The influence of Leucidal, a natural cosmetic preservative, on fibroblast and keratinocytes. Studies on cells and on model membrane systems.

The aim of this work was to investigate the influence of Leucidal® Liquid (abbr. Leucidal), which is recommended as a natural cosmetic ingredient of antimicrobial properties, on model membranes of keratinocytes and fibroblasts. The toxicity tests on cell lines were also performed to allow for a more detailed discussion of the results. As model membrane systems the lipid Langmuir monolayers were applied. During the investigations, the surface pressure/area measurements, penetration studies and Brewster Angle Microscopy (BAM) visualization were performed for one component and mixed lipid monolayers. It was evidenced that at the membrane - corresponding conditions, the components of Leucidal do not penetrate either model keratinocyte and fibroblast membranes or one component films composed of the major lipids of skin cell membranes. Leucidal makes these systems slightly more expanded and less stable, however this is not reflected in the changes in the film morphology. Only the ceramide systems were sensitive to the presence of Leucidal, i.e. the incorporation of Leucidal components manifested well in the decrease of the films' condensation and alterations in their morphology. The tests on cells demonstrated that Leucidal is non toxic for these types of cells at the concentrations suggested by the producer. A thorough comparison of these results with those published for bacteria model membranes enabled us to discuss them in the context of the mechanism of action of Leucidal components. It was concluded that Leucidal components are of low affinity to the skin cellular model membranes of low content of Leucidal-sensitive ceramides and are not toxic for fibroblast and keratinocyte cell lines. Moreover, the lipid composition of the membrane and its molecular organization can be important targets for Leucidal components, decisive from the point of view of the activity and selectivity of the studied composition.

The new preservative-free ophthalmic formulation of bilastine 0.6% preserves the ocular surface epithelial integrity in a comparative in vitro study.

Allergic conjunctivitis (AC) is the most common form of allergic eye disease and an increasingly prevalent condition. Topical eye drop treatments are the usual approach for managing AC, although their impact on the ocular surface is not frequently investigated. The aim of this study was to perform a comparative physicochemical characterization, and in vitro biological evaluations in primary conjunctival and corneal epithelial cells of the new multidose preservative-free bilastine 0.6% and main commercially available eye drops. MTT assay was used to measure cell viability; oxidative stress was analyzed with a ROS-sensitive probe; and apoptosis was evaluated monitoring caspase 3/7 activation. Differences in pH value, osmolarity, viscosity and phosphate levels were identified. Among all formulations, bilastine exhibited pH, osmolarity and viscosity values closer to tear film (7.4, 300 mOsm/l and ~ 1.5-10 mPa·s, respectively), and was the only phosphates-free solution. Single-dose ketotifen did not induce ROS production, and single-dose azelastine and bilastine only induced a mild increase. Bilastine and single-dose ketotifen and azelastine showed high survival rates attributable to the absence of preservative in its formulation, not inducing caspase-3/7-mediated apoptosis after 24 h. Our findings support the use of the new bilastine 0.6% for treating patients with AC to preserve and maintain the integrity of the ocular surface.

Machine learning assisted rational design of antimicrobial peptides based on human endogenous proteins and their applications for cosmetic preservative system optimization.

Preservatives are essential components in cosmetic products, but their safety issues have attracted widespread attention. There is an urgent need for safe and effective alternatives. Antimicrobial peptides (AMPs) are part of the innate immune system and have potent antimicrobial properties. Using machine learning-assisted rational design, we obtained a novel antibacterial peptide, IK-16-1, with significant antibacterial activity and maintaining safety based on ß-defensins. IK-16-1 has broad-spectrum antimicrobial properties against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, and has no haemolytic activity. The use of IK-16-1 holds promise in the cosmetics industry, since it can serve as a preservative synergist to reduce the amount of other preservatives in cosmetics. This study verified the feasibility of combining computational design with artificial intelligence prediction to design AMPs, achieving rapid screening and reducing development costs.

Antibacterial and antibiofilm effects of Camellia oleifera seed dreg extract and its application in cosmetics.

