Solvent-free enzymatic synthesis and evaluation of vanillyl propionate as an effective and biocompatible preservative.

Preservatives are chemicals added to protect products against microbial spoilage, and thus are indispensable for pharmaceuticals, cosmetics, and foods. Due to growing concerns about human health and environments in conventional chemical preservatives, many companies have been seeking safe and effective alternatives that can be produced through environment-friendly processes. In this work, in order to develop effective and safe preservatives from plants, we attempt solvent-free lipase-catalyzed transesterification of vanillyl alcohol with ethyl propionate for the first time. The reaction product, vanillyl propionate was efficiently obtained in a high yield. Unlike vanillyl alcohol and ethyl propionate, vanillyl propionate showed antimicrobial activity. The minimal inhibitory concentration test showed that it exhibited high and broad antimicrobial activity against all the tested microorganisms (Gram-negative and Gram-positive bacteria, yeasts, and molds), which was overall comparable to that of propyl paraben, which is one of the most effective preservatives. It was also found to have even higher antioxidant capacity and biocompatibility with human cells than propyl paraben. Vanillyl propionate, which is a plant-based preservative produced through a green bioprocess, is expected to be successfully applied to various industries thanks to its high antimicrobial and antioxidant effect, and high biocompatibility.

Disruption of type I interferon pathway and reduced production of IFN-α by parabens in virus-infected dendritic cells.

BACKGROUND: Parabens are widely used preservatives commonly found in foods, cosmetics, and industrial products. Several studies have examined the effects of parabens on human health owing to widespread and continuous exposure to them in daily life. However, little is known about their immune-regulatory effects. OBJECTIVE: Here, we aimed to investigate whether methylparaben, ethylparaben, and propylparaben affect the function of dendritic cells (DCs) as the most potent antigen-presenting cells that play a critical role in the initiation of adaptive immune responses. METHODS: Bone-marrow derived DCs (BMDCs) were treated with three types of parabens (methylparaben, ethylparaben, and propylparaben) for 12 h. Subsequently, the transcriptomic profile was analyzed using RNA sequencing with further gene set enrichment analysis based on commonly regulated differentially expressed genes (DEGs). To test whether parabens suppress the production of type-I interferons (IFN-I) in BMDCs during viral infection, BMDCs or paraben-treated BMDCs were infected with Lymphocytic Choriomeningitis Virus (LCMV) at 10 multiplicity of infection (MOI) and measured the production of IFN-α1. RESULTS: Transcriptomic analyses revealed that all three types of parabens reduced the transcription levels of genes in virus infection-associated pathways, such as IFN-I responses in BMDCs. Furthermore, parabens considerably reduced IFN-α1 production in the virus-infected BMDCs. CONCLUSION: Our study is the first to show that parabens may modulate anti-viral immune responses by regulating DCs.

In vitro reciprocal interactions between yeasts from human cutaneous mycobiota and parabens used in cosmetics.

Parabens are substances with antifungal and antibacterial properties, suspected to be endocrine disruptors and widely used as preservatives in cosmetics. In this case, exposure to these compounds is mainly dermal and interactions may occur with skin components including cutaneous mycobiota. In this work, we have explored the in vitro reciprocal interactions between three parabens (methylparaben, ethylparaben, and propylparaben) and yeasts from the human cutaneous mycobiota (Candida parapsilosis, Cryptococcus uniguttulatus, and Rhodotorula mucilaginosa) by studying the effect of these parabens on fungal growth and the fungal ability to metabolize the tested compounds. Our results showed that, at the tested concentrations, the growth of three strains of C. parapsilosis was not influenced by the presence of parabens. Whereas, using the same parabens concentrations, growth of C. uniguttulatus and R. mucilaginosa was completely inhibited by ethylparaben since the first day of contact, whereas these same fungi were not sensitive to the two other parabens, even after seven days of incubation. The presence of a lamellar wall in these basidiomycete fungi as well as the physico-chemical properties of ethylparaben could explain this selective inhibition. Additionally, C. parapsilosis and R. mucilaginosa degraded 90% to 100% of propylparaben after seven days of incubation but had no effect on the other tested parabens. Thus, their enzymes seem to only degrade long chain parabens. In the same conditions, C. uniguttulatus did not degrade any paraben. This inability may be due to the absence of fungal enzymes able to degrade parabens or to the possible inaccessibility of intracellular enzymes due to the polysaccharide capsule. Our work has shown that parabens can act differently from one fungus to another within the cutaneous mycobiota. These preliminary results have evidenced that in vitro parabens, contained in cosmetic products, could be involved in the occurrence of a state of dysbiosis. The tested yeasts from the cutaneous mycobiota can also be involved in the degradation of parabens and thereby reduce, according to the produced metabolites and their activities, the risk of endocrine disruption they can induce.

