Structural and thermal analyses of zinc and lactose in homeopathic triturated systems.

BACKGROUND: A series of different experimental approaches was applied in Zincum metallicum (Zinc met.) samples and lactose controls. Experiments were designed to elucidate the effect of zinc trituration and dynamization on physicochemical properties of homeopathic formulations, using lactose as excipient. METHODS: Zinc met. potencies (Zinc met 1-3c) were triturated and dynamized using lactose as excipient, according to Brazilian Homeopathic Pharmacopoeia. Lactose samples (LAC 1-3c) were also prepared following the same protocol and used as controls. The samples were analyzed structurally by Atomic Absorption Spectroscopy (AAS), X-ray Diffraction (XRD), Transmission Electron Microscopy (TEM) with Energy Dispersive X-ray Spectroscopy (EDX) and Scanning Electron Microscopy (SEM), and thermodynamically by Thermogravimetry (TG) and Differential Scanning Calorimetry (DSC). RESULTS: AAS analysis detected 97.0 % of zinc in the raw material, 0.75 % (Zinc met 1c) and 0.02% (Zinc met 2c). XRD analysis showed that inter-atomic crystalline spacing of lactose was not modified by dynamization. Amorphous and crystalline lactose spheres and particles, respectively, were observed by TEM in all samples, with mean size from 200 to 800 nm. EDX obtained with TEM identified zinc presence throughout the amorphous matter but individualized zinc particles were not observed. SEM images obtained from dynamized samples (LAC 1c and Zinc met 1c) with electron backscattering could not identify zinc metal grains. The dynamization process induced Derivatives of Thermal Gravimetric (DTg) peak modification, which was previously centered near 158°C to lactose, to a range from 140 to 170°C, suggesting the dynamization process modifies the temperature range of water aggregation. Thermal phenomena were analyzed and visualized by Analysis of Variance (ANOVA) and Principal Component Analysis (PCA) statistics. Both indicated that fusion enthalpy of dynamized samples (DynLAC 1-3c; DynZn 1-3c) increased 30.68 J/g in comparison to non-dynamized lactose (LAC; p < 0.05). CONCLUSIONS: Our results suggested no structural changes due to the trituration and dynamization process. However, TG and DSC analyses permit the differentiation of dynamized and non-dynamized groups, suggesting the dynamization process induced a significant increase in the degradation heat. These results call for further calorimetric studies with other homeopathic dilutions and other methodologies, to better understand the dynamics of these systems.

Homeopathic medicines for prevention of influenza and acute respiratory tract infections in children: blind, randomized, placebo-controlled clinical trial.

BACKGROUND: Influenza and its complications are common at all ages, especially in children. Vaccines and anti-influenza drugs aim to prevent it. Preventative approaches with favorable risk profiles should be considered for flu, particularly since the evidence of the efficacy of anti-viral drugs is debated. METHODS: This pragmatic clinical trial was conducted in the Brazilian Public Health System in Petrópolis (BPHSP) with children aged from 1 to 5 years old. The medications used were mainly selected based on in vitro experiments (InfluBio), and in successful qualitative clinical experiences (Homeopathic Complex). Following informed parental consent, subjects were randomly distributed, in a blind manner, to three experimental groups: Homeopathic Complex, Placebo, and InfluBio. BPHSP health agents collected flu and acute respiratory infection symptomatic episodes monthly following the established protocol. The number of these episodes was registered in one year (2009-2010). RESULTS: Out of the 600 children recruited, 445 (74.17%) completed the study (149: Homeopathic complex; 151: Placebo; 145: InfluBio). The number of flu and acute respiratory infection symptomatic episodes detected in this clinical trial was low; however, it was different between homeopathic groups and placebo (p < 0.001). In the first year post-intervention, 46/151 (30.5%) of children in the placebo group developed 3 or more flu and acute respiratory infection episodes, while there was no episode in the group of 149 children who used Homeopathic Complex, and only 1 episode in the group of 145 (1%) children who received InfluBio. CONCLUSION: These results suggested that the use of homeopathic medicines minimized the number of flu and acute respiratory infection symptomatic episodes in children, signalizing that the homeopathic prophylactic potential should be investigated in further studies.

Efeitos in vitro de Natrum muriaticum nas linhagens renais MDCK e LLC-PK1K1 / In Vitro Effects of Natrum muriaticum in Kidney Cell Lines MDCK and LLC-PK1

