Lippia origanoides essential oil possesses anticonvulsant effect in pentylenetetrazol-induced seizures in rats: a behavioral, electroencephalographic, and electromyographic study.

Epilepsy is a neuronal disorder characterized by abnormal excitability of the brain, leading to seizures. Only around 66% of the epileptic patients respond adequately to treatment with existing conventional anticonvulsants, making it necessary to investigate new antiepileptic drugs. The growing research into natural products and their pharmacological properties has become increasingly promising, particularly in the study of essential oils, which are already widely used in popular culture for treating various diseases. The present study evaluated the anticonvulsant effects of Lippia origanoides essential oil (LOEO) (100 mg/kg i. p.) compared to diazepam (DZP) (5 mg/kg i. p.), and the combined administration of these two substances to control convulsions induced by pentylenetetrazol (PTZ) (60 mg/kg i. p.). This evaluation was carried out using 108 male Wistar rats, which were divided into two experiments. Experiment 1-Behavioral assessment: The animals were divided into 4 groups (n = 9): (I) saline solution + PTZ, (II) DZP + PTZ, (III) LOEO + PTZ, (IV) LOEO + DZP + PTZ. The convulsive behavior was induced 30 min after the administration of the tested anticonvulsant drugs, and the observation period lasted 30 min. Experiment 2- Electrocorticographic evaluation: The animals were divided into 8 groups (n = 9): (I) saline solution; (II) LOEO; (III) DZP; (IV) LOEO + DZP; (V) saline + PTZ, (VI) DZP + PTZ (VII) LOEO + PTZ, (VIII) LOEO + DZP + PTZ. PTZ was administered 30 min after LOEO and DZP treatments and electrocorticographic activity was assessed for 15 min. For the control groups, electromyographic recordings were performed in the 10th intercostal space to assess respiratory rate. The results demonstrated that Lippia origanoides essential oil increased the latency time for the appearance of isolated clonic seizures without loss of the postural reflex. The animals had a more intense decrease in respiratory rate when combined with LOEO + DZP. EEG recordings showed a reduction in firing amplitude in the LOEO-treated groups. The combining treatment with diazepam resulted in increased anticonvulsant effects. Therefore, treatment with Lippia origanoides essential oil was effective in controlling seizures, and its combination with diazepam may represent a future option for the treatment of difficult-to-control seizures.

Lippia origanoides Essential Oil or Thymol in Combination with Fluconazole Produces Damage to Cells and Reverses the Azole-Resistant Phenotype of a Candida tropicalis Strain.

Candida tropicalis is one of the most pathogenic species within the genus. Increased antifungal resistance has been reported, which is in part due to the organism's ability to form biofilms. In natural products derived from plants, such as essential oils (EOs) or their major components, there is significant potential to develop new antifungals or to both enhance the efficacy and reduce the toxicity of conventional antifungals. This study aimed to evaluate the effect of combining an EO of Lippia origanoides or thymol with fluconazole on an azole-resistant C. tropicalis strain. Synergism was observed in the combination of fluconazole with the EO and with thymol, and minimal inhibitory concentrations for fluconazole decreased at least 32-fold. As a consequence of the synergistic interactions, mitochondrial membrane potential was reduced, and mitochondrial superoxide production increased. Alteration in nuclear morphology, cell surface, and ultrastructure was also observed. In conclusion, the synergistic interaction between L. origanoides EO or thymol with fluconazole reverted the azole-resistant C. tropicalis phenotype. These findings suggest that L. origanoides EO or thymol alone, or in combination with fluconazole, have the potential for development as antifungal therapies for this yeast, including resistant strains.

Preparation, Physicochemical Characterization, and Stability Study of Lippia origanoides Essential Oil-Based Nanoemulsion as a Topical Delivery System.

INTRODUCTION: Fungal diseases are a priority in research, development, and health care, according to the WHO, mainly due to Candida spp. Essential oils (EOs) of the genus Lippia have demonstrated broad antimicrobial biological activity. Previous studies identified the anti-Candida potential of a thymol/p-cymene chemotype EO from Lippia origanoides H.B.K coded "0018". Nanoemulsions favor the biological activity of EOs and overcome limitations such as low solubility, instability against oxidizing agents, pH, light, and low permeability. To develop, characterize, and adjust a prototype of an O/W nanoemulsion containing the "0018" EO from Lippia origanoides for its evaluation in an In vitro permeability study. METHOD: Nanoemulsions were obtained using a high energy high shear method. Their particle size distribution, Z potential, viscosity, pH, encapsulation efficiency (EE), thermodynamic stability and the Turbiscan Stability Index (TSI) were evaluated. The nanoemulsion prototype was adjusted to improve performance characteristics and microbiological efficacy. Thymol was used as an analyte in the EO quantification using UHPLC-DAD. RESULTS: An O/W nanoemulsion with hydrodynamic diameter <200 nm and polydispersity index <0.3, EE >95%, with TSI < 1.5, anti-Candida albicans efficiency >95% was obtained; permeable with a flow of 6.0264 µg/cm2/h and permeability coefficient of 1.3170x10-3 cm/h. CONCLUSION: A pharmaceutical formulation prototype is obtained that maintains the physical and physicochemical characteristics over time. Permeability is verified in an in-vitro model. It is proposed to evaluate its antifungal activity in preclinical or clinical studies as a contribution to the treatment of topical fungal diseases caused by Candida spp., through the use of biological resources and Colombian biodiversity.

Effect of Essential Oil from Lippia origanoides on the Transcriptional Expression of Genes Related to Quorum Sensing, Biofilm Formation, and Virulence of Escherichia coli and Staphylococcus aureus.

Microbial infections resistant to conventional antibiotics constitute one of the most important causes of mortality in the world. In some bacterial species, such as Escherichia coli and Staphylococcus aureus pathogens, biofilm formation can favor their antimicrobial resistance. These biofilm-forming bacteria produce a compact and protective matrix, allowing their adherence and colonization to different surfaces, and contributing to resistance, recurrence, and chronicity of the infections. Therefore, different therapeutic alternatives have been investigated to interrupt both cellular communication routes and biofilm formation. Among these, essential oils (EO) from Lippia origanoides thymol-carvacrol II chemotype (LOTC II) plants have demonstrated biological activity against different biofilm-forming pathogenic bacteria. In this work, we determined the effect of LOTC II EO on the expression of genes associated with quorum sensing (QS) communication, biofilm formation, and virulence of E. coli ATCC 25922 and S. aureus ATCC 29213. This EO was found to have high efficacy against biofilm formation, decreasing-by negative regulation-the expression of genes involved in motility (fimH), adherence and cellular aggregation (csgD), and exopolysaccharide production (pgaC) in E. coli. In addition, this effect was also determined in S. aureus where the L. origanoides EO diminished the expression of genes involved in QS communication (agrA), production of exopolysaccharides by PIA/PNG (icaA), synthesis of alpha hemolysin (hla), transcriptional regulators of the production of extracellular toxins (RNA III), QS and biofilm formation transcriptional regulators (sarA) and global regulators of biofilm formation (rbf and aur). Positive regulation was observed on the expression of genes encoding inhibitors of biofilm formation (e.g., sdiA and ariR). These findings suggest that LOTCII EO can affect biological pathways associated with QS communication, biofilm formation, and virulence of E. coli and S. aureus at subinhibitory concentrations and could be a promising candidate as a natural antibacterial alternative to conventional antibiotics.

Essential Oils Distilled from Colombian Aromatic Plants and Their Constituents as Penetration Enhancers for Transdermal Drug Delivery.

The study aimed to determine the enhanced effects of essential oils (EOs) and plant-derived molecules (PDMs) as penetration enhancers (PEs) for transdermal drug delivery (TDD) of caffeine. A 1% w/w solution of eight EOs and seven PDMs was included in the 1% caffeine carbopol hydrogel. Franz diffusion cell experiments were performed using mice with full-thickness skin. At various times over 24 h, 300 µL of the receptor were withdrawn and replaced with fresh medium. Caffeine was analyzed spectrophotometrically at 272 nm. The skin irritation effects of the hydrogels applied once a day for 21 days were investigated in mice. The steady-state flux (JSS) of the caffeine hydrogel was 30 ± 19.6 µg cm-2 h-1. An increase in caffeine JSS was induced by Lippia origanoides > Turnera diffusa > eugenol > carvacrol > limonene, with values of 150 ± 14.1, 130 ± 47.6, 101 ± 21.7, 90 ± 18.4, and 86 ± 21.0 µg cm-2 h-1, respectively. The Kp of caffeine was 2.8 ± 0.26 cm h-1, almost 2-4 times lower than that induced by Lippia origanoides > Turnera diffusa > limonene > eugenol > carvacrol, with Kp values of 11 ± 1.7, 8.8 ± 4.2, 6.8 ± 1.7, 6.3 ± 1.2, and 5.15 ± 1.0 cm h-1, respectively. No irritating effects were observed. Lippia origanoides, Turnera diffusa, eugenol, carvacrol, and limonene improved caffeine's skin permeation. These compounds may be as effective as the PE in TDD systems.

Antifungal susceptibility profile of Trichosporon inkin: About three cases of White Piedra.

Trichosporon spp. usually cause systemic or superficial infections. Three cases of White Piedra produced by Trichosporon inkin are described. The in vitro antifungal activity to fluconazole, amphotericin B, ketoconazole and caspofungin against the three clinical isolates were evaluated. Sensitivity to fluconazole and ketoconazole was evidenced. However, the treatment of this mycosis is still a challenge.

Lippia origanoides derivatives in vitro evaluation on polymicrobial biofilms: Streptococcus mutans, Lactobacillus rhamnosus and Candida albicans.

OBJECTIVE: This work evaluated the Lippia origanoides derivatives in vitro effect on polymicrobial biofilms of Streptococcus mutans, Lactobacillus rhamnosus and Candida albicans. Additionally, the cytotoxic effect of the oils on human skin keratinocytes (HaCaT) and fibroblasts of the periodontal ligament (FLP) cell lines was evaluated. DESIGN: The minimum inhibitory concentration, the inhibitory activity on monomicrobial (S. mutans) and polymicrobial biofilm (S. mutans, L. rhamnosus and C. albicans) of L. origanoides four essential oils and terpenes (thymol and carvacrol) were evaluated. The cytotoxic effect of each one of the compounds was measured, and all the tests were compared against chlorhexidine. RESULTS: All the evaluated compounds reached an inhibition percentage of S. mutans monomicrobial biofilms formation of 100 % at 600 µg/mL (p < 0.0001). The highest concentration (2 MIC) eradicated 100 % of S. mutans-preformed biofilms after 5 min L. origanoides carvacrol + thymol and thymol chemotypes showed marked reductions in topography, the number of microbial cells and extracellular matrix on polymicrobial biofilm. The cytotoxic effect of the compounds was very similar to chlorhexidine. CONCLUSIONS: L. origanoides essential oils have an inhibitory effect on mono and polymicrobial biofilms. The oils present a similar cytotoxic effect to chlorhexidine on HaCaT and FLP cell lines. However, including these compounds in formulations for clinical use is an exciting proposal yet to be investigated.

Alternative Controlling Agent of Theobroma grandiflorum Pests: Nanoscale Surface and Fractal Analysis of Gelatin/PCL Loaded Particles Containing Lippia origanoides Essential Oil.

A new systematic structural study was performed using the Atomic Force Microscopy (AFM) reporting statistical parameters of polymeric particles based on gelatin and poly-ε-caprolactone (PCL) containing essential oil from Lippia origanoides. The developed biocides are efficient alternative controlling agents of Conotrachelus humeropictus and Moniliophtora perniciosa, the main pests of Theobroma grandiflorum. Our results showed that the particles morphology can be successfully controlled by advanced stereometric parameters, pointing to an appropriate concentration of encapsulated essential oil according to the particle surface characteristics. For this reason, the absolute concentration of 1000 µg·mL-1 (P1000 system) was encapsulated, resulting in the most suitable surface microtexture, allowing a faster and more efficient essential oil release. Loaded particles presented zeta potential around (-54.3 ± 2.3) mV at pH = 8, and particle size distribution ranging from 113 to 442 nm. The hydrodynamic diameter of 90% of the particle population was found to be up to (405 ± 31) nm in the P1000 system. The essential oil release was evaluated up to 80 h, with maximum release concentrations of 63% and 95% for P500 and P1000, respectively. The best fit for the release profiles was obtained using the Korsmeyer-Peppas mathematical model. Loaded particles resulted in 100% mortality of C. humeropictus up to 48 h. The antifungal tests against M. perniciosa resulted in a minimum inhibitory concentration of 250 µg·mL-1, and the P1000 system produced growth inhibition up to 7 days. The developed system has potential as alternative controlling agent, due to its physical stability, particle surface microtexture, as well as pronounced bioactivity of the encapsulated essential oil.

In Vitro Pharmacological Screening of Essential Oils from Baccharis parvidentata and Lippia origanoides Growing in Brazil.

In this study, the in vitro antimicrobial, antiparasitic, antiproliferative and cytotoxic activities of essential oil from Baccharis parvidentata Malag. (EO-Bp) and Lippia origanoides Kunth (EO-Lo) were explored. The relevant effects were observed against the parasitic protozoans Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei and Leishmania amazonensis (ranging 0.6 to 39.7 µg/mL) and malignant MCF-7, MCF-7/HT, 22Rv1, and A431 cell lines (ranging 6.1 to 31.5 µg/mL). In parallel, EO-Bp showed better selective indexes in comparison with EO-Lo against peritoneal macrophages from BALB/c mice and MRC-5 cell line. In conclusion, EO-Lo is known to show a wide range of health benefits that could be added as another potential use of this oil with the current study. In the case of EO-Bp, the wide spectrum of its activities against protozoal parasites and malignant cells, as well as its selectivity in comparison with non-malignant cells, could suggest an interesting candidate for further tests as a new therapeutic alternative.