Investigation of combined effects of propyl paraben and methyl paraben on the hypothalamic-pituitary-adrenal axis in male rats.

The aim of this study was to investigate the endocrine-disrupting effects of methyl paraben (MeP) and propyl paraben (PrP) mixture on the hypothalamic-pituitary-adrenal axis (HPA). In this study, six experimental groups were designated. These groups included three control groups (control, corn oil control, and positive control (50 mg/kg/day BPA)) and three dose groups (10, 100, and 500 mg/kg/day MeP+PrP). MeP with PrP were mixed in a 1:1 ratio and administered to the 42-day-old male rats by oral gavage for 30 days. At the end of the experiment, adrenocorticotropic hormone (ACTH), corticosterone and aldosterone hormones were analyzed in serum. Effects of MeP+PrP on the adrenal glands were investigated by immunohistochemical staining of 11ß hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) enzymes involved in the synthesis steps of corticosterone and aldosterone. Also, pituitary and adrenal glands were examined histopathologically. In the histopathological findings, cortical nodule, congestion, and edema were found in the tissues. In the pituitary gland, cytokeratin rings were detected in all MeP+PrP dose groups, supporting the increase of corticosterone and ACTH. Serum corticosterone, aldosterone, and ACTH hormone levels were increased in the 100 mg/kg/day MeP+PrP and BPA groups. Results obtained from immunohistochemical staining showed that increased staining parallelled increased corticosterone and aldosterone hormone levels. In summary, the results showed that exposure to the MeP+PrP mixture caused a significant increase in ACTH and corticosterone. Also, the MeP+PrP mixture caused a significant increase of CYP11B1 and CYP11B2. MeP+PrP exposure disrupts the normal HPA axis.

The SH-SY5Y human neuroblastoma cell line, a relevant in vitro cell model for investigating neurotoxicology in human: Focus on organic pollutants.

Investigation of the toxicity triggered by chemicals on the human brain has traditionally relied on approaches using rodent in vivo models and in vitro cell models including primary neuronal cultures and cell lines from rodents. The issues of species differences between humans and rodents, the animal ethical concerns and the time and cost required for neurotoxicity studies on in vivo animal models, do limit the use of animal-based models in neurotoxicology. In this context, human cell models appear relevant in elucidating cellular and molecular impacts of neurotoxicants and facilitating prioritization of in vivo testing. The SH-SY5Y human neuroblastoma cell line (ATCC® CRL-2266™) is one of the most used cell lines in neurosciences, either undifferentiated or differentiated into neuron-like cells. This review presents the characteristics of the SH-SY5Y cell line and proposes the results of a systematic review of literature on the use of this in vitro cell model for neurotoxicity research by focusing on organic environmental pollutants including pesticides, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), flame retardants, PFASs, parabens, bisphenols, phthalates, and PAHs. Organic environmental pollutants are widely present in the environment and increasingly known to cause clinical neurotoxic effects during fetal & child development and adulthood. Their effects on cultured SH-SY5Y cells include autophagy, cell death (apoptosis, pyroptosis, necroptosis, or necrosis), increased oxidative stress, mitochondrial dysfunction, disruption of neurotransmitter homeostasis, and alteration of neuritic length. Finally, the inherent advantages and limitations of the SH-SY5Y cell model are discussed in the context of chemical testing.

Evaluation of the Effect of Isobutyl Paraben and 2-ethyl Hexyl Paraben on P-glycoprotein Functional Expression in Rats: A Pharmacokinetic Study.