Trabalhos prévios têm indicado que soluções homeopáticas modificam os aspectos celulares e bioquímicos de células mantidas em cultura. No presente estudo, os efeitos de Natrum muriaticum, medicamento usado na clínica homeopática para o tratamento de hipertensão, foram avaliados em células renais das linhagens MDCK e LLC-PK1. Os seguintes parâmetros celulares foram analisados: viabilidade, morfologia e expressão da (Na+-K+)-ATPase e dos receptores AT1 e AT2 da angiotensina II. As linhagens celulares foram plaqueadas (5,0 x 104 células/mL) em DMEM suplementado com 10% de soro fetal bovino (SFB). Após 24 horas a 37°C, o meio DMEM foi substituído com a adição de 10% (v/v) e 1% (v/v) das seguintes amostras: Natrum muriaticum 30CH, água dinamizada 30CH e água destilada estéril, para a medida de viabilidade celular por MTT. As absorbâncias quantificadas em leitor de ELISA (490 nm) foram comparadas entre si e com os valores obtidos com o grupo controle (células isentas de tratamento) Os valores obtidos de quatro experimentos independentes realizados em quintuplicata foram tabulados e analisados estatisticamente pelo Software Sigma Plot v.11 (Jandel Scientific). A morfologia das células MDCK foi avaliada por microscópio óptico após coloração de Giemsa e a expressão da (Na+-K+)-ATPase e AT1/AT2 de células LLC-PK1 por Western Blot (WB). Para ambos experimentos, 5,0 x 104 células/mL foram incubadas em DMEM suplementado com 10% SFB sendo o meio de cultura diariamente substituído por um novo, contendo 1% de Natrum muriaticum 30CH e água dinamizada 30CH, por 5, 10 e 15 dias. A quantificação de proteínas totais foi realizada pelo método de Lowry, após lise celular. As amostras foram analisadas por eletroforese em SDS-PAGE (12%) e transferidos para membrana de nitrocelulose. Esta membrana foi incubada com anticorpos específicos primário (anti-(Na+-K+)-ATPase, anti-AT1 e AT2 ou anti-anti-beta-actina). A detecção foi realizada utilizando o sistema ECL e Hyperfilm. Os ensaios de MTT mostraram uma redução estatisticamente significativa na atividade mitocondrial celular (p <0,001), indicando um efeito osmótico decorrente da incubação com as soluções teste na concentração de 10% (v/v) Não foram detectadas alterações morfológicas significativas quando ambas as linhagens foram submetidas aos diferentes tratamentos . A análise por WB indicou um aumento proporcional da quantidade (Na+-K+)-ATPase expressa na linhagem LLC-PK1 em função do número de estímulos homeopáticos aplicado. Embora preliminares estes resultados evidenciam, pela primeira vez, que o estímulo homeopático parece modificar a expressão de importantes marcadores fisiológicos da linhagem LLC-PK1 que estão diretamente envolvidos na gênese da hipertensão arterial.(AU)

Previous papers have indicated that homeopathic solutions modify the cellular and biochemical aspects of cells maintained in culture. In this study, the effects of Natrum muriaticum, a medicine used in the homeopathic clinic for the treatment of hypertension, were evaluated in kidney MDCK and LLC-PK1 cell lines. The following cellular parameters were analyzed: viability, morphology and expression of the (Na+-K+)-ATPase and the angiotensin II receptors AT1 and AT2. The cell lines were plated (5.0 x 104 cells/mL) in DMEM supplemented with 10% fetal calf serum (FCS). After 24 hours at 37°C, DMEM was re-fed with the addition of 10% (V/V) and 1% (V/V) of the following samples: Natrum muriaticum 30CH, water 30CH and non-dynamized sterile distilled water to do the MTT assay. The results obtained from these groups were compared to those obtained by incubation of the cells in culture medium free of these solutions (Control). Cell viability was assessed by a colorimetric MTT ELISA assay (490nm). The values from four independent experiments performed in quintuplicate were plotted and statistically analyzed by Sigma Plot v.11 (Jandel Scientific). The morphology of MDCK cells was evaluated by optical microscope after Giemsa?s staining. The expression of the (Na++K+)-ATPase and AT1/AT2 of LLC-PK1 cells was evaluated by Western Blot (WB) analysis. For this experimental set, 5.0x104 cells/mL were incubated in DMEM supplemented with 10% FCS and daily culture medium was replaced by a new one, containing: Natrum muriaticum 30CH and water 30CH. Additionally, cells were treated for 5, 10 and 15 days with 1% of specific solutions and the total protein was measured by the Lowry method, after cell lysis. The samples were analyzed by electrophoresis in SDS-PAGE (12% gel) and transferred to nitrocellulose membrane. This membrane was incubated with specific primary antibodies (anti-(Na++K+)-ATPase, anti-AT1 and AT2 or anti-anti-beta-actin). The detection was performed using ECL system and Hyperfilm. MTT assays showed a statistically significant reduction in cellular mitochondrial activity (p<0.001) probably attributed to an osmotic effect due to the use of 10% (V/V) concentration rather than 1% (V/V). The optical microscopy analysis revealed no significant morphological changes in MDCK and LLC-PK1 cells submitted to the different treatments when compared to controls groups. The WB analysis indicated a proportional increase in (Na++K+)-ATPase content according to an increase in the homeopathic stimuli. Although preliminary, these results show for the first time, that the homeopathic medicine is able to modify the expression of important physiological markers for LLC-PK1 cells, which are directly involved in the genesis of hypertension.(AU)

Respostas celulares e bioquímicas induzidas por bioterápicos preparados a partir de influenza A (H3N2) íntegro e influenza A (H3N2) inativado nas potências 12x e 30x na linhagem MDCK / Cellular and biochemical responses induced by Biotherapics prepared from intact influenza A (H3N2) and inactivated influenza A (H3N2) virus at 12x and 30x in the MDCK cells

Biotherapics are homeopathic remedies prepared from organic products that are chