Humulus lupulus aqueous extract and hydrolate as a potential ingredient for cosmetics: chemical characterization and in vitro antimicrobial, cytotoxicity, antioxidant and anti-inflammatory assessment.

Humulus lupulus extracts have in their composition different molecules, such as polyphenols, α-acids, ß-acids, and hydrocarbons, which contribute to the plant's medicinal properties. These molecules are associated with antimicrobial, antioxidant and anti-inflammatory activities. OBJECTIVE: This work focuses on the evaluation of H. lupulus biological activities, with the aim of evaluating its potential for inclusion in cosmetic formulations. METHODS: Two distinct aqueous extracts and two hydrolates obtained via hydrodistillation were evaluated. These include the flower parts (FE, FH) and the mix of aboveground parts (ME, MH). The chemical profiles for both aqueous extracts and hydrolates were identified by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). Antimicrobial, antioxidant, cytotoxicity, and anti-inflammatory activity were tested in vitro using standard methods. RESULTS: Rutin was the major compound found in FE (40.041 µg mg-1 of extract) and ME (2.909 µg mg-1 of extract), while humulenol II was the most abundant compound in hydrolates (FH: 20.83%; MH: 46.80%). Furthermore, FE was able to inhibit the growth of Staphylococcus aureus and Staphylococcus epidermis with MIC values of 50% and 25% (v/v), respectively. FH showed the same effect in Staphylococcus aureus (50% v/v). FH evidenced poor antioxidant potential in DPPH scavenging test and demonstrated significant antioxidant and anti-inflammatory effects by reducing (***p < 0.001) intracellular reactive oxygen species (ROS), NO (nitric oxide) levels (***p < 0.001) and cyclooxygenase-2 (COX-2) protein expression (***p < 0.001) in lipopolysaccharide (LPS)-stimulated macrophages. Nevertheless, it is important to note that FH exhibited cytotoxicity at high concentrations in 3T3 fibroblasts and RAW 264.7 macrophages. CONCLUSION: The studied H. lupulus aqueous extracts and hydrolates revealed that FH stands out as the most promising bioactive source for cosmetic formulations. However, future research addressing antimicrobial activity is necessary to confirm its potential incorporation into dermatological and cosmetic formulations.

Vaginal Sheets with Thymbra capitata Essential Oil for the Treatment of Bacterial Vaginosis: Design, Characterization and In Vitro Evaluation of Efficacy and Safety.

We aimed to incorporate Thymbra capitata essential oil (TCEO), a potent antimicrobial natural product against bacterial vaginosis (BV)-related bacteria, in a suitable drug delivery system. We used vaginal sheets as dosage form to promote immediate relief of the typical abundant vaginal discharge with unpleasant odour. Excipients were selected to promote the healthy vaginal environment reestablishment and bioadhesion of formulations, while the TCEO acts directly on BV pathogens. We characterized vaginal sheets with TCEO in regard to technological characterization, predictable in vivo performance, in vitro efficacy and safety. Vaginal sheet D.O (acid lactic buffer, gelatine, glycerine, chitosan coated with TCEO 1% w/w) presented a higher buffer capacity and ability to absorb vaginal fluid simulant (VFS) among all vaginal sheets with EO, showing one of the most promising bioadhesive profiles, an excellent flexibility and structure that allow it to be easily rolled for application. Vaginal sheet D.O with 0.32 µL/mL TCEO was able to significantly reduce the bacterial load of all in vitro tested Gardnerella species. Although vaginal sheet D.O presented toxicity at some concentrations, this product was developed for a short time period of treatment, so this toxicity can probably be limited or even reversed when the treatment ends.

