuration by means of vibrational circular dichroism and density functional theory calculations. Full article

(This article belongs to the Special Issue Chirality in Health and Environment: Recent developments)

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11 pages, 902 KiB

Open AccessArticle

Triterpenoid Saponins from Anemone rivularis var. Flore-Minore and Their Anti-Proliferative Activity on HSC-T6 Cells

by Xiao-Yang Wang, Hui Gao, Xiao-Jie Xie, Jirimubatu Jurhiin, Mu-Zi-He Zhang, Yan-Ping Zhou, Rui Liu, Meng Ning, Jin Han and Hai-Feng Tang

Molecules 2018, 23(2), 491; https://doi.org/10.3390/molecules23020491 - 23 Feb 2018

Cited by 6 | Viewed by 4462

Abstract

Five previously undescribed triterpenoid saponins (1–5), along with eight known ones (6–13), were isolated from the whole plants of Anemone rivularis var. flore-minore. Their structures were clarified by extensive spectroscopic data and chemical evidence. [...] Read more.

Five previously undescribed triterpenoid saponins (1–5), along with eight known ones (6–13), were isolated from the whole plants of Anemone rivularis var. flore-minore. Their structures were clarified by extensive spectroscopic data and chemical evidence. For the first time, the lupane-type saponins (3 and 12) were reported from the Anemone genus. The anti-proliferative activity of all isolated saponins was evaluated on hepatic stellate cells (HSC-T6). Saponins 12 and 13, which possess more monosaccharides than the others, displayed potent anti-proliferative activity, with IC₅₀ values of 18.21 and 15.56 μM, respectively. Full article

(This article belongs to the Section Natural Products Chemistry)

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10 pages, 3168 KiB

Open AccessArticle

Structural Dynamics of DPP-4 and Its Influence on the Projection of Bioactive Ligands

by Simone Queiroz Pantaleão, Eric Allison Philot, Pedro Túlio De Resende-Lara, Angélica Nakagawa Lima, David Perahia, Maria Atanassova Miteva, Ana Ligia Scott and Kathia Maria Honorio

Molecules 2018, 23(2), 490; https://doi.org/10.3390/molecules23020490 - 23 Feb 2018

Cited by 30 | Viewed by 9208

Abstract

Dipeptidyl peptidase-4 (DPP-4) is a target to treat type II diabetes mellitus. Therefore, it is important to understand the structural aspects of this enzyme and its interaction with drug candidates. This study involved molecular dynamics simulations, normal mode analysis, binding site detection and [...] Read more.

Dipeptidyl peptidase-4 (DPP-4) is a target to treat type II diabetes mellitus. Therefore, it is important to understand the structural aspects of this enzyme and its interaction with drug candidates. This study involved molecular dynamics simulations, normal mode analysis, binding site detection and analysis of molecular interactions to understand the protein dynamics. We identified some DPP-4 functional motions contributing to the exposure of the binding sites and twist movements revealing how the two enzyme chains are interconnected in their bioactive form, which are defined as chains A (residues 40–767) and B (residues 40–767). By understanding the enzyme structure, its motions and the regions of its binding sites, it will be possible to contribute to the design of new DPP-4 inhibitors as drug candidates to treat diabetes. Full article

(This article belongs to the Special Issue Computational Analysis for Protein Structure and Interaction)

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(A) Dimeric representation of the DPP-4 enzyme (PDB code: 4A5S [<a href="#B14-molecules-23-00490" class="html-bibr">14</a>,<a href="#B15-molecules-23-00490" class="html-bibr">15</a>]); (B) Representation of three binding sites of the DPP-4 enzyme found by FTSite [<a href="#B18-molecules-23-00490" class="html-bibr">18</a>,<a href="#B19-molecules-23-00490" class="html-bibr">19</a>,<a href="#B20-molecules-23-00490" class="html-bibr">20</a>] and FTMap [<a href="#B18-molecules-23-00490" class="html-bibr">18</a>,<a href="#B20-molecules-23-00490" class="html-bibr">20</a>,<a href="#B21-molecules-23-00490" class="html-bibr">21</a>,<a href="#B22-molecules-23-00490" class="html-bibr">22</a>] where regions 1 and 2 (colored in salmon and green, respectively) correspond to the active sites described in the literature (sites 1 and 2) and region 3 (colored in blue) is an alternative binding site (site 3) not described in the literature. (C) Key residues of the active site of DPP-4. Region 3 is a possible candidate as an allosteric binding site. Full article ">Figure 2
Indicative vectors of the direction of movement of the DPP-4 enzyme: (A) this figure corresponds to mode 7 (twisting motion between the chains); (B) this figure corresponds to mode 8 (active site exposure). The pink region is formed by Glu91, Asn92, Ser93, Thr94, Phe95, Asp96 and Glu97; blue is: Ser745, Thr746, Ala747, His748, Gln749, His750, Ile751, Tyr752, Thr753, His754, Met755, Ser756, His757, Phe758, Ile759, Lys760, Gln761, Cys762 and Phe763; and the green region is formed by: Phe713, Gln714, Gln715, Ser716, Ala717, Gln718, Ile719, Ser720, Lys721, Ala722, Leu723, Val724, Asp725 and Val726. Full article ">Figure 3
Fluctuation of α-carbon from DPP-4 (PDB: 4A5S [<a href="#B20-molecules-23-00490" class="html-bibr">20</a>,<a href="#B21-molecules-23-00490" class="html-bibr">21</a>]) at the presence and absence of inhibitor in the chains, with overlap of the two DPP-4 chains for the following systems: (a) dimer with inhibitor only in the chain A; (b) dimer with inhibitor only in the chain B; (c) dimer with inhibitor in both chains; (d) dimer without inhibitor. Full article ">Figure 3 Cont.
Fluctuation of α-carbon from DPP-4 (PDB: 4A5S [<a href="#B20-molecules-23-00490" class="html-bibr">20</a>,<a href="#B21-molecules-23-00490" class="html-bibr">21</a>]) at the presence and absence of inhibitor in the chains, with overlap of the two DPP-4 chains for the following systems: (a) dimer with inhibitor only in the chain A; (b) dimer with inhibitor only in the chain B; (c) dimer with inhibitor in both chains; (d) dimer without inhibitor. Full article ">Figure 4
Clusters containing the molecule probes anchored in the A-chain of DPP-4 (PDB 4A5S). Site 1 in salmon, site 2 in green and site 3 in blue. Full article ">

13 pages, 3320 KiB

Open AccessArticle

Fast Determination of Yttrium and Rare Earth Elements in Seawater by Inductively Coupled Plasma-Mass Spectrometry after Online Flow Injection Pretreatment

by Zuhao Zhu and Airong Zheng

Molecules 2018, 23(2), 489; https://doi.org/10.3390/molecules23020489 - 23 Feb 2018

Cited by 29 | Viewed by 5442

Abstract

A method for daily monitoring of yttrium and rare earth elements (YREEs) in seawater using a cheap flow injection system online coupled to inductively coupled plasma-mass spectrometry is reported. Toyopearl AF Chelate 650M^® resin permits separation and concentration of YREEs using a [...] Read more.

A method for daily monitoring of yttrium and rare earth elements (YREEs) in seawater using a cheap flow injection system online coupled to inductively coupled plasma-mass spectrometry is reported. Toyopearl AF Chelate 650M^® resin permits separation and concentration of YREEs using a simple external calibration. A running cycle consumed 6 mL sample and took 5.3 min, providing a throughput of 11 samples per hour. Linear ranges were up to 200 ng kg⁻¹ except Tm (100 ng kg⁻¹). The precision of the method was <6% (RSDs, n = 5), and recoveries ranged from 93% to 106%. Limits of detection (LODs) were in the range 0.002 ng kg⁻¹ (Tm) to 0.078 ng kg⁻¹ (Ce). Good agreement between YREEs concentrations in CASS-4 and SLEW-3 obtained in this work and results from other studies was observed. The proposed method was applied to the determination of YREEs in seawater from the Jiulong River Estuary and the Taiwan Strait. Full article

(This article belongs to the Special Issue Innovative Extraction Techniques and Hyphenated Instrument Configuration for Complex Matrices Analysis)

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12 pages, 2342 KiB

Open AccessArticle

An Interaction of Rhamnolipids with Cu²⁺ Ions

by Jolanta Cieśla, Magdalena Koczańska and Andrzej Bieganowski

Molecules 2018, 23(2), 488; https://doi.org/10.3390/molecules23020488 - 23 Feb 2018

Cited by 15 | Viewed by 4195

Abstract

This study was focused on the description of interaction between Cu²⁺ ions and the 1:1 mono- and dirhamnolipid mixtures in the premicellar and aggregated state in water and 20 mM KCl solution at pH 5.5 and 6.0. The critical micelle concentration of [...] Read more.

This study was focused on the description of interaction between Cu²⁺ ions and the 1:1 mono- and dirhamnolipid mixtures in the premicellar and aggregated state in water and 20 mM KCl solution at pH 5.5 and 6.0. The critical micelle concentration of biosurfactants was determined conductometrically and by the pH measurements. Hydrodynamic diameter and electrophoretic mobility were determined in micellar solutions using dynamic light scattering and laser Doppler electrophoresis, respectively. The copper immobilization by rhamnolipids, methylglycinediacetic acid (MGDA), and ethylenediaminetetraacetic acid (EDTA) was estimated potentiometrically for the Cu²⁺ to chelating agent molar ratio from 16:100 to 200:100. The degree of ion binding and the complex stability constant were calculated at a 1:1 metal to chelant molar ratio. The aggregates of rhamnolipids (diameter of 43–89 nm) were negatively charged. Biosurfactants revealed the best chelating activities in premicellar solutions. For all chelants studied the degree of metal binding decreased with the increasing concentration of the systems. The presence of K⁺ lowered Cu²⁺ binding by rhamnolipids, but did not modify the complex stability significantly. Immobilization of Cu²⁺ by biosurfactants did not cause such an increase of acidification as that observed in MGDA and EDTA solutions. Rhamnolipids, even in the aggregated form, can be an alternative for the classic chelating agents. Full article

(This article belongs to the Section Nanochemistry)

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The structures of (a) monorhamnolipid; (b) dirhamnolipid; (c) ethylenediaminetetraacetic acid (EDTA); and (d) methylglycinediacetic acid (MGDA). Full article ">Figure 2
Dependence of electrolytic conductivity (EC) and pH on the rhamnolipids (Rh) concentration in water (a,c) and KCl solution (b,d) at pH 5.5 (a,b) and 6.0 (c,d); bars represent standard deviation. Full article ">Figure 3
(a) Hydrodynamic diameter (dh,app); (b) polydispersity index (PDI); (c) aggregation index (AI); and (d) electrophoretic mobility (EM), which were determined in micellar solutions of rhamnolipids (Rh) in water and 20 mM KCl at pH 5.5 and 6.0 without and with the presence of Cu2+ ions (at the 1:1 Cu2+ to rhamnolipid molar ratio). Full article ">Figure 4
Dependence of pH and pCufixed (the negative logarithm of the concentration of Cu2+ ions fixed by chelating agent) on the Cu2+ to chelating agent molar ratio (nCu:nchelating agent) in water (a,c,e) and KCl solution (b,d,f) at pH 5.5; (a,b) rhamnolipids (Rh); (c,d) methylglycinediacetic acid (MGDA); and (e,f) ethylenediaminetetraacetic acid (EDTA); CMCRh means the rhamnolipids’ critical micelle concentration. Full article ">Figure 5
Dependence of pH and pCufixed (the negative logarithm of the concentration of Cu2+ ions fixed by chelating agent) on the Cu2+ to chelating agent molar ratio (nCu:nchelating agent) in water (a,c,e) and KCl solution (b,d,f) at pH 6.0; (a,b) rhamnolipids (Rh); (c,d) methylglycinediacetic acid (MGDA); and (e,f) ethylenediaminetetraacetic acid (EDTA); CMCRh means the rhamnolipids’ critical micelle concentration. Full article ">

9 pages, 430 KiB

Open AccessArticle

Fast and Simple Analytical Method for Direct Determination of Total Chlorine Content in Polyglycerol by ICP-MS

by Agata Jakóbik-Kolon, Andrzej Milewski, Piotr Dydo, Magdalena Witczak and Joanna Bok-Badura

Molecules 2018, 23(2), 487; https://doi.org/10.3390/molecules23020487 - 23 Feb 2018

Cited by 6 | Viewed by 4055

Abstract

The fast and simple method for total chlorine determination in polyglycerols using low resolution inductively coupled plasma mass spectrometry (ICP-MS) without the need for additional equipment and time-consuming sample decomposition was evaluated. Linear calibration curve for ³⁵Cl isotope in the concentration range [...] Read more.