BACKGROUND: Pharmaceutical excipients have been shown to influence drug disposition through modulating transport protein. OBJECTIVES: This study assessed the effect of single dose administration of parabens on the pharmacokinetics (PK) of digoxin, a probe substrate of p-glycoprotein (p-gp), in vivo. Also, the effect of multiple dosing of parabens on p-gp expression was examined. METHODS: Rats were randomized into four groups that received either the vehicle, 25 mg/ kg verapamil, 100 mg/ kg isobutyl paraben, or 100 mg/ kg 2-ethyl hexyl paraben, which was followed by giving 0.2 mg/ kg digoxin via oral gavage. Blood samples were collected at different time points, digoxin concentration was measured using LC/MS-MS, and digoxin PK parameters were estimated. Another set of rats received multiple doses of parabens for 14 days, followed by measuring intestinal and hepatic mRNA expression of p-gp using qRT-PCR. RESULTS: Single dose administration of verapamil significantly increased Cmax (by 60.4 %) and AUC0-t (by 61.7 %) of digoxin compared to the control group, while the PK parameters of digoxin in rats exposed to parabens were not significantly different from the control. Consistently, the mRNA expression of p-gp in the intestine and liver was not affected by parabens treatment. CONCLUSIONS: The lack of isobutylparaben and 2-ethylhexyl paraben effect on p-gp may suggest the insignificant interaction of parabens with p-gp drug substrates, which could be considered for safety when designing pharmaceutical formulations.

Óleo essencial de Pataqueira (Conobea scoparioides Cham. & Schltdl.) como conservante natural em Cosméticos / Pataqueira essential oil (Conobea scoparioides Cham. & Schltdl.) as a natural preservative in cosmetics

Um dos principais grupos de conservantes utilizados na maioria dos cosméticos são os parabenos que em muitos estudos demonstraram que podem provocar reações alérgicas como dermatite de contato, entre outras sensibilizações cutâneas. A fim de minimizar esses problemas, a indústria está produzindo cosméticos livres de conservantes ou de origem natural e em associações aos sintéticos. Dentre os conservantes naturais utilizados, podemos citar os óleos essenciais como uma alternativa viável. Diante deste contexto o presente trabalho visa avaliar experimentalmente o potencial antimicrobiano do óleo essencial de Conobea scoparioides Cham. & Schltdl., conhecida popularmente como pataqueira, o efeito de sua associação com parabenos e de sua eficácia como conservante em bases cosméticas. A composição do óleo essencial foi avaliada, indicando que este é composto em sua maior parte por terpenos, tendo éter metílico do timol (39,2%), timol (33,8 %) e α-felandreno (15,9%) como compostos majoritários. A atividade antimicrobiana do óleo essencial e do timol foi acessada através da concentração inibitória mínima (CIM), cujos resultados em µg/mL para o óleo essencial e o timol foram respectivamente: Staphylococcus aureus 650,70 e 284,90, Escherichia coli 721,53 e 271,20, Pseudomonas aeruginosa 1748,00 e > 2.000, Burkholderia cepacia 833,03 e 1.077,70, Candida albicans 521,43 e 172,61 e Aspergillus brasiliensis 300 e 400. O efeito sinérgico da associação do óleo essencial com os parabenos foi realizado através de um delineamento experimental centroide simplex para uma mistura de metilparabeno, propilparabeno e óleo essencial frente aos mesmos micro-organismos utilizados na determinação da atividade antimicrobiana. As concentrações ideais obtidas pela análise estatística para cada componente em µg/mL foram: 1120 para o metilparabeno, 350 para o propilparabeno e 675 para o óleo essencial. O teste de eficácia do sistema conservante em formulação cosmética foi efetuado empregando as concentrações ideais e mais duas concentrações superiores e uma abaixo do ideal. Para todas as cepas microbianas desafiadas o resultado do teste foi de redução total da carga microbiana inoculada nos sete dias de ensaio e nenhum aumento até o vigésimo oitavo dia o que demonstra a eficácia da associação do óleo essencial com os conservantes sintéticos. O óleo essencial de C. scoparioides apresentou um potencial antimicrobiano importante tanto sozinho como em associação com conservantes sintéticos. Estes resultados sugerem que esse óleo pode ser usado para compor um sistema conservante para formulações cosméticas contendo uma menor quantidade de sintéticos