Morphometry of bovine blastocysts produced in vitro in culture media with antioxidants cysteamine or oily extract of Lippia origanoides / [Morfometria de blastocistos bovinos produzidos in vitro em meio de cultura com antioxidantes cisteamina ou extrato oleoso de Lippia origanoides]

This study aimed to evaluate the ultrastructural morphometry of bovine embryos produced in vitro grown at different concentrations of antioxidants. After in vitro maturation and fertilization, the presumptive zygotes were assigned into five treatments. T1) without the addition of any antioxidants (negative control); T2) addition of 50µM/mL cysteamine; and T3, T4 and T5) adding 2.5µg/mL, 5.0µg/mL or 10.0µg/mL of the antioxidants derived from the oily extract from Lippia origanoides, respectively. On D7 of culture, the embryos in the blastocyst stage were fixed and prepared for electron transmission microscopy. These were evaluated for the proportion of cytoplasm-to-nucleus, cytoplasm-to-mitochondria, cytoplasm-to-vacuoles, cytoplasm-to-autophagic vacuoles and cytoplasm-to-lipid droplets. Blastocysts cultured in media containing oily extract of Lippia origanoides presented morphological characteristics such as high cell:mitochondria ratio and low cell:vacuoles and cell:autophagic vacuole ratio, possibly been morphological indicators of embryonic quality. Inner cell mass (ICM) from blastocysts cultured in media without any antioxidants had the highest cell:vacuole ratio. Similar results were found in the trophectoderm (TE) cells of blastocysts from treatment 2. Embryo culture media supplemented with antioxidants derived from Lippia origanoides oil produced embryos with a higher cytoplasmic proportion of organelles, such as mitochondria. Also, treatments without any antioxidants or with the addition of cysteamine presented cytoplasmic vacuolization, a characteristic related to production of poor-quality embryos.(AU)

Este estudo teve como objetivo avaliar a morfometria ultraestrutural de embriões bovinos produzidos in vitro e cultivados em diferentes concentrações de antioxidantes. Após a maturação e a fertilização in vitro, os possíveis zigotos foram divididos em cinco tratamentos: T1) sem adição de antioxidantes (controle negativo); T2) adição de 50µM/mL de cisteamina; e T3, T4 e T5) adição de 2,5µg/mL, 5,0µg/mL ou 10,0µg/mL dos antioxidantes derivados do extrato oleoso de Lippia origanoides, respectivamente. No D7 de cultivo, os embriões em estágio de blastocisto foram fixados e preparados para microscopia eletrônica de transmissão. Estes foram avaliados para a proporção entre citoplasma e núcleo, citoplasma e mitocôndria, citoplasma e vacúolos, citoplasma e vacúolos autofágicos e citoplasma e gotículas lipídicas. Blastocistos cultivados em meio contendo extrato oleoso de Lippia origanoides apresentaram características morfológicas como alta relação célula:mitocôndria e baixa relação célula:vacúolos e célula:vacúolo autofágico, possíveis indicadores morfológicos de qualidade embrionária. A massa celular interna (MCI) de blastocistos cultivados em meio sem quaisquer antioxidantes teve a maior razão célula:vacúolo. Resultados semelhantes foram encontrados nas células do trofectoderma (TE) de blastocistos do tratamento 2. Portanto, o meio de cultivo embrionário suplementado com antioxidantes derivados do óleo de Lippia origanoides produziu embriões com maior proporção citoplasmática de organelas, como mitocôndrias. Além disso, tratamentos sem antioxidantes ou com adição de cisteamina apresentaram vacuolização citoplasmática, característica relacionada à produção de embriões de baixa qualidade.(AU)

Evaluation of Three Formulations of Essential Oils in Broiler Chickens under Cyclic Heat Stress.

The objective of the present research was to assess the dietary supplementation of three formulations of essential oils (EO) in chickens under heat stress (HS). Day-of-hatch Cobb 500 chicks (n = 500) were randomly distributed into four groups: 1. HS control + control diets; 2. HS + control diets supplemented with 37 ppm EO of Lippia origanoides (LO); 3. HS + control diets supplemented with 45 ppm LO + 45 ppm EO of Rosmarinus officinalis (RO) + 300 ppm red beetroot; 4. HS + 45 ppm LO + 45 ppm RO + 300 ppm natural betaine. Chickens that received the EO showed significant (p < 0.05) improvement on BW, BWG, FI, and FCR compared to control HS chickens. Average body core temperature in group 3 and group 4 was significantly (p < 0.05) reduced compared with the HS control group and group 2. Experimental groups showed a significant reduction in FITC-d at 42 days, a significant increase in SOD at both days but a significant reduction of IFN-γ and IgA compared with HS control (p < 0.05). Bone mineralization was significantly improved by EO treatments (p < 0.05). Together these data suggest that supplemental dietary EO may reduce the harmful effects of HS.

Efficacy of novel antiseptic product containing essential oil of Lippia origanoides to reduce intramammary infections in cows.

BACKGROUND AND AIM: The use of antimicrobials in the control of mastitis is of concern in public health due to their inefficiency in targeting microorganisms. Studies with medicinal plants have risen as an alternative to the use of conventional products. The objective of this study was to evaluate the efficacy of an experimental disinfectant based on the essential oil (EO) from Lippia origanoides in preventing the development of new intramammary infections (IMI) in Holstein cows. MATERIALS AND METHODS: The conventional protocol of pre- and post-milking was used and the control (Conventional treatment [CNV]) and experimental (Experimental treatment [PEX]) products containing EO at 120 µL/mL were applied by immersion. Individual milk samples were analyzed using sheep blood agar methodologies and biochemical tests. The efficiency of the treatment was defined by the presence or absence of Staphylococcus aureus, coagulase-negative Staphylococcus, and Streptococcus spp. RESULTS: There were no clinical and subclinical mastitis cases, no lesions in the mucosal of teats, nor dirt score between groups in this study. Both treatments did not influence the occurrence of IMI. CONCLUSION: The results revealed that PEX acts efficiently against microorganisms compared to the disinfection by the conventional product demonstrating the efficacy of the alternative product on the prevention of new IMIs in dairy cows.

Bacillus cereus bacteria endófita promotora de crecimiento vegetal / Bacillus cereus endophytic bacterial plant growth promoter

RESUMEN La planta Lippia origanoides ha sido ampliamente estudiada debido al efecto antimicrobiano y antifúngico que poseen sus extractos y aceites esenciales, los cuales han sido probados contra un gran número de microorganismos patógenos. Sin embargo, es escasa la literatura que registra la diversidad de bacterias endófitas asociadas a esta especie de plantas. El objetivo del trabajo fue evaluar in vitro la capacidad de promoción de crecimiento vegetal de bacterias endófitas de Lippia origanoides en el municipio de Sincelejo -Sucre, Colombia. En este estudio se aislaron bacterias endófitas en medio de cultivo agar R2A a partir de diferentes tejidos, se evaluó la densidad poblacional (UFC/g de tejido) por conteo en superficie y la promoción de crecimiento vegetal de forma cualitativa en medios selectivos específicos. Se observaron diferencias significativas para la densidad poblacional de bacterias endófitas respecto al tipo de tejido, con mayores valores en la raíz (2,0 x 1010/g raíz), seguido del tallo (1,3 x 1010/g tallo) y hojas (9,2 x 109/g hoja). Se obtuvieron un total de 20 bacterias endófitas, los cuales dos mostraron capacidad solubilizadora de fosfato, fijación biológica de nitrógeno, producción de sideróforos y ACC desaminasa. Los morfotipos TLO5 y RLO4 fueron identificados molecularmente como Bacillus cereus, mostrando buenos resultados de promoción de crecimiento vegetal.

ABSTRACT The Lippia origanoides plant has been widely studied due to the antimicrobial and antifungal effect of its extracts and essential oils, which have been tested against a large number of pathogenic microorganisms. However, there is little literature that records the diversity of endophytic bacteria associated with this plant species. The objective of the work was to evaluate in vitro the plant growth promotion capacity of endophytic Lippia origanoides bacteria in the municipality of Sincelejo-Sucre, Colombia. In this study, endophytic bacteria were isolated in R2A agar culture medium from different tissues, population density (CFU / g of tissue) was evaluated by surface counting and the promotion of plant growth qualitatively in specific selective media. Significant differences were observed for the population density of endophytic bacteria regarding tissue type, with higher values in the root (2.0 x 1010 / g root), followed by the stem (1.3 x 1010 / g stem) and leaves (9.2 x 109 / g sheet). A total of 20 endophytic bacteria were obtained, which two showed phosphate solubilizing capacity, biological nitrogen fixation, production of siderophores and ACC deaminase. The TLO5 and RLO4 morphotypes were molecularly identified as Bacillus cereus, showing good results in promoting plant growth.