Thymus mastichina (L.) L. and Cistus ladanifer L. for skin application: chemical characterization and in vitro bioactivity assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: Thymus mastichina (L.) L. (TM) and Cistus ladanifer L. (CL) are two Portuguese autochthonous species with traditional skin application in folk medicine. TM is majorly known for its antiseptic and wound healing properties, as an external anti-inflammatory agent and for its application in folk cosmetics and hygiene products. Its use in acne vulgaris has also been reported. CL is traditionally used in remedies for wounds, ulcers and other skin ailments such as psoriasis and eczema. Its application has been found useful due to its anti-inflammatory, astringent, wound healing and antiseptic properties. AIM OF THE STUDY: With this work, we aimed to investigate relevant bioactivities related with the traditional application of TM and CL essential oils (EOs) and hydrolates (by-products of EO production) in skin ailments. Specifically their in vitro antioxidant, anti-inflammatory, cytotoxic, wound healing and antimicrobial properties were evaluated. The chemical composition of both EOs and respective hydrolates was also characterized. MATERIALS AND METHODS: Chemical characterization of EOs and hydrolates was performed by GC-FID and GC-MS. Cellular biocompatibility was evaluated using the MTT assay in macrophages (RAW 264.7) and fibroblasts (L929) cell lines. Anti-inflammatory activity was investigated by studying nitric oxide (NO) production by macrophages with Griess reagent. Wound healing potential was evaluated with the scratch-wound assay. The antioxidant potential was studied by the DPPH scavenging method. Antimicrobial activity was evaluated by broth microdilution assay against relevant microbial strains and skin pathogens, namely Staphylococcus aureus, Staphylococcus epidermidis, Cutibacterium acnes, Pseudomonas aeruginosa, Escherichia coli, Candida albicans and Aspergillus brasiliensis. RESULTS: The major compounds present in TM and CL EOs were 1,8-cineole and α-pinene, respectively. 1,8-cineole and E-pinocarveol were the major compounds in the correspondent hydrolates. CL EO presented the highest anti-inflammatory potential [EC50 = 0.002% (v/v)], still with significant cytotoxicity [IC50 = 0.012% (v/v)]. TM preparations presented anti-inflammatory potential, also presenting higher biocompatibility. The same profile was present on fibroblasts regarding biocompatibility of the tested preparations. CL EO and hydrolate increased fibroblasts' migration by 155.7% and 148.4%, respectively. TM hydrolate presented a milder activity than CL hydrolate, but wound healing potential was still present, increasing cell migration by 125.1%. All preparations presented poor antioxidant capacity. CL EO presented higher antimicrobial activity, with MICs ranging from 0.06% (v/v) to 2% (v/v), against different microorganisms. CONCLUSIONS: Anti-inflammatory and skin repairing potential were present for CL preparations. TM hydrolate presented an interesting biocompatible profile on both cell lines, also presenting anti-inflammatory potential. Furthermore, EOs from both species presented antimicrobial activity against a panel of different microorganisms. These in vitro bioactivities support some of their traditional skin applications, specifically regarding their antiseptic, wound healing and anti-inflammatory uses.

Iodine Supplementation in Pregnancy in an Iodine-Deficient Region: A Cross-Sectional Survey.

Iodine deficiency is a common problem in pregnant women and may have implications for maternal and child health. Iodine supplementation during pregnancy has been recommended by several scientific societies. We undertook a cross-sectional survey to assess the efficacy of these recommendations in a European iodine-deficient region. Urinary iodine concentrations (UIC) were determined in pregnant women before (n = 203) and after (n = 136) the implementation of guidelines for iodine supplementation in pregnancy. Iodine supplementation (200 µg/day) reduced the proportion of pregnant women with severe iodine deficiency (37.4% to 18.0%, p = 0.0002). The median UIC increased from 67.6 µg/L to 106.8 µg/L but remained below the recommended target level (>150 µg/L) for pregnant women. In conclusion, iodine supplementation in pregnant women improved iodine status in this iodine-deficient region but was insufficient to achieve recommended iodine levels in pregnancy. Additional measures, such as the adjustment of the dose or timing of supplementation, or universal salt iodization, may be needed.

Drug Formulations for Localized Treatment of Human Papillomavirus-Induced Lesions.

BACKGROUND: The human papillomavirus (HPV) is responsible for over 90% of all cervical cancer cases. The use of vaginal gels is often indicated for local vaginal drug delivery. Previous studies have shown that Thymus vulgaris essential oil (TEO) exhibits anticancer properties besides antifungal and antibacterial properties. Its activity derives from a specific increase in free radicals and oxidative stress caused in cancer cells. Furthermore, mitoxantrone (MTX), an anthracenedione, and C8, an acridine orange derivative, were shown to inhibit the growth of the cervical cancer cell line HeLa. RESULTS: The results showed that TEO + C8 is the most promising formulation in terms of viscosity and osmolality properties in vaginal fluid simulant (VFS). The combined action of TEO with the compounds MTX and C8 resulted in HeLa cell viability reduction compared with the effect obtained with the individual formulations containing each one of the compounds. CONCLUSIONS: The formulation TEO + C8 holds promise in terms of cost-benefit and topical application of the active compound for the HeLa cells.

Chemical characterization and bioactive potential of Thymus×citriodorus (Pers.) Schreb. preparations for anti-acne applications: Antimicrobial, anti-biofilm, anti-inflammatory and safety profiles.

ETHNOPHARMACOLOGICAL RELEVANCE: Thymus × citriodorus (Pers.) Schreb. is an interspecific hybrid between Thymus pulegioides and Thymus vulgaris, known for its pharmacological activities as diaphoretic, deodorant, antiseptic and disinfectant, the last mostly related with its antimicrobial activity. The folk use of other extracts, as hydrolates, have also been disseminated, as regulators of oily skin with anti-acne effect. AIM OF THE STUDY: We aimed to evaluate the anti-acne potential of two Thymus x citriodorus (TC) preparations, the essential oil (EO) and the hydrolate, to be used as active ingredients for skin applications. Specifically, we intend to validate their anti-acne potential by describing their activity on acne related bacteria, bacterial virulence, anti-oxidant and anti-inflammatory potential, and biocompatibility on inflammatory cells. Additionally, we aimed to report their ecotoxicity under the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), thus focusing not only on the consumer, but also on environmental safety assessment. MATERIALS AND METHODS: Minimum inhibitory concentration (MIC) against C. acnes, S. aureus and S. epidermidis was evaluated. Minimum lethal concentration (MLC) was also determined. The effect on C. acnes biofilm formation and disruption was evaluated with crystal violet staining. Anti-inflammatory activity was investigated on LPS-stimulated mouse macrophages (RAW 264.7), by studying nitric oxide (NO) production (Griess reagent) and cellular biocompatibility through MTT assay. In-vitro NO and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging potential were also evaluated. The ecotoxicity was evaluated using Daphnia magna acute toxicity assays. RESULTS: EO presented direct antimicrobial activity, with visual MICs ranging from 0.06% for S. epidermidis and C. acnes to 0.125% for S. aureus. MLCs were higher than the obtained MICs. Hydrolate revealed visual MIC only for C. acnes. TC essential oil was effective in preventing biofilm formation and disrupting preformed biofilms even at sub-inhibitory concentrations. Hydrolate showed a more modest anti-biofilm effect. Regarding anti-inflammatory activity, TC hydrolate has a higher cellular biocompatibility. Still, both plant preparations were able to inhibit at least 50% of NO production at non-cytotoxic concentrations. Both EO and hydrolate have poor anti-oxidant activities. Regarding the ecotoxicity, TC essential oil was classified under acute 3 category, while the hydrolate has proved to be nontoxic, in accordance to the GHS. CONCLUSIONS: These results support the anti-acne value of different TC preparations for different applications. TC hydrolate by presenting higher biocompatibility, anti-inflammatory potential and the ability to modulate C. acnes virulence, can be advantageous in a product for everyday application. On the other hand, EO by presenting a marked antimicrobial, anti-biofilm and anti-inflammatory activities, still with some cytotoxicity, may be better suited for application in acute flare-ups, for short treatment periods.

Virulence Factors as Promoters of Chronic Vulvovaginal Candidosis: A Review.

INTRODUCTION: The vast majority of the species of the genus Candida spp. is commensal in humans; however, some are opportunistic pathogens that can cause infection, called candidosis. Among the different types of candidosis, we highlight the vulvovaginal (VVC) which can occur in two main clinical variants: chronic (cVVC) and episodic or sporadic. The incidence of cVVC has been worrying the scientific community, promoting the research on genotypic and phenotypic causes of its occurrence. We summarize important findings on factors that favor chronic vulvovaginal candidosis with respect to molecular epidemiology and the expression of various virulence factors, while clarifying the terminology involving these infections. AIM AND METHODOLOGY: The aim of this review was to gather research that linked virulence factors to VVC and its persistence and recurrence, using two databases (Pubmed and Google Scholar). Predisposing factors in women for the occurrence of cVVC and some studies that refer new preventive and alternative therapies were also included, where appropriate. RESULTS AND DISCUSSION: Several studies have been shedding light on the increasing number of persistence and recurrences of VVC. The expression of virulence factors has been related to both chronic forms of VVC and antifungal resistance. Other studies report mutations occurring in the genome of Candida spp. during the infection phase which may be important indications for new therapies. The introduction of preventive therapies and new therapies has revealed great importance and is also highlighted here.

Chemical signature and antimicrobial activity of Central Portuguese Natural Mineral Waters against selected skin pathogens.

The common therapeutic indications of Portuguese Natural Mineral Waters (NMWs) are primarily for respiratory, rheumatic and musculoskeletal systems. However, these NMWs have been increasingly sought for dermatologic purposes. Opposing to what is observed in the major European Thermal Centres, there are few scientific evidences supporting the use of Portuguese NMWs for clinical applications. The aim of this study was to characterize the antimicrobial profile of individual NMWs from the central region of Portugal and correlate the results with their physicochemical characterization. An extensive multivariate analysis (principal component analysis) was also performed to further investigate this possible correlation. Six collection strains representing skin microbiota, namely Staphylococcus aureus, Escherichia coli, Corynebacterium amycolatum, Candida albicans, Staphylococcus epidermidis and Cutibacterium acnes, were analysed, and their antimicrobial profile was determined using Clinical and Laboratory Standards Institute M07-A10, M45-A2, M11-A6 and M27-A3 microdilution methods. Different NMWs presented different antimicrobial profiles against the strains used; the physicochemical composition of NMWs seemed to be correlated with the different susceptibility profiles. Cutibacterium acnes showed a particularly high susceptibility to all NMWs belonging sulphurous/bicarbonated/sodic ionic profile, exhibiting microbial reductions up to 65%. However, due to the complex physicochemical composition of each water an overall conclusion regarding the effect of a specific ion on the growth of different microorganisms is yet to be known.

Thymbra capitata essential oil as potential therapeutic agent against Gardnerella vaginalis biofilm-related infections.

AIM: To evaluate the antibacterial activity of Thymbra capitata essential oil and its main compound, carvacrol, against Gardnerella vaginalis grown planktonically and as biofilms, and its effect of vaginal lactobacilli. MATERIALS & METHODS: Minimal inhibitory concentration, minimal lethal concentration determination and flow cytometry analysis were used to assess the antibacterial effect against planktonic cells. Antibiofilm activity was measured through quantification of biomass and visualization of biofilm structure by confocal laser scanning microscopy. RESULTS: T. capitata essential oil and carvacrol exhibited a potent antibacterial activity against G. vaginalis cells. Antibiofilm activity was more evident with the essential oil than carvacrol. Furthermore, vaginal lactobacilli were significantly more tolerant to the essential oil. CONCLUSION: T. capitata essential oil stands up as a promising therapeutic agent against G. vaginalis biofilm-related infections.

New strategies for local treatment of vaginal infections.

Vaginal infections are extremely prevalent, particularly among women of reproductive age. Although they do not result in high mortality rates, these infections are associated with high levels of anxiety and reduction of quality of life. In most cases, topical treatment of vaginal infections has been shown to be at least as effective as oral treatment, resulting in higher local drug concentrations, with fewer drug interactions and adverse effects. Furthermore, the emergence of microbial resistance to chemotherapeutics and the difficulties in managing infection recurrences sustain the need for more effective local treatments. However, conventional dosage forms have been associated with low retention in the vagina and discomfort. Formulation strategies such as the development of bioadhesive, thermogelling systems and microtechnological or nanotechnological approaches have been proposed to improve delivery of traditional drugs, and other treatment modalities such as new drugs, plant extracts, and probiotics are being studied. This article reviews the recent strategies studied to improve the treatment and prevention of the commonest vaginal infections-namely, vaginal bacteriosis, aerobic vaginitis, vulvovaginal candidosis, and trichomoniasis-through the intravaginal route.

Studies and methodologies on vaginal drug permeation.

The vagina stands as an important alternative to the oral route for those systemic drugs that are poorly absorbed orally or are rapidly metabolized by the liver. Drug permeation through the vaginal tissue can be estimated by using in vitro, ex vivo and in vivo models. The latter ones, although more realistic, assume ethical and biological limitations due to animal handling. Therefore, in vitro and ex vivo models have been developed to predict drug absorption through the vagina while allowing for simultaneous toxicity and pathogenesis studies. This review focuses on available methodologies to study vaginal drug permeation discussing their advantages and drawbacks. The technical complexity, costs and the ethical issues of an available model, along with its accuracy and reproducibility will determine if it is valid and applicable. Therefore every model shall be evaluated, validated and standardized in order to allow for extrapolations and results presumption, and so improving vaginal drug research and stressing its benefits.

Helichrysum italicum: from traditional use to scientific data.

ETHNOPHARMACOLOGICAL RELEVANCE: Helichrysum italicum (Roth) G. Don fil. (family Asteraceae) has been used for its medicinal properties for a long time and, even nowadays, continues to play an important role in the traditional medicine of Mediterranean countries. Based on this traditional knowledge, its different pharmacological activities have been the focus of active research. This review aims to provide an overview of the current state of knowledge of the pharmacological activities of Helichrysum italicum, as well as its traditional uses, toxicity, drug interactions and safety. MATERIALS AND METHODS: The selection of relevant data was made through a search using the keywords "Helichrysum italicum" and "H. italicum" in "Directory of Open Access Journals", "Google Scholar", "ISI Web of Knowledge", "PubMed", "ScienceDirect" and "Wiley Online Library". Information obtained in local and foreign books and other sources was also included. RESULTS: There are reports on the traditional use of Helichrysum italicum in European countries, particularly Italy, Spain, Portugal and Bosnia and Herzegovina. In these countries, its flowers and leaves are the most used parts in the treatment of health disorders such as allergies, colds, cough, skin, liver and gallbladder disorders, inflammation, infections and sleeplessness. In order to validate some of the traditional uses of Helichrysum italicum and highlight other potential applications for its extracts and isolated compounds, several scientific studies have been conducted in the last decades. In vitro studies characterized Helichrysum italicum as an antimicrobial and anti-inflammatory agent. Its flavonoids and terpenes were effective against bacteria (e.g. Staphylococcus aureus), its acetophenones, phloroglucinols and terpenoids displayed antifungal action against Candida albicans and its flavonoids and phloroglucinols inhibited HSV and HIV, respectively. Helichrysum italicum acetophenones, flavonoids and phloroglucinols demonstrated inhibitory action in different pathways of arachidonic acid metabolism and other pro-inflammatory mediators. Regarding Helichrysum italicum in vivo activity, the highlight goes to the anti-erythematous and photoprotective activities of its flavonoids, demonstrated both in animals and humans, and to the anti-inflammatory properties exhibited by its flavonoids, acetophenones and phloroglucinols, as seen in animal models. Concerning its safety and adverse effects, while Helichrysum italicum does not display significant levels of cytotoxicity or genotoxicity, it should be noticed that one of its flavonoids inhibited some CYP isoforms and a case has been reported of an allergic reaction to its extracts. CONCLUSIONS: Helichrysum italicum is a medicinal plant with promising pharmacological activities. However, most of its traditionally claimed applications are not yet scientifically proven. Clinical trials are needed to further confirm these data and promote Helichrysum italicum as an important tool in the treatment of several diseases.