BACKGROUND: Cosmetic care products contain a high proportion of water and nutrients. Therefore, preventing bacterial growth is an important issue to ensure product quality and safety. The application of antibacterial natural ingredients derived from plants is considered to have the potential to maintain product quality and reduce the use of chemicals in formulations. Additionally, chemically synthesized antiseptic and antibacterial agents are widely used in the industry at present. However, some preservative ingredients have been reported that may cause skin irritation, redness, allergies, and even dermatitis. AIMS: This study aimed to prepare extract from Camellia oleifera tea seed dregs (CTSD), investigate the antibacterial effects on two pathogenic bacteria and evaluate the product preservative ability. METHODS: Ethanol extraction was prepared and subjected to characterize their triterpenoid contents. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) were determined for Pseudomonas aeruginosa and Staphylococcus aureus. The product's stability and preservative qualities, along with its ability to scavenge free radicals through antioxidant activity, were also assessed. RESULTS: The gram-positive S. aureus showed greater susceptibility to the treatment. In additional, CTSD possessed significant free radical scavenging activity in vitro and cultured normal human skin fibroblast CCD-966SK cells under nontoxic concentration. The challenge test and accelerated storage test confirmed the CTSD containing formulated emulsion is eligible for commercialization. CONCLUSIONS: CTSD has the potential to be developed as an alternative agent to control microbial biofilm formation, or can be used as an adjuvant compound for infectious disease control.

Comparing the effect of benzalkonium chloride-preserved, polyquad-preserved, and preservative-free prostaglandin analogue eye drops on cultured human conjunctival goblet cells.

PURPOSE: To investigate the effect of benzalkonium chloride (BAK)-preserved latanoprost and bimatoprost, polyquad (PQ)-preserved travoprost, and preservative-free (PF) latanoprost and tafluprost, all prostaglandin analogues (PGAs), on human conjunctival goblet cell (GC) survival. Furthermore, to investigate the effect of BAK-preserved and PF latanoprost on the cytokine secretion from GC. METHODS: Primary human conjunctival GCs were cultivated from donor tissue. Lactate dehydrogenase (LDH) and tetrazolium dye colorimetric (MTT) assays were used for the assessment of GC survival. A cytometric bead array was employed for measuring secretion of interleukin (IL)-6 and IL-8 from GC. RESULTS: BAK-preserved latanoprost and bimatoprost reduced cell survival by 28% (p = 0.0133) and 20% (p = 0.0208), respectively, in the LDH assay compared to a negative control. BAK-preserved latanoprost reduced cell proliferation by 54% (p = 0.003), BAK-preserved bimatoprost by 45% (p = 0.006), PQ-preserved travoprost by 16% (p = 0.0041), and PF latanoprost by 19% (p = 0.0001), in the MTT assay compared to a negative control. Only PF tafluprost did not affect the GCs in either assay. BAK-preserved latanoprost caused an increase in the secretion of pro-inflammatory IL-6 and IL-8 (p = 0.0001 and p = 0.0019, respectively) compared to a negative control, which PF latanoprost did not. CONCLUSION: BAK-preserved PGA eye drops were more cytotoxic to GCs than PQ-preserved and PF PGA eye drops. BAK-preserved latanoprost induced an inflammatory response in GC. Treatment with PF and PQ-preserved PGA eye drops could mean better tolerability and adherence in glaucoma patients compared to treatment with BAK-preserved PGA eye drops.

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.

New Candidate Preservative in Ophthalmic Solution Instead of Benzalkonium Chloride: 1,3-Didecyl-2-methyl Imidazolium Chloride.

Benzalkonium chloride (BAC) is a useful preservative for ophthalmic solutions but has some disadvantageous effects on corneal epithelium, especially keratinocytes. Therefore, patients requiring the chronic administration of ophthalmic solutions may suffer from damage due to BAC, and ophthalmic solutions with a new preservative instead of BAC are desired. To resolve the above situation, we focused on 1,3-didecyl-2-methyl imidazolium chloride (DiMI). As a preservative for ophthalmic solutions, we evaluated the physical and chemical properties (absorption to a sterile filter, solubility, heat stress stability, and light/UV stress stability), and also the anti-microbial activity. The results indicated that DiMI was soluble enough to prepare ophthalmic solutions, and was stable under severe heat and light/UV conditions. In addition, the anti-microbial effect of DiMI as a preservative was considered to be stronger than BAC. Moreover, our in vitro toxicity tests suggested that DiMI is safer to humans than BAC. Considering the test results, DiMI may be an excellent candidate for a new preservative to replace BAC. If we can overcome manufacturing process issues (soluble time and flushing volume) and the insufficiency of toxicological information, DiMI may be widely adopted as a safe preservative, and immediately contribute to the increased well-being of all patients.