The fast and simple method for total chlorine determination in polyglycerols using low resolution inductively coupled plasma mass spectrometry (ICP-MS) without the need for additional equipment and time-consuming sample decomposition was evaluated. Linear calibration curve for ³⁵Cl isotope in the concentration range 20–800 µg/L was observed. Limits of detection and quantification equaled to 15 µg/L and 44 µg/L, respectively. This corresponds to possibility of detection 3 µg/g and determination 9 µg/g of chlorine in polyglycerol using studied conditions (0.5% matrix-polyglycerol samples diluted or dissolved with water to an overall concentration of 0.5%). Matrix effects as well as the effect of chlorine origin have been evaluated. The presence of 0.5% (m/m) of matrix species similar to polyglycerol (polyethylene glycol—PEG) did not influence the chlorine determination for PEGs with average molecular weights (MW) up to 2000 Da. Good precision and accuracy of the chlorine content determination was achieved regardless on its origin (inorganic/organic). High analyte recovery level and low relative standard deviation values were observed for real polyglycerol samples spiked with chloride. Additionally, the Combustion Ion Chromatography System was used as a reference method. The results confirmed high accuracy and precision of the tested method. Full article

(This article belongs to the Special Issue Green Analytical Chemistry)

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16 pages, 7229 KiB

Open AccessArticle

Reconstructing Phylogeny by Aligning Multiple Metabolic Pathways Using Functional Module Mapping

by Yiran Huang, Cheng Zhong, Hai Xiang Lin, Jianyi Wang and Yuzhong Peng

Molecules 2018, 23(2), 486; https://doi.org/10.3390/molecules23020486 - 23 Feb 2018

Cited by 2 | Viewed by 4422

Abstract

Comparison of metabolic pathways provides a systematic way for understanding the evolutionary and phylogenetic relationships in systems biology. Although a number of phylogenetic methods have been developed, few efforts have been made to provide a unified phylogenetic framework that sufficiently reflects the metabolic [...] Read more.

Comparison of metabolic pathways provides a systematic way for understanding the evolutionary and phylogenetic relationships in systems biology. Although a number of phylogenetic methods have been developed, few efforts have been made to provide a unified phylogenetic framework that sufficiently reflects the metabolic features of organisms. In this paper, we propose a phylogenetic framework that characterizes the metabolic features of organisms by aligning multiple metabolic pathways using functional module mapping. Our method transforms the alignment of multiple metabolic pathways into constructing the union graph of pathways, builds mappings between functional modules of pathways in the union graph, and infers phylogenetic relationships among organisms based on module mappings. Experimental results show that the use of functional module mapping enables us to correctly categorize organisms into main categories with specific metabolic characteristics. Traditional genome-based phylogenetic methods can reconstruct phylogenetic relationships, whereas our method can offer in-depth metabolic analysis for phylogenetic reconstruction, which can add insights into traditional phyletic reconstruction. The results also demonstrate that our phylogenetic trees are closer to the classic classifications in comparison to existing classification methods using metabolic pathway data. Full article

(This article belongs to the Special Issue Selected Papers from the Second CCF Bioinformatics Conference (CBC 2017))

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7 pages, 1046 KiB

Open AccessFeature PaperArticle

Epitaxially Grown Ultra-Flat Self-Assembling Monolayers with Dendrimers

by Takane Imaoka, Noriko Bukeo and Kimihisa Yamamoto

Molecules 2018, 23(2), 485; https://doi.org/10.3390/molecules23020485 - 23 Feb 2018

Viewed by 3768

Abstract

Mono-molecular films formed by physical adsorption and dendrimer self-assembly were prepared on various substrate surfaces. It was demonstrated that a uniform dendrimer-based monolayer on the subnanometer scale can be easily constructed via simple dip coating. Furthermore, it was shown that an epitaxially grown [...] Read more.