Potencial terapêutico de extractos de plantas em infecçoes genitais / Therapeutic potential of plant extracts on genital infections

Os compostos naturais tem servido de base para o deselvolvimento de novos productos terapéuticos. A actividades antimicrobiana destes productos e objecto de diversos estudos. As infecçoes vaginais sao a principa razao pela qual as mulheres procuram ajuda especializada. Os produtos naturais tem sido bastante procurados pela mulheres que sofrem de infecçoes vaginais crónicas, dado o aumento das resistencias dos microrganismos aos antimicrobianos clásicos, traduzindo-se na ineficacia clínica das estratégias terapéuticas disponíveis. Neste artigo apresentamos uma revisao dos estudos mais relevantes que reportam a actividade antimicrobiana de extractos naturais e seus compostos isolados em microrganismos responsáveis por algunas das mais prevalentes infecçoes genitais, nomeadamente Candida spp., Gardnerella vaginalis, Trichoma vaginales e virus do papiloma humano. A relacao entre a composiçao dos extractos e a suea actividade antimicrobiana e realçada e apresentam-se em cada caso as mais importantes formulaçoes terapéuticas desenvolvidas com base nestes productos naturais(AU)

Natural products remain an important basis for the development os new formulations. Teir antimicrobial activity has been the subject of various studies. Infections are one of the main reasons women visit a gynecologist. Due to the increases microorganism resistance to classic antimicrobials, natural products are in demand by women suffering from genital chronic infectious. In this article we review the most relevant studies on the antimicrobial activity of natural extracts and its individual constituents against microorganisms responsible for most frequent genital infections in women: Candida spp. Gardnerella vaginalis, Trichomonas vaginalis and human papilloma virus. We also describe the relationship between the extracts chemical composition and its antimicrobial activity as well as the most important therapeutic formulations developed(AU)

Antifungal activity of the essential oil of Thymus pulegioides on Candida, Aspergillus and dermatophyte species.

The composition of the essential oil of Thymus pulegioides and its antifungal activity on Candida, Aspergillus and dermatophyte fungal strains were studied. Essential oil from the aerial parts of the plant was obtained by hydrodistillation and analysed by GC and GC-MS. The oil showed high contents of carvacrol and thymol. The MIC and minimal lethal concentration were used to evaluate the antifungal activity against Candida (seven clinical isolates and four ATCC type strains), Aspergillus [five clinical isolates, and two Colección Española de Cultivos Tipo (CECT) and two ATCC type strains] and five clinical dermatophyte strains. Antifungal activity was evaluated for the essential oil and for its main components. To clarify its mechanism of action on yeasts and filamentous fungi, flow-cytometric studies of cytoplasmic membrane integrity were performed, and the effect on the amount of ergosterol was investigated. Results showed that T. pulegioides essential oil exhibited a significant activity against clinically relevant fungi, mainly due to lesion formation in the cytoplasmic membrane and a considerable reduction of the ergosterol content. The present study indicates that T. pulegioides essential oil has considerable antifungal activity, deserving further investigation for clinical applications.