Effect of leave-on cosmetic antimicrobial preservatives on healthy skin resident Staphylococcus epidermidis.

BACKGROUND: The effect of high doses preservatives in the leave-on cosmetic products to the skin microbiota is not clear. Studies have shown that the preservatives might alter the balance of the skin microbiota. AIM: In this study, we aimed to evaluate antimicrobial effect of nine cosmetic chemical preservatives. MATERIAL & METHOD: A total of 77 Staphylococcus epidermidis isolates from 46 healthy zygomatic skin samples were characterized by multilocus sequence typing (MLST). Nine preservatives used in leave-on cosmetics were analyzed by testing the minimal inhibitory concentrations (MICs) against S. epidermidis isolates. We also determined the mutant prevention concentration (MPC) and bactericidal kinetics on selected isolates. RESULTS: More than 17 sequence types were recognized among 77 S. epidermidis isolates. Our data demonstrated that the maximum permitted doses of 2-bromo-2-nitro-1,3-propanediol, ethyl 4-hydroxybenzoate, hexadecyltrimethylammonium bromide, and imidazolidinyl urea were significantly higher than both their MICs and MPCs. We showed that, at the maximum permitted doses, two preservatives could completely kill 107 CFU/mL S. epidermidis in less than 1 h in MH broth. CONCLUSION: Our data demonstrated that certain preservatives of the leave-on cosmetics might inhibit or kill S. epidermidis cells and perturb the skin microbiota balance. The determination of the maximum permitted doses of the preservatives should not only be based on the toxicological data, but also antimicrobial susceptibility analysis. This comprehensive evaluation would ensure a balanced and healthy skin microbiota.

Influence of BAKs on tear film lipid layer: In vitro and in silico models.

Benzalkonium chloride (BAK) compounds are commonly used in topical ophthalmic products as preservatives and stabilizers. BAK mixtures containing several compounds with different alkyl chain lengths are typically used. However, in chronic eye conditions, such as dry eye disease and glaucoma, the accumulation of adverse effects of BAKs was observed. Hence, preservative-free eye drops formulations are preferred. On the other hand, selected long-chain BAKs, particularly cetalkonium chloride, exhibit therapeutic functions, promoting epithelium wound healing and tear film stability. Nevertheless, the mechanism of BAKs influence on the tear film is not fully understood. By employing in vitro experimental and in silico simulation techniques, we elucidate the action of BAKs and demonstrate that long-chain BAKs accumulate in the lipid layer of the tear film model, stabilizing it in a concentration-dependent fashion. In contrast, short-chain BAKs interacting with the lipid layer compromise the tear film model stability. These findings are relevant for topical ophthalmic drug formulation and delivery in the context of selecting proper BAK species and understanding the dose dependency for tear film stability.

[Impact of preservatives in topicals on the cutaneous microbiota]. / Einfluss von Konservierungsmitteln in Topika auf die kutane Mikrobiota.

Preservatives are used to stabilize topical preparations and protect the user from the influence of pathogenic microbes. After the application of a topical preparation, the matrix undergoes a metamorphosis, and by proportional evaporation of the hydrophilic phase the preservative may accumulate on the skin surface. This is believed to lead to antiseptic effects and may influence the diversity of the cutaneous microbiota. The regulation of the cutaneous microbiome and the associated influencing factors is a complex system that results in highly individualized conditions. Therefore, investigations on the influence of defined interventions are methodologically difficult. In the present proof-of-concept study, potential antiseptic effects of preservatives were investigated in a combination of in vitro and in vivo methods using microbiological culture tests. In addition, the investigations served to develop a clinical study design to answer further questions and use of an extended range of methods. The results support the hypothesis of an antiseptic effect of the tested preservatives (methyl-4-hydroxybenzoate and propyl-4-hydroxybenzoate, potassium sorbate and propylene glycol) on prominent reference bacteria, which could also be observed in clinical settings.

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.

Difference in Anti-microbial Activity of Propan-1,3-diol and Propylene Glycol.

Propan-1,3-diol (PD) and propan-1,2-diol (propylene glycol, PG) are very similar compounds because their structures, safety data, and anti-microbial activities are almost the same. Actually, both compounds are made up of three carbon atoms and two hydroxyl groups. Regarding their safety, they do not have serious hazard data for animals, and LD50 values (in rats) of both are similar. As for the anti-microbial activity, minimum inhibitory concentration (MIC) values of both PD and PG are approximately 10% (v/v). In this study, we used the preservatives-effectiveness test (PET) to evaluate the anti-microbial activities of PD and PG, because both compounds are used in cosmetics as preservatives. The results indicated that PD was more effective as an anti-microbial agent compared with PG, and the effect of PD was marked against Escherichia coli and Pseudomonas aeruginosa. Scanning electron microscopy (SEM) images showed that the membrane of Escherichia coli was injured by PD and PG, but the damage by PD was more marked. The damage of the cell membrane may be the cause of high anti-microbial activity of PD in PET. These results suggest that PD has greater potential as a preservative, and PD should be recommended as an additive for food and medicine.

Ethylated analogue of Zingerone: A new and eco-respectful preservative in cosmetics.

OBJECTIVE: This article describes the eco-design of a new preservative in cosmetics based on bio-inspiration of natural extracts from traditional medicines. In order to reach the multiple specification targets, various structures have been synthesized and evaluated to select the one demonstrating broad antimicrobial spectrum with favorable environmental profile and application potential to a wide variety of formulas. METHODS: In order to evaluate the antimicrobial activity of the synthesized structures, the method of the challenge test consisting in an artificial contamination of the sample with collection microbial strains and evaluation of the number of revivable microorganisms was used to select the most promising candidate. Validation of its antimicrobial potential was later confirmed on Gram positive and Gram negative bacteria, yeast and mold with measurement of its Minimum Inhibitory Concentration (MIC) in comparison with known preservatives. Environmental impact assessment of the selected candidate was achieved with the help of ready biodegradability and aquatic ecotoxicity tests performed according to appropriate Organization tes a strfor Economic Co-operation and Development (OECD) and European Union (EU) guidelinesy. RESULTS: Bio-inspired from turmeric and ginger extracts, an ethylated analog of Zingerone (EZ) with the chemical name 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one demonstrates the strongest activity on Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa and Candida albicans. Moreover, EZ shows a solubility in water two times higher than that of Zingerone thus increasing its interest as a potential preservative. Finally, its assessment of ready biodegradability and aquatic ecotoxicity in OECD-EU tests with a favorable environmental profile confirms its unique interest and fully justifies its use in cosmetic formulas as an eco-respectful preservative. CONCLUSION: Bio-inspiration based on technologies without noteworthy side effects but also on eco-design, particularly through the use of measures of potential environmental impact very upstream of a development, are two fundamental elements for the launching of new eco-friendly cosmetic ingredients. This approach has thus validated the strong potential of EZ as a preservative of eco-respectful formulas. The selection of EZ is also a very good example of the achievement of two key objectives targeted by cosmetic companies for the development of a novel active ingredient: environmental performance and technical performance.