Mono-molecular films formed by physical adsorption and dendrimer self-assembly were prepared on various substrate surfaces. It was demonstrated that a uniform dendrimer-based monolayer on the subnanometer scale can be easily constructed via simple dip coating. Furthermore, it was shown that an epitaxially grown monolayer film reflecting the crystal structure of the substrate (highly ordered pyrolytic graphite (HOPG)) can also be formed by aligning specific conditions. Full article

(This article belongs to the Special Issue Dendrimers: A Themed Issue in Honor of Professor Donald A. Tomalia on the Occasion of His 80th Birthday)

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15 pages, 263 KiB

Open AccessArticle

Impact of Storage Conditions on the Stability of Predominant Phenolic Constituents and Antioxidant Activity of Dried Piper betle Extracts

by Ameena Ali, Chien Hwa Chong, Siau Hui Mah, Luqman Chuah Abdullah, Thomas Shean Yaw Choong and Bee Lin Chua

Molecules 2018, 23(2), 484; https://doi.org/10.3390/molecules23020484 - 23 Feb 2018

Cited by 94 | Viewed by 7214

Abstract

The phenolic constituents in Piper betle are well known for their antioxidant potential; however, current literature has very little information on their stability under the influence of storage factors. Present study evaluated the stability of total phenolic content (TPC) and antioxidant activity together [...] Read more.

The phenolic constituents in Piper betle are well known for their antioxidant potential; however, current literature has very little information on their stability under the influence of storage factors. Present study evaluated the stability of total phenolic content (TPC) and antioxidant activity together with individual phenolic constituents (hydroxychavicol, eugenol, isoeugenol and allylpyrocatechol 3,4-diacetate) present in dried Piper betle’s extract under different storage temperature of 5 and 25 °C with and without light for a period of six months. Both light and temperature significantly influenced TPC and its corresponding antioxidant activity over time. More than 95% TPC and antioxidant activity was retained at 5 °C in dark condition after 180 days of storage. Hydroxychavicol demonstrated the best stability with no degradation while eugenol and isoeugenol displayed moderate stability in low temperature (5 °C) and dark conditions. 4-allyl-1,2-diacetoxybenzene was the only compound that underwent complete degradation. A new compound, 2,4-di-tert-butylphenol, was detected after five weeks of storage only in the extracts exposed to light. Both zero-order and first-order kinetic models were adopted to describe the degradation kinetics of the extract’s antioxidant activity. Zero-order displayed better fit with higher correlation coefficients (R² = 0.9046) and the half-life was determined as 62 days for the optimised storage conditions (5 °C in dark conditions). Full article

(This article belongs to the Section Natural Products Chemistry)

13 pages, 1232 KiB

Open AccessArticle

Persicaline, A New Antioxidant Sulphur-Containing Imidazoline Alkaloid from Salvadora persica Roots

by Mohamed Farag, Wael M. Abdel-Mageed, Omer Basudan and Ali El-Gamal

Molecules 2018, 23(2), 483; https://doi.org/10.3390/molecules23020483 - 23 Feb 2018

Cited by 26 | Viewed by 6103

Abstract

Salvadora persica L. is a popular chewing stick commonly known as “miswak”. During our ongoing research activities on the chemical constituents of Salvadora persica roots, which is a new sulphur-containing imidazoline alkaloid 1,3-Dibenzyl-4-(1,2,3,4-tetrahydroxy-butyl)-1,3-dihydro-imidazole-2-thione, persicaline, (1) along with five known compounds ( [...] Read more.

Salvadora persica L. is a popular chewing stick commonly known as “miswak”. During our ongoing research activities on the chemical constituents of Salvadora persica roots, which is a new sulphur-containing imidazoline alkaloid 1,3-Dibenzyl-4-(1,2,3,4-tetrahydroxy-butyl)-1,3-dihydro-imidazole-2-thione, persicaline, (1) along with five known compounds (2–6) are identified. Compounds (2, 3) were reported for the first time from the family Salvadoraeceae. The structure of the new compound was established by extensive spectroscopic data and HR-MS. The antioxidant activities of the fractions and isolates were evaluated using different in vitro methods, such as DPPH, superoxide anion and nitric oxide radicals scavenging assays. Compound (1) showed a promising antioxidant activity with IC₅₀ 0.1, 0.08, and 0.09 µM in the three assays, respectively, comparable to ascorbic acid. Full article

(This article belongs to the Collection Bioactive Compounds)

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13 pages, 1333 KiB

Open AccessFeature PaperArticle

Antimicrobial and Antibiofilm Activity and Machine Learning Classification Analysis of Essential Oils from Different Mediterranean Plants against Pseudomonas aeruginosa

by Marco Artini, Alexandros Patsilinakos, Rosanna Papa, Mijat Božović, Manuela Sabatino, Stefania Garzoli, Gianluca Vrenna, Marco Tilotta, Federico Pepi, Rino Ragno and Laura Selan

Molecules 2018, 23(2), 482; https://doi.org/10.3390/molecules23020482 - 23 Feb 2018

Cited by 66 | Viewed by 8591

Abstract

Pseudomonas aeruginosa is a ubiquitous organism and opportunistic pathogen that can cause persistent infections due to its peculiar antibiotic resistance mechanisms and to its ability to adhere and form biofilm. The interest in the development of new approaches for the prevention and treatment [...] Read more.

Pseudomonas aeruginosa is a ubiquitous organism and opportunistic pathogen that can cause persistent infections due to its peculiar antibiotic resistance mechanisms and to its ability to adhere and form biofilm. The interest in the development of new approaches for the prevention and treatment of biofilm formation has recently increased. The aim of this study was to seek new non-biocidal agents able to inhibit biofilm formation, in order to counteract virulence rather than bacterial growth and avoid the selection of escape mutants. Herein, different essential oils extracted from Mediterranean plants were analyzed for their activity against P. aeruginosa. Results show that they were able to destabilize biofilm at very low concentration without impairing bacterial viability. Since the action is not related to a bacteriostatic/bactericidal activity on P. aeruginosa, the biofilm change of growth in presence of the essential oils was possibly due to a modulation of the phenotype. To this aim, application of machine learning algorithms led to the development of quantitative activity–composition relationships classification models that allowed to direct point out those essential oil chemical components more involved in the inhibition of biofilm production. The action of selected essential oils on sessile phenotype make them particularly interesting for possible applications such as prevention of bacterial contamination in the community and in healthcare environments in order to prevent human infections. We assayed 89 samples of different essential oils as P. aeruginosa anti-biofilm. Many samples inhibited P. aeruginosa biofilm at concentrations as low as 48.8 µg/mL. Classification of the models was developed through machine learning algorithms. Full article

(This article belongs to the Special Issue Essential Oils as Antimicrobial and Anti-infectious Agents)

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15 pages, 13188 KiB

Open AccessArticle

Antioxidant Activity and Protective Effects of Enzyme-Extracted Oudemansiella radiata Polysaccharides on Alcohol-Induced Liver Injury

by Xiuxiu Wang, Min Liu, Chen Zhang, Shangshang Li, Qihang Yang, Jianjun Zhang, Zhiyuan Gong, Jiandong Han and Le Jia

Molecules 2018, 23(2), 481; https://doi.org/10.3390/molecules23020481 - 23 Feb 2018

Cited by 31 | Viewed by 5782

Abstract

This work was to examine the antioxidation in vitro and hepatoprotective effects of enzyme-extracted Oudemansiella radiata polysaccharides (En-OPS) on alcohol-induced liver damage in mice. The antioxidant activities were determined according to the scavenging effects of En-OPS on hydroxyl, superoxide, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, [...] Read more.

This work was to examine the antioxidation in vitro and hepatoprotective effects of enzyme-extracted Oudemansiella radiata polysaccharides (En-OPS) on alcohol-induced liver damage in mice. The antioxidant activities were determined according to the scavenging effects of En-OPS on hydroxyl, superoxide, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, and the level of reducing power. En-OPS showed hepatoprotective activities on decreasing the serum levels of aspertate aminotransferase (AST), alamine aminotransferase (ALT), and alkaline phosphatase (ALP), as well as hepatic lipid levels of total cholesterol (TC) and triacylglycerols (TG). En-OPS treatment reversed the acute impairment induced by alcohol consumption, including reactive oxygen species (ROS) generation, malondialdehyde (MAD), and lipid peroxide (LPO) elevation; and superoxide dismutase (SOD), GSH peroxide (GSH-Px), catalase (CAT), and total antioxidant capacity (T-AOC) impairment. The En-OPS effectively ameliorated alcohol metabolism by activating alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), and reducing cytochrome P450 2E1 (CYP2E1) levels. Furthermore, the histopathological observations also displayed that En-OPS could alleviate liver damage. These results indicated that En-OPS could be suitable to be an ingredient of preventing alcoholic liver diseases (ALD). In addition, the preliminary structure characteristics of En-OPS were also analyzed by Fourier transform infrared (FT-IR) spectroscopy and a gas chromatography-flame ionization detector (GC-FID). Full article

(This article belongs to the Special Issue Advances in Natural Polysaccharides Research)