One of the main groups of preservatives used in most cosmetics are parabens, that many studies have shown that they can cause allergic reactions such as contact dermatitis, among other skin sensitizations. To minimize these problems, the industry is producing cosmetics preservative free or using natural products instead and their combination with the synthetics. Among the natural preservatives used, we can mention essential oils as a viable alternative. In this context, the present work aims to experimentally evaluate the antimicrobial potential of the Conobea scoparioides Cham. & Schltdl. essential oil, popularly known as pataqueira, the effect of its association with parabens and its effectiveness as a preservative in cosmetic bases. The essential oil composition was analyzed, indicating that it is composed mostly of terpenes, with thymol methyl ether (39.2%), thymol (33.8%) and -phelandrene (15.9%) as major compounds. The antimicrobial activity of essential oil and thymol was accessed through the minimum inhibitory concentration (MIC), whose results in µg/mL for essential oil and thymol were respectively: Staphylococcus aureus 650.70 and 284.90, Escherichia coli 721, 53 and 271.20, Pseudomonas aeruginosa 1748.00 and > 2,000, Burkholderia cepacia 833.03 and 1,077.70, Candida albicans 521.43 and 172.61 and Aspergillus brasiliensis 300 and 400. The synergistic effect of the association of essential oil with parabens was performed through a centroid simplex experimental design for a mixture of methylparaben, propylparaben and essential oil against the same microorganisms used in the antimicrobial activity evaluation The ideal concentrations obtained by statistical analysis for each component in µg/mL were: 1120 for methylparaben, 350 for propylparaben and 675 for essential oil. The effectiveness test of the preservative system in cosmetic formulation was carried out using the ideal concentrations plus two higher concentrations and one below the ideal. For all challenged microbial strains, the test result was a total reduction of the inoculated microbial load in the seven days of testing and no increase until the twenty-eighth day, which demonstrates the effectiveness of the association of essential oil with synthetic preservatives. C. scoparioides essential oil showed an important antimicrobial potential both alone and in association with parabens. These results demonstrated that it can be used to compose a preservative system for cosmetic formulations containing lower amounts of synthetics

Parabens disrupt non-canonical inflammasome activation.

Parabens are synthetic chemicals widely used as preservatives in cosmetics, pharmaceuticals, and foods. Although parabens, i.e., ethyl- and methyl-parabens, are considered relatively safe, study of possible health hazards has been undertaken due to the frequent exposure to parabens and their accumulation in the body. In this study, we elucidated the effect of parabens on inflammasome induction of inflammatory responses in innate immunity, such as interleukin (IL)-1ß maturation and gasdermin D (GSDMD)-mediating pyroptosis. Parabens attenuated the inflammatory responses to intracellular lipopolysaccharide (LPS) triggering of non-canonical (NC) inflammasome activation, but did not alter canonical inflammasome (i.e., NLRP3, NLRC4 and AIM2) responses. The NC inflammasome is assembled by the interaction of murine caspase (Casp)-11 (Casp4/5 in human) with cytosolic LPS, inducing endotoxin sepsis. Parabens selectively inhibited NC inflammasome activation in both human and murine macrophages and diminished the peritoneal IL-1ß production in LPS-injected mice. Parabens blocked the cleavage of GSDMD, Casp1, and Casp4, but did not change the expression of Casp11 or the activity of Casp1. Taken together, the results indicate that parabens could disrupt Gram-negative pathogen infection through the inhibition of NC inflammasome activation.

A Novel In Vitro Assay Using Human iPSC-Derived Sensory Neurons to Evaluate the Effects of External Chemicals on Neuronal Morphology: Possible Implications in the Prediction of Abnormal Skin Sensation.

Neuronal morphological changes in the epidermis are considered to be one of causes of abnormal skin sensations in dry skin-based skin diseases. The present study aimed to develop an in vitro model optimised for human skin to test the external factors that lead to its exacerbation. Human-induced pluripotent stem cell-derived sensory neurons (hiPSC-SNs) were used as a model of human sensory neurons. The effects of chemical substances on these neurons were evaluated by observing the elongation of nerve fibers, incidence of blebs (bead-like swellings), and the expression of nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2). The nerve fiber length increased upon exposure to two common cosmetic preservatives-methylparaben and phenoxyethanol-but not to benzo[a]pyrene, an air pollutant at the estimated concentrations in the epidermis. Furthermore, the incidence of blebs increased upon exposure to benzo[a]pyrene. However, there was a decrease in the expression of NMNAT2 in nerve fibers, suggesting degenerative changes. No such degeneration was found after methylparaben or phenoxyethanol at the estimated concentrations in the epidermis. These findings suggest that methylparaben and phenoxyethanol promote nerve elongation in hiPSC-SNs, whereas benzo[a]pyrene induces nerve degeneration. Such alterations may be at least partly involved in the onset and progression of sensitive skin.