OBJECTIF: Cet article décrit l'éco-conception d'un nouveau conservateur en cosmétique à partir de la bio-inspiration d'extraits naturels utilisées dans les médecines traditionnelles. Afin d'atteindre les cibles de spécification multiples, diverses structures ont été synthétisées et évaluées pour choisir celle qui présente un large spectre antimicrobien avec un profil environnemental favourable et un potentiel d'application pour une grande variété de formules. MÉTHODES: Afin d'évaluer l'activité antimicrobienne des structures synthétisées, la méthode du challenge test consistant en une contamination artificielle de l'échantillon par des souches microbiennes de collection suivie de l'évaluation du nombre de microorganismes revivifiables a été utilisée pour sélectionner le candidat le plus prometteur. La validation de son potentiel antimicrobien a par la suite été confirmée sur des bactéries à Gram positif et à Gram négatif, des levures et des moisissures grâce à la mesure de sa Concentration Minimale Inhibitrice (CMI) par rapport aux agents de conservation connus. L'évaluation de l'impact sur l'environnement du candidat sélectionné a été réalisée à l'aide de tests de biodégradabilité facile et d'écotoxicité aquatique réalisés selon les lignes directrices appropriées de l'Organization de Coopération et de Développement Economiques (OCDE). RESULTATS: Bio-inspiré des extraits de curcuma et de gingembre, un analogue éthylé de Zingerone nommé ETHYLZINGERONE (EZ) avec pour nom chimique 4-(3-ethoxy-4-hydroxyphenyl)butan-2-one démontre l'activité la plus forte sur Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa et Candida albicans. De plus, EZ montre une solubilité dans l'eau 2 fois supérieure à celle de la Zingerone renforçant ainsi son intérêt en tant que conservateur potentiel. Enfin, son évaluation dans les tests OCDE de la biodégradabilité facile et de l'écotoxicité aquatique avec un profil environnemental favourable confirme son intérêt unique et justifie pleinement son utilization dans les formules cosmétiques comme conservateur respectueux de l'environnement. CONCLUSION: La bio-inspiration basée sur des technologies sans effet secondaire notoire mais aussi l'éco-conception par l'utilization de mesures de l'impact environnemental potentiel très en amont d'un développement sont deux éléments fondamentaux pour la mise sur le marché de nouveaux ingrédients cosmétiques éco-respectueux. Cette approche a ainsi permis de valider le fort potentiel de EZ comme agent de conservation de formules respectueuses de l'environnement. La sélection de EZ est également un très bon exemple de l'atteinte de deux objectifs-clés recherchés par les entreprises cosmétiques pour le développement d'un nouvel actif: la performance environnementale et la performance technique.

Cytotoxicity, Mitochondrial Functionality, and Redox Status of Human Conjunctival Cells after Short and Chronic Exposure to Preservative-Free Bimatoprost 0.03% and 0.01%: An In Vitro Comparative Study.

Prostaglandin analogues (PGAs), including bimatoprost (BIM), are generally the first-line therapy for glaucoma due to their greater efficacy, safety, and convenience of use. Commercial solutions of preservative-free BIM (BIM 0.03% and 0.01%) are already available, although their topical application may result in ocular discomfort. This study aimed to evaluate the in vitro effects of preservative-free BIM 0.03% vs. 0.01% in the human conjunctival epithelial (HCE) cell line. Our results showed that long-term exposure to BIM 0.03% ensues a significant decrease in cell proliferation and viability. Furthermore, these events were associated with cell cycle arrest, apoptosis, and alterations of ΔΨm. BIM 0.01% does not exhibit cytotoxicity, and no negative influence on conjunctival cell growth and viability or mitochondrial activity has been observed. Short-time exposure also demonstrates the ability of BIM 0.03% to trigger reactive oxygen species (ROS) production and mitochondrial hyperpolarisation. An in silico drug network interaction was also performed to explore known and predicted interactions of BIM with proteins potentially involved in mitochondrial membrane potential dissipation. Our findings overall strongly reveal better cellular tolerability of BIM 0.01% vs. BIM 0.03% in HCE cells.

Cytotoxic, Apoptotic, and Oxidative Effects of Preserved and Preservative-Free Brimonidine in a Corneal Epithelial Cell Line.