Antimicrobial and Antibiofilm Activities of Essential Oils against Escherichia coli O157:H7 and Methicillin-Resistant Staphylococcus aureus (MRSA).

The emergence of multidrug resistant microorganisms represents a global challenge due to the lack of new effective antimicrobial agents. In this sense, essential oils (EOs) are an alternative to be considered because of their anti-inflammatory, antiviral, antibacterial, and antibiofilm biological activities. Therefore, multiple efforts have been made to consider the potential use of EOs in the treatment of infections which are caused by resistant microorganisms. In this study, 15 EOs of both Colombian and introduced aromatic plants were evaluated against pathogenic strains of E. coli O157:H7 and methicillin resistant Staphylococcus aureus (MRSA) in planktonic and sessile states in order to identify relevant and promising alternatives for the treatment of microbial infections. Forty different compounds were identified in the 15 EO with nine of them constituted mainly by oxygenated monoterpenes (OM). EOs from Lippia origanoides, chemotypes thymol, and carvacrol, displayed the highest antibacterial activity against E. coli O157:H7 (MIC50 = 0.9 and 0.3 mg/mL, respectively) and MRSA (MIC50 = 1.2 and 0.6 mg/mL, respectively). These compounds from EOs had also the highest antibiofilm activity (inhibition percentage > 70.3%). Using scanning electron microscopy (SEM), changes in the size and morphology of both bacteria were observed when they were exposed to sub-inhibitory concentrations of L. origanoides EO carvacrol chemotype. EOs from L. origanoides, thymol, and carvacrol chemotypes represented a viable alternative for the treatment of microbial infections; however, the Selectivity Index (SI ≤ 3) indicated that it was necessary to study alternatives to reduce its in vitro cytotoxicity.

Lippia sidoides and Lippia origanoides essential oils affect the viability, motility and ultrastructure of Trypanosoma cruzi.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is considered a public health problem. The current chemotherapy for this illness causes serious side effects and its use in the chronic phase of the disease is still controversial. In this regard, the investigation of novel therapeutic strategies remains a priority. The essential oils (EOs) from aromatic plants emerge as a promising source of bioactive compounds. In a previous work we reported the trypanocidal activity of the essential oils from the medicinal plants Lippia sidoides (LSEO) and Lippia origanoides (LOEO) against T. cruzi. Herein, we aimed to further investigate, in more details, the mode of action of LSEO and LOEO on the different developmental stages of this parasite. We showed that Lippia sidoides (LSEO) and Lippia origanoides (LOEO) induced a significant reduction in the percentage of macrophages infected by T. cruzi and in the number of intracellular parasites. Ultrastructural analysis showed that the treatment with both oils caused morphological changes consistent with loss of viability and cell death. The reduced staining with calcein and the increase in the proportion of HE-positive cells also demonstrated that LSEO and LOEO caused loss of parasite viability and membrane integrity. A considerable decrease in Rhodamine 123 and an increase in fluorescence intensity of MitoSox in LOEO were indicative of loss of mitochondrial potential and generation of reactive oxygen species, which ultimately lead to parasite death. Moreover, the optical tweezer analysis indicated that LOEO was more effective in reducing the motility of the epimastigotes. Taken together, our results demonstrated that the LSEO and LOEO are active against T. cruzi and constitute a promising drugs for the therapy of Chagas disease.

Lipid nanoparticles as carriers of cyclodextrin inclusion complexes: A promising approach for cutaneous delivery of a volatile essential oil.

Solid inclusion complexes with cyclodextrins (CD) may be used to overcome volatility and solubility problems of essential oils of pharmacological interest. However, they lack the many dermatological advantages of lipid nanoparticles. This study intends to evaluate the ability of nanostructured lipid carriers (NLC) to encapsulate hydroxypropyl-ß-cyclodextrin inclusion complexes of Lippia origanoides essential oil (EO) and to maintain the desirable aspects of lipid colloids interaction with the skin, specifically follicular accumulation and controlled delivery. CD and NLC were also evaluated separately. Thymol (TML) was used as the essential oil marker and to produce control formulations. As expected, CD alone, though effective in overcoming volatility and low aqueous solubility of TML, were ineffective in controlling marker release (˜50% of EO released after 3 h, Hixson-Crowell kinetics). Even though NLC controlled drug release (˜20% EO released after 12 h, zero-order kinetics) enabling TML penetration into the skin (> 40 µg/cm2after 12 h), NLC alone were not efficient in preventing TML volatility, especially at higher temperatures (calculated shelf-life of 2 days at 35 °C). The combined approach resulted in a synergistic effect (˜20% EO released after 12 h; shelf life of 6 days). The lack of statistical difference of TML skin penetration from NLC and NLC-CD suggests the developed system maintained all skin interaction aspects of lipid colloids, including follicular accumulation forming a depot for controlled delivery. In conclusion, lipid nanoparticles demonstrated to be promising carriers for inclusion complexes of this particular volatile essential oil.

Antioxidant activity of oily extract obtained from Lippia origanoides improves the quality of bovine embryos produced in vitro / Atividade antioxidante de óleo essencial obtido de Lippia origanoides melhora a qualidade de embriões bovinos produzidos in vitro

The aim of this study was to evaluate the supplementation of embryo culture medium with antioxidant obtained from oily extract of Lippia origanoides on in vitro blastocyst development and quality. Oocytes collected from slaughterhouse ovaries were matured and fertilized in vitro following standard laboratory procedures. Zygotes were cultured in SOF medium supplemented according to the following treatments: T1 embryo culture medium without antioxidant supplementation; T2)50µM/mL Cysteamine; T3)2.5µg/mL; T4)5.0µg/mL and T5)10.0µg/mL of antioxidant obtained from oily extract of Lippia origanoides. On the seventh day of culture, the blastocysts were fixed and evaluated for apoptosis rates, number of total cell and inner cell mass cells by means of the TUNEL Test. The use of antioxidants during cultivation did not increase (P> 0.05) the final blastocyst production rate. The treatments T2, T3, T4 and T5 had the lowest (P< 0.05) apoptotic indexes (4.5±1.1%, 8.4±2.5%, 3.4±1.1% and 5.5±0.9%, respectively) when compared to T1 treatment (10.0±1.4%). The number of inner cell mass did not differ (P> 0.05) among embryos from different treatments. The addition of antioxidant obtained from oily extract of Lippia origanoides reduces the apoptosis rate and improves the quality without increasing the total in vitro production of bovine embryos.(AU)

O objetivo desse estudo foi avaliar a suplementação de meio de cultura de embriões com antioxidante obtido do extrato oleoso da Lippia origanoides no desenvolvimento e na qualidade de blastocistos produzidos in vitro. Oócitos coletados de ovários de matadouros foram maturados e fertilizados in vitro segundo procedimento laboratorial padrão. Zigotos foram cultivados em meio SOF suplementado de acordo com os seguintes tratamentos: T1) meio de cultivo embrionário sem suplementação antioxidantes; T2) 50µM/mL Cisteamina; T3) 2,5µg/mL; T4) 5,0µg/mL e T5) 10,0µg/mL do antioxidante obtido do extrato oleoso de Lippia origanoides. No sétimo dia de cultivo, os blastocistos foram fixados e avaliados para taxa de apoptose, número total de células e massa celular interna através do teste TUNEL. O uso de antioxidantes durante cultivo não aumentou (P>0,05) a taxa de produção final de blastócitos. Os tratamentos T2, T3, T4 e T5 tiverem menor índice apoptótico (p>0,05 - 4,5±1,1%, 8,4±2,5%, 3,4±1,1% e 5,5±0,9%, respectivamente) quando comparados a T2 (10,0±1,4%). O valor de massa celular interna não diferenciou (p>0,05) entre embriões de diferentes tratamentos. A adição de antioxidante obtido do extrato oleoso de Lippia origanoides reduziu a taxa de apoptose e melhorou a qualidade sem aumentar a produção in vitro de embriões bovinos.(AU)

Genetic diversity and chemical variability of Lippia spp. (Verbenaceae).

BACKGROUND: The genus Lippia comprises 150 species, most of which have interesting medicinal properties. Lippia sidoides (syn. L. origanoides) exhibits strong antimicrobial activity and is included in the phytotherapy program implemented by the Brazilian Ministry of Health. Since species of Lippia are morphologically very similar, conventional taxonomic methods are sometimes insufficient for the unambiguous identification of plant material that is required for the production of certified phytomedicines. Therefore, genetic and chemical analysis with chemotype identification will contribute to a better characterization of Lippia species. METHODS: Amplified Length Polymorphism and Internal Transcribed Spacer molecular markers were applied to determine the plants' genetic variability, and the chemical variability of Lippia spp. was determined by essential oil composition. RESULTS: Amplified Length Polymorphism markers were efficient in demonstrating the intra and inter-specific genetic variability of the genus and in separating the species L. alba, L. lupulina and L. origanoides into distinct groups. Phylogenetic analysis using Amplified Length Polymorphism and markers produced similar results and confirmed that L. alba and L. lupulina shared a common ancestor that differ from L. origanoides. Carvacrol, endo-fenchol and thymol were the most relevant chemical descriptors. CONCLUSION: Based on the phylogenetic analysis it is proposed that L. grata should be grouped within L. origanoides due to its significant genetic similarity. Although Amplified Length Polymorphism and Internal Transcribed Spacer markers enabled the differentiation of individuals, the genotype selection for the production of certified phytomedicines must also consider the chemotype classification that reflects their real medicinal properties.

Drying temperature changes trichome integrity, chemical content and composition of the essential oil of pepper-rosmarin / Temperatura de secagem altera integridade de tricomas, teor e composição química doóleo essencial de alecrim-pimenta

ABSTRACT: Medicinal plants are generally commercialized dried. However, temperatures used in their drying processes may interfere with the content and chemical composition of their essential oils. The Lippia origanoides possesses thymol in the essential oil that is stored in glandular trichomes. Thymol is a major component of economic importance in the chemical and pharmaceutical industry. The objective of this study was to evaluate the effect of the drying temperatures of L. origanoides leaves regarding trichome integrity, content, and chemical composition of the essential oil. The experimental design was completely randomized with four treatments defined by oven drying temperatures (40, 50, 60, and 70ºC) and four replications. Essential oil was extracted by hydro distillation and the essential oil contents were determined (%). Chemical composition of the oil was analyzed by gas chromatography coupled to mass spectrometry. Scanning electron microscopy was carried out to determine trichome integrity. Drying the L. origanoides leaves in a forced ventilation oven at 40°C minimized the loss of extracted essential oil content (17.5g kg-1) and relative thymol percentage. Leaves dried at the temperatures of 60 and 70ºC exhibited a higher percentage of ruptured trichomes and reduced essential oil content to 13.7g kg-1 and 11.8g kg-1, respectively.

RESUMO: As plantas medicinais são geralmente comercializadas secas, no entanto, as temperaturas utilizadas nos processos de secagem podem interferir no teor e composição química dos óleos essenciais destas plantas. A Lippia origanoides possui timol em seu óleo essencial, que se encontra armazenado em tricomas glandulares. O timol é um componente majoritário de importância econômica na indústria química e farmacêutica. O objetivo do trabalho foi estudar o efeito das temperaturas de secagem de folhas de L. origanoides na integridade dos tricomas, teor e composição química do óleo essencial. O delineamento experimental foi inteiramente casualizado, com quatro tratamentos definidos por temperaturas de secagem em estufa (40ºC, 50ºC, 60ºC e 70ºC) e quatro repetições. Foram realizadas extrações por hidrodestilação e determinação dos teores dos óleos essenciais (%), análise de composição química do óleo por cromatografia gasosa acoplada à espectrometria de massas e microscopia eletrônica de varredura para determinação da integridade dos tricomas. A secagem das folhas de L. origanoides em estufa de ventilação forçada a 40ºC minimizou a perda do teor de óleo essencial extraído (17,5g kg-1) e porcentagem relativa de timol. Folhas secas em temperatura de 60 e 70ºC exibiram maior porcentagem de tricomas rompidos e redução do teor de óleo essencial para 13,7g kg-1 e 11,8g kg-1, respectivamente.

Pharmaceutical development of tablets containing a spray-dried optimized extract from Lippia origanoides H. B. K.: influence of excipients and toxicological assessment

ABSTRACT Lippia origanoides is a honey shrub which has showed hypotensive potential assessed by in vivo studies. The aim of this work is the development of a pharmaceutical formulation composed by an optimized extract obtained from aerial parts of L. origanoides. The quantification of the naringenin marker in the dry extract and tablets developed was performed, as well as the assessment of the oral acute toxicity in rats. The hydroalcoholic extract of L. origanoides was spray-dried with the addition of colloidal silicon dioxide (Lo-HAE/CSD), and then applied in the preparation of eight different lots of tablets. The influence of the diluent (cellulose or babassu mesocarp), the presence of binder, and the percentage of lubricant, as well as organoleptic and physicochemical characteristics were screened. For the quantification of the marker content both in Lo-HAE/CSD and in the tablets, an analytical curve of the naringenin standard was fitted, and the samples were then analyzed in UFLC. The toxicological assessment was performed in female Wistar rats according to the Acute Toxic Class Method from OECD. The developed tablets produced meet acceptable macroscopic characteristics, and the presence of babassu as diluent provided improved physicochemical properties. The best content of Lo-HAE/CSD in the tablet (100.27%) was identified for the lot containing babassu, composed by 1.0% magnesium stearate, without PVP binder in its formulation. Moreover, Lo-HAE/CSD showed no signs of toxicity. Therefore, the babassu mesocarp powder is a promising pharmaceutical excipient for the development of herbal tablets containing the Lippia origanoides extract.