Design of Multifaceted Antioxidants: Shifting towards Anti-Inflammatory and Antihyperlipidemic Activity.

Oxidative stress and inflammation are two conditions that coexist in many multifactorial diseases such as atherosclerosis and neurodegeneration. Thus, the design of multifunctional compounds that can concurrently tackle two or more therapeutic targets is an appealing approach. In this study, the basic NSAID structure was fused with the antioxidant moieties 3,5-di-tert-butyl-4-hydroxybenzoic acid (BHB), its reduced alcohol 3,5-di-tert-butyl- 4-hydroxybenzyl alcohol (BHBA), or 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), a hydrophilic analogue of α-tocopherol. Machine learning algorithms were utilized to validate the potential dual effect (anti-inflammatory and antioxidant) of the designed analogues. Derivatives 1-17 were synthesized by known esterification methods, with good to excellent yields, and were pharmacologically evaluated both in vitro and in vivo for their antioxidant and anti-inflammatory activity, whereas selected compounds were also tested in an in vivo hyperlipidemia protocol. Furthermore, the activity/binding affinity of the new compounds for lipoxygenase-3 (LOX-3) was studied not only in vitro but also via molecular docking simulations. Experimental results demonstrated that the antioxidant and anti-inflammatory activities of the new fused molecules were increased compared to the parent molecules, while molecular docking simulations validated the improved activity and revealed the binding mode of the most potent inhibitors. The purpose of their design was justified by providing a potentially safer and more efficient therapeutic approach for multifactorial diseases.

Staphylococcus aureus membrane-damaging activities of four phenolics.

The membrane-damaging activities of four phenolics chosen for their bactericidal activity against Staphylococcus aureus CNRZ3 were investigated: 5,7-dihydroxy-4-phenylcoumarin (DHPC), 5,8-dihydroxy-1,4-naphthoquinone (DHNQ), epigallocatechin gallate (EGCG) and isobutyl 4-hydroxybenzoate (IBHB). Staphylococcus aureus CNRZ3 cells, as well as model liposomes mimicking its membrane phospholipids composition, were treated with each phenolic at its minimal bactericidal concentration. Membrane integrity, intracellular pH and intracellular esterase activity were examined by flow cytometric analysis of S. aureus cells stained with propidium iodide and SYTO® 9, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, and 5(6)-carboxyfluorescein diacetate, respectively. While intracellular pH was affected by the foyr phenolics, only DHNQ and to a lesser extent EGCG, caused a loss of membrane integrity. Flow cytometric analysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and DPPC/POPG (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphoglycerol) liposomes stained with Coumarin 6 (which penetrates the lipid bilayer) or 5-N(octadecanoyl)-amino-fluorescein (which binds to the liposome shell) suggested that only EGCG and DHNQ penetrated the bilayer of phospholipids of liposomes. Taken together, these findings support the hypothesis that EGCG and DHNQ bactericidal activity results from their accumulation in the phospholipid bilayer of S. aureus cells membrane causing its disruption.

Promising anticancer activity of polysaccharides and other macromolecules derived from oyster mushroom (Pleurotus sp.): An updated review.

Cancer dominates among many causes of mortality worldwide. Traditional chemotherapeutic agents are powerful anti-cancer agents employed for treatment of this deadly disease. However, they are always associated with toxic side effects and immunosuppression making person more vulnerable to tumor relapse and fatalities. A promising alternative could be identification, isolation and transfer of naturally occurring bioactive macromolecules to the tumorigenic population. Oyster mushroom, a major source of nutraceuticals, belonging to class basidiomycetes of kingdom Mycota is known to have immense therapeutic properties. It is a reservoir of macromolecules like ß-glucan, α-glucan, resveratrol, concanavalin A, cibacron blue affinity protein, p-hydroxybenzoic acid, ergosterol, linoleic acid etc. that are responsible for mediating anti-tumor, immunomodulatory, antioxidant, and anti-diabetic roles. Various studies have shown that extracts derived from oyster mushroom is rich in polysaccharides like ß-glucan and other macro molecules which have an anti-proliferative effect against cancer cell lines, without harming the normal cells. This review presents a brief highlight of the work covering the overall significance of oyster mushroom in different types of cancer treatment. It also explores the immunomodulatory effects of polysaccharides, proteoglycans and polypeptides derived from oyster mushroom that boosts the immune system to overcome the limitation of traditional cancer therapies.