Purpose: This study aims to compare the cytotoxic, apoptotic, and oxidative effects of preserved and preservative-free forms of brimonidine 0.15% on the human corneal epithelial cell (HCEC) line. Methods: Time-dependent cytotoxicity studies were performed with the Alamar Blue method. For apoptotic studies, PE Annexin V and 7-amino-actinomycin (7-AAD) staining and flow cytometry were performed. Messenger RNA (mRNA) expressions of Bax, Bcl-2, and caspase-3, -9, -12, and protein expressions of Bax and Bcl-2 were evaluated by quantitative real-time polymerase chain reaction and Western blot method, respectively. Results: Cell viability was 76.4% with the preserved solution and 36.05% with the preservative-free solution at the fifth minute. No significant difference was observed with either solution at the 15-min mark, whereas cell viability did not change significantly after 1 h. In the apoptosis evaluation, it was observed that the preservative-free solution increased the early apoptotic activity to a greater degree (P < 0.05). Preservative-free solution also induced gene expression of proapoptotic Bax, caspase-9 and -12, and protein expression of Bax while reducing the protein expression of anti-apoptotic Bcl-2 (P < 0.0001). Preserved solution induced only the gene expression of caspase-12, and reduced the protein expression of Bcl-2 (P < 0.0001). No significant difference was observed in the reactive oxygen species (ROS) levels of either solution compared with the control group (P > 0.05). Conclusion: It was demonstrated that the preserved solution is less cytotoxic to the HCEC line in the early period, has less early apoptotic activity, and does not significantly increase ROS levels.

Generic benzalkonium chloride-preserved travoprost eye drops are not identical to the branded polyquarternium-1-preserved travoprost eye drop: Effect on cultured human conjunctival goblet cells and their physicochemical properties.

PURPOSE: To investigate the effect of polyquaternium-1 (PQ)-preserved and benzalkonium chloride (BAK)-preserved travoprost eye drops on viability of primary human conjunctival goblet cell (GC) cultures and on secretion of mucin and cytokines. Furthermore, to evaluate the physicochemical properties of the branded travoprost eye drop Travatan® and available generics. METHODS: The effect of travoprost eye drops was evaluated on GC cultures. Cell viability was assessed through lactate dehydrogenase (LDH) and tetrazolium dye (MTT) colorimetric assays. Mucin secretion was evaluated by immunohistochemical staining. Secretion of interleukin (IL)-6 and IL-8 was measured using BD Cytometric Bead Arrays. pH, viscosity, droplet mass, osmolality and surface tension were measured for all included eye drops. RESULTS: In the LDH assay, BAK travoprost caused significant GC loss after 2 hrs of incubation compared to the control. PQ travoprost caused no GC loss at any time point. Both PQ- and BAK travoprost caused secretion of mucin to the cytoplasma. No difference in IL-6 and IL-8 secretion was identified compared to controls. The pH values for the generics were lower (pH 6.0) than the pH value for Travatan (pH 6.7; p < 0.0001). The viscosity was lowest for Travatan, while the mean droplet mass was higher for Travatan (35 mg) than the generics (28-30 mg; p ≤ 0.0318). The osmolality and surface tension did not differ between the eye drops investigated. CONCLUSION: BAK travoprost caused GC loss, indicating that PQ preservation may be preferable in treatment of glaucoma. Furthermore, physicochemical properties of branded and generic travoprost eye drops can not be assumed to be identical.

Real-time qPCR to evaluate bacterial contamination of cosmetic cream and the efficiency of protective ingredients.

AIMS: The absence of objectionable micro-organisms in cosmetics and the efficiency of preservatives are still mainly assessed by time-consuming cultivation-based methods. We explored the applicability of real-time quantitative polymerase chain reaction (qPCR) and reported on the behaviour of different bacteria in artificially contaminated creams. METHODS AND RESULTS: Real-time qPCR on DNA from Burkholderia cepacia, Pluribacter gergoviae, Pseudomonas aeruginosa and Sphingomonas paucimobilis identified specific primer pairs that amplify accurately and efficiently two strains/isolates of each species. Using DNeasy mericon Food Kit, we detected bacterial growth in an inoculated cosmetic cream and persistency of DNA from heat-inactivated bacteria. We were also able to monitor the growth inhibitory effect of caprylyl glycol and EDTA, also showing how different bacterial species interact depending on the presence/absence of these ingredients. Finally, creams supplemented with the protective cosmetic ingredients revealed the various behaviour of five strains/isolates from P. aeruginosa. CONCLUSIONS: Successfully extracting bacterial DNA from artificially contaminated cosmetic creams, we could perform real-time qPCR to identify and follow the growth of various strains of 4 bacteria species under different conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time qPCR appears as a promising method to detect bacterial contamination in cosmetic creams and/or to monitor growth inhibition by ingredients.

Benzalkonium Chloride-Induced Corneal Epithelial Injury in Rabbit Reduced by Rebamipide.