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Antioxidant activities of En-OPS in vitro. (A) hydroxyl radical-scavenging abilities; (B) superoxide radical-scavenging abilities; (C) 1,1-diphenyl-2-picrylhydrazyl radical-scavenging abilities; and (D) reducing power. “#” indicated that the values differ significantly at p < 0.05 (En-OPS compared with BHT at different concentration). The statistical significance of the differences between the groups were analyzed using Tukey’s tests in the software package (SAS 9.3). A value of p < 0.05 was regarded as statistically significant. Full article ">Figure 2
Effects of En-OPS on the enzymic activities in serum of mice. (A) aspertate aminotransferase; (B) alamine aminotransferase; and (C) alkaline phosphatase. The values were reported as the mean ± S.D. of 10 mice per group. Bars with different letters were significantly different (p < 0.05). The statistical significance of the differences between the groups were analyzed using Tukey’s tests in the software package (SAS 9.3). A value of p < 0.05 was regarded as statistically significant. Full article ">Figure 3
Effects of En-OPS on biochemical parameters in liver of mice. (A) superoxide dismutase; (B) GSH peroxide; (C) catalase; and (D) total antioxidant capacity, and contents of (E) lipid peroxide and (F) malondialdehyde. The values were reported as the mean ± S.D. of 10 mice per group. Bars with different letters were significantly different (p < 0.05). The statistical significance of the differences between the groups were analyzed using Tukey’s tests in the software package (SAS 9.3). A value of p < 0.05 was regarded as statistically significant. Full article ">Figure 4
Effects of En-OPS on lipid levels and enzymic activities in liver of mice. (A) total cholesterol levels; (B) triacylglycerols levels; (C) alcohol dehydrogenase activities; (D) aldehyde dehydrogenase activities; and (E) cytochrome P450 2E1 levels. The values were reported as the mean ± S.D. of 10 mice per group. Bars with different letters were significantly different (p < 0.05). The statistical significance of the differences between the groups were analyzed using Tukey’s tests in the software package (SAS 9.3). A value of p < 0.05 was regarded as statistically significant. Full article ">Figure 5
Effects of En-OPS on hepatic cells in liver tissue of alcohol-intoxicated mice. (A) normal control groups; (B) model control groups; (C) positive control groups; and (D–F) groups treated with 200, 400, and 800 mg/kg En-OPS (magnification 400×). Cellular degeneration ( <img alt="Molecules 23 00481 i001" src="/molecules/molecules-23-00481/article_deploy/html/images/molecules-23-00481-i001.png"/>), lipid droplet accumulation ( <img alt="Molecules 23 00481 i002" src="/molecules/molecules-23-00481/article_deploy/html/images/molecules-23-00481-i002.png"/>). Full article ">Figure 6
Fourier transform infrared (FT-IR) spectrum of En-OPS. Full article ">Figure 7
Gas chromatography (GC) chromatograms of monosaccharide. (A) Standard sugars; (B) En-OPS. Peaks: (1) rhamnose, (2) fucose, (3) ribose, (4) arabinose, (5) xylose, (6) mannose, (7) galactose, and (8) glucose. Full article ">

11 pages, 760 KiB

Open AccessReview

Sulphated Flavonoids: Biosynthesis, Structures, and Biological Activities

by Yanna C. F. Teles, Maria Sallett R. Souza and Maria De Fátima Vanderlei de Souza

Molecules 2018, 23(2), 480; https://doi.org/10.3390/molecules23020480 - 23 Feb 2018

Cited by 119 | Viewed by 9091

Abstract

The great diversity of enzymatic reactions in plant secondary metabolism allows the continuous discovery of new natural compounds and derivatives. Flavonoids, for example, can be found as aglycone or as several sorts of glycosylated, acetylated, methylated, and sulphated derivatives. This review focuses on [...] Read more.

The great diversity of enzymatic reactions in plant secondary metabolism allows the continuous discovery of new natural compounds and derivatives. Flavonoids, for example, can be found as aglycone or as several sorts of glycosylated, acetylated, methylated, and sulphated derivatives. This review focuses on sulphated flavonoids, an uncommon group of flavonoid derivatives found in some plant families. This work presents a compilation of sulphated flavonoids and their natural sources reported in the literature. Biosynthetic aspects and biological activities have also been reviewed, showing that these particular kinds of natural compounds play an interesting role in plant metabolism, as well as being potential candidates for the development of new drugs. Full article

(This article belongs to the Collection Phytochemicals: Biosynthesis, Metabolism and Biological Activities)

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