Antibacterial Activity of the Mixed Systems Containing 1,2-Dodecanediol against Staphylococcus aureus and Staphylococcus epidermidis.

1,2-Alkanediols are characteristic cosmetic ingredients because these moisturizers exhibit the antibacterial activity against Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). However, the antimicrobial behavior in mixed systems containing several active ingredients is unclear because previous reports focus on an antibacterial system containing only 1,2-alkanediol. In this study, the minimal inhibitory concentration (MIC) and the fractional inhibitory concentration (FIC) were evaluated for 1,2-dodecanediol/lactic acid, 1,2-dodecanediol/myristic acid, 1,2-dodecanediol/methylparaben, and 1,2-dodecanediol/isopropyl methylphenol mixed systems to show the effect of the addition of other antimicrobial components to 1,2-dodecanediol. The antibacterial property of 1,2-dodecanediol/lactic acid mixed system was almost similar compared to 1,2-dodecanediol monomeric system. On the other hand, the antimicrobial activity of 1,2-dodecanediol against S. epidermidis was inhibited in the 1,2-dodecanediol/myristic acid mixed system. Because the selective antimicrobial activity of myristic acid against S. aureus was demonstrated in the mixed system. The present findings are useful for designing formulations of cosmetics and body cleansers containing 1,2-dodecanediol.

Assessment of butylparaben (4-hydroxybenzoic acid butyl ester)-assisted heat treatment against Escherichia coli O157:H7 and Salmonella enterica serotype Typhimurium in meat and bone meal.

Heat-resistant foodborne pathogens have been a concern in low-moisture foods and ingredients (LMFs). Due to low thermal conductivity of low moisture materials, thermal treatment is not efficient and may cause nutritional loss. This study investigated the enhancement of thermal treatment of meat and bone meal (MBM) at low water activity (aw ) by inclusion of butylparaben (BP) as a model antimicrobial compound. Stationary phase Escherichia coli O157:H7 (Shiga toxin-negative) or Salmonella enterica serotype Typhimurium was inoculated into MBM containing 0-2000 ppm BP and incubated at 55 or 60°C for up to 5 hr. A biphasic inactivation pattern was observed for both pathogens, indicating existence of potentially thermal resistant subpopulations. Addition of 1000 ppm BP to MBM (aw  = 0.4) significantly lowered the D-value at 55°C for E. coli O157:H7 (2.6 ± 0.5 hr) compared to thermal treatment alone (5.1 ± 0.6 h) during the treatment after the first 1 hr (p < 0.05), indicating that addition of BP accelerated the inactivation of thermal-resistant subpopulation of E. coli O157:H7 in MBM. Interestingly, similar enhancement in thermal inactivation upon addition of BP was not observed in either the sensitive or resistant subpopulation of S. Typhimurium at aw of 0.4 or 0.7, which is likely caused by the higher thermal resistance developed by S. Typhimurium within a low aw environment (aw  < 0.85). These results suggest that addition of certain antimicrobial compounds can improve the thermal processing efficiency in LMFs, while their efficiency against different pathogens may vary. PRACTICAL APPLICATION: Addition of appropriate food-grade compounds may help to improve thermal treatment efficiency in low moisture foods with varied efficiency against different pathogens. This approach has the potential to reduce the required heat treatment intensity while minimizing food safety risk.

Effect of chain length and branching on the in vitro metabolism of a series of parabens in human liver S9, human skin S9, and human plasma.

Parabens are antimicrobial compounds used as preservatives in cosmetics, foods, and pharmaceuticals. Paraben exposure occurs through a variety of routes including dermal absorption, ingestion, and inhalation. Ester bond hydrolysis has been shown to be the predominant biotransformation for this chemical class. Here we evaluated a series of parabens of increasing alkyl chain length and branching in addition to the aryl side chain of phenyl paraben (PhP). We evaluated the parabens under full Michaelis-Menten (MM) parameters to obtain intrinsic clearance values and found different trends between human liver and skin, which correlate with the predominant esterase enzymes in those matrices, respectively. In liver, where carboxylesterase 1 (CES1) is the predominant esterase enzyme, the shorter chain parabens were more readily metabolized, while in skin, where carboxylesterase 2 (CES2) is the predominant esterase enzyme, the longer chain parabens were more readily metabolized. Alkyl chain branching reduced the hydrolysis rates relative to those for the straight chain compounds, while the addition of a phenyl group, as in PhP, showed an increase in hydrolysis, producing the highest observed hydrolysis rate for skin. These data summarize the structure-metabolism relationship for a series of parabens and contribute to the safety assessment of this class of compounds.

Identification of a bacteria-produced benzisoxazole with antibiotic activity against multi-drug resistant Acinetobacter baumannii.

The emergence of multi-drug resistant pathogenic bacteria represents a serious and growing threat to national healthcare systems. Most pressing is an immediate need for the development of novel antibacterial agents to treat Gram-negative multi-drug resistant infections, including the opportunistic, hospital-derived pathogen, Acinetobacter baumannii. Herein we report a naturally occurring 1,2-benzisoxazole with minimum inhibitory concentrations as low as 6.25 µg ml-1 against clinical strains of multi-drug resistant A. baumannii and investigate its possible mechanisms of action. This molecule represents a new chemotype for antibacterial agents against A. baumannii and is easily accessed in two steps via de novo synthesis. In vitro testing of structural analogs suggest that the natural compound may already be optimized for activity against this pathogen. Our results demonstrate that supplementation of 4-hydroxybenzoate in minimal media was able to reverse 1,2-benzisoxazole's antibacterial effects in A. baumannii. A search of metabolic pathways involving 4-hydroxybenzoate coupled with molecular modeling studies implicates two enzymes, chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyltransferase, as promising leads for the target of 3,6-dihydroxy-1,2-benzisoxazole.

Metabolism and plasma protein binding of 16 straight- and branched-chain parabens in in vitro liver and skin models.

Parabens are alkyl esters of 4-hydroxybenzoic acid (4-HBA), with short-chain parabens used as antimicrobials in cosmetics. We investigated the impact of chain structure on skin and liver metabolism. Incubations with primary human hepatocytes and human liver S9 indicated that methyl-, ethyl-, propyl- and butylparaben were rapidly metabolized to similar metabolites, including 4-HBA plus the corresponding alcohols. Liver and EpiSkin™ S9 were used to investigate the metabolism of 16 short and long straight- and branched-chain parabens. The rate of hydrolysis generally decreased with increasing chain length in liver S9, whereas the reverse was true for EpiSkin™ S9. Chain length also correlated with the number of metabolites, with more oxidized metabolites detected from longer chain parabens. The identity of the alcohol group impacted metabolism the most, in terms of the rate of metabolism and the contribution of cofactors. The majority of parabens (13/16) exhibited high plasma protein binding (PPB) (>90%); whereas, 4-HBA PPB was 38%. PPB was related to the LogP of the parabens. In conclusion, the major and common paraben metabolite in PHH, liver S9 and EpiSkin™ S9 was 4-HBA. The rate of metabolism, type of metabolite and contribution of hydrolysis was tissue-specific (liver, skin) and was influenced by the chain length (and hence LogP), structural isomeric form (straight vs branched), and/or the identity of the alkyl group. SHORT ABSTRACT: We investigated how the chain structure of parabens affects their metabolism by liver and EpiSkin™ S9. The major and common metabolite in primary human hepatocytes, liver S9 and EpiSkin™ S9 was 4-HBA plus the corresponding alcohols. The rate of metabolism, type of metabolite and contribution of hydrolysis was tissue-specific and influenced by the chain length, structural isomeric form (straight vs branched), and/or the identity of the alkyl group. Most parabens exhibited high PPB (>90%), whereas the PPB of 4-HBA was 38%.

Mycobacterial para-Hydroxybenzoic Acid-Derivatives (pHBADs) and Related Structures Induce Macrophage Innate Memory.