Purpose: We assessed the effect of rebamipide ophthalmic solution on corneal epithelial injury due to benzalkonium chloride (BAK) by fluorescein (FL) staining and corneal resistance (CR). Methods: After determining the absence of corneal epithelial damage by FL and CR, rebamipide ophthalmic solution (50 µL) was instilled five times, each interspaced by 5 min, into one eye of mature New Zealand white rabbits, and likewise physiological saline was instilled into the contralateral eye as the control. After 30 min, eyes were similarly treated with one of the following solutions: BAK solution 0.02%, latanoprost ophthalmic solution (0.02% BAK), or latanoprost ophthalmic solution without BAK. The presence of corneal epithelial damage was quantitated at 10, 30, and 60 min by CR after the last instillation. FL staining was also performed at 60 min after the last instillation. Results: CR ratios (%) at 60 min after the last instillation in rebamipide/BAK and rebamipide/latanoprost (0.02% BAK) groups were significantly increased by 18.3% and 25.6% compared with saline/BAK and saline/latanoprost (0.02% BAK) groups, respectively (P < 0.05). Findings by FL staining were consistent with those by CR; BAK and latanoprost with BAK groups were positive, and eyes with the most severe area and density of corneal epithelial damage (A2D2) were in the saline/BAK group. Conclusion: The rebamipide ophthalmic solution reduces the severity of corneal epithelial injury caused by BAK, an ophthalmic solution preservative.

Captopril oral solution for pediatric use: formulation, stability study and palatability assessment in vivo

Abstract he aim of this work was to develop an oral solution of captopril at 5 mg/mL preservative-free. Two formulations were prepared, one containing sweetener (formulation 1) and the other without this excipient (formulation 2). The results found of validation parameters from analytical method performed by HPLC for captopril were, linearity 0.9998, the limit of detection 15.71 µg/mL, the limit of quantification 47.60 µg/mL, repeatability 1.05%, intermediate precision 2.42%, accuracy intraday 101,53%, accuracy inter-day 99.85%. Moreover, the results found for captopril disulfide were, linearity 0.9999, limit of detection 0.65 µg/mL, limit of quantification 1.96 µg/mL, repeatability 2.28%, intermediate precision 1.51%, accuracy intraday 101.36%, accuracy inter-day 100.29%. The appearance of formulations was clear and colorless, pH measures were 3.12 and 3.04, dosage of captopril and captopril disulfide were 99.45% and 99.82%, 0.24% and 0.12% for formulation 1 and formulation 2, respectively. The stability study demonstrated that the concentration of captopril and captopril disulfide in the formulations was > 90% and below 3%, respectively. The in vivo palatability study in animals and humans showed that Formulation 1 containing the sweetener had better acceptance. Thus, the sweetener was able to improve the unpleasant taste of the formulation

Development of Hinoline® as a natural preservative for cosmetic product using bioinspiration and Greenpharma Database.

AIMS: The cosmetic industry needs new preservatives that are effective, natural, safe, cost effective, sustainable and compliant with regulatory standards. This necessity has posed challenges requiring obligations, bioinformatics and bioinspiration as driving forces. METHODS AND RESULTS: Twenty natural extracts were selected from the Greenpharma Database with parameter filters corresponding to development constraints and antimicrobial properties. We confirmed using minimum inhibition concentration (MIC) assays that eight of the extracts have good bactericidal properties and that one has a high antifungal activity. The latter was purified hinokitiol, a bioproduct from Aomori Hiba wood. This substance provides high resistance against putrefaction; for instance, old Japanese temples were made of Aomori Hiba wood. The combination of hinokitiol with levulinic acid, another bioproduct, demonstrated complementary antimicrobial activities and synergistic effects in MIC studies and measurements according to Kull synergy index. Further, the mixture Hinoline® was tested at 2% in challenge tests and fulfilled criteria A of different standards. It also exerted complementary preservative effects with potassium sorbate and beneficial effects in unbalanced skin microbiota. CONCLUSION: Hinoline, a new effective preservative from renewable bioresources, was developed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study accelerates the development of a preservative solution for cosmetics selected from Greenpharma Database, through bioinspiration and the identification of cost-effective investments and resources.