Macrophages are key immune cells for combatting Mycobacterium tuberculosis. However, M. tuberculosis possesses means to evade macrophage bactericidal responses by, for instance, secretion of the immunomodulatory para-hydroxybenzoic acid derivatives (pHBADs). While these molecules have been implicated in inhibiting macrophage responses in an acute context, little is known about their ability to reprogram macrophages via induction of long-term innate memory. Since innate memory has been highlighted as a promising strategy to augment bactericidal immune responses against M. tuberculosis, investigating corresponding immune evasion mechanisms is highly relevant. Our results reveal for the first time that pHBAD I and related molecules (unmethylated pHBAD I and the hexose l-rhamnose) reduce macrophage bactericidal mechanisms in both the short- and the long-term. Moreover, we demonstrate how methyl-p-anisate hinders bactericidal responses soon after exposure yet results in enhanced pro-inflammatory responses in the long-term. This work highlights new roles for these compounds in M. tuberculosis pathogenesis.

Parabens inhibit hNaV 1.2 channels.

Propylparaben, a commonly used antimicrobial preservative, has been reported as an anticonvulsant agent targeting neuronal Na+ channels (NaV). However, the specific features of the NaV channel inhibition by this agent have so far not been extensively studied. Moreover, it is still unclear if it shares this pharmacological activity with other parabens. Here, we fully characterized the mechanism of action of the inhibitory effect that propylparaben and benzylparaben induce on human NaV 1.2 channel isoform (hNaV1.2). We established a first approach to know the parabens structural determinants for this channel inhibition. The parabens effects on hNaV1.2 channel mediated currents were recorded using the patch-clamp whole-cell configuration on hNaV1.2 stably transfected HEK293 cells. Propylparaben induced a typical state-dependent inhibition on hNaV1.2 channel carried current, characterized by a left-shift in the steady-state inactivation curve, a prolongation in the time needed for recovery from fast inactivation and a frequency-dependent blocking behavior. The state-dependent inhibition is increased for butylparaben and benzylparaben and diminished for methylparaben, ethylparaben and p-hydroxybenzoic acid (the major metabolite of parabens hydrolysis). Particularly, butylparaben and benzylparaben shift the steady-state inactivation curve 2- and 3-times more than propylparaben, respectively. Parabens are blockers of hNaV1.2 channels, sharing the mechanism of action of most of sodium channel blocking antiseizure drugs. The potency of this inhibition increases with the size of the lipophilic alcoholic residue of the ester group. These results provide a basis for rational drug design directed to generate new potential anticonvulsant agents.

The varying effects of a range of preservatives on Myoviridae and Siphoviridae bacteriophages formulated in a semi-solid cream preparation.

Bacteriophages may be formulated into semi-solid bases for therapeutic delivery. This work investigated the effects of a range of preservatives on the viability of Myoviridae and Siphoviridae bacteriophages when these were formulated into a standard semi-solid cream base. The six preservatives tested included: benzoic acid (0·1%), chlorocresol (0·1%), combination hydroxybenzoates (propyl 4-hydroxybenzoates with methyl 4-hydroxybenzoates) (0·1%), methyl 4-hydroxybenzoate (0·08%), 2-phenoxyethanol (1%) and propyl 4-hydroxybenzoate (0·02%). These were each formulated into cetomacrogol cream aqueous to generate six individual semi-solid bases into which Myoviridae and Siphoviridae bacteriophages were added and tested for stability. Optimal bacteriophage stability was seen when the preservative chlorocresol was used. Bacteriophage in the acidic benzoic acid were the least stable, resulting in complete loss of viability after 4-5 weeks. Of the bacteriophages tested, the Myoviridae KOX1 was significantly more stable than the Siphoviridae PAC1 after 91 days in formulations with each of the preservatives. Our results suggest the need for individual testing of specific bacteriophages in pharmaceutical formulations, as their efficacy when exposed to preservatives and excipients in these delivery forms may vary. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages are being increasingly investigated as alternatives to antibiotics. While bacteriophages can be formulated in diverse ways for therapeutic delivery, there has been scant work on how excipients and preservatives in these formulations affect stability of different bacteriophages. We demonstrate that the nature of preservatives in formulations will affect bacteriophage stability, and that in these formulations, viability of bacteriophage differs according to their morphology. Our work highlights the need for individual testing of specific bacteriophages in pharmaceutical formulations, as efficacy when exposed to preservatives and excipients in these delivery forms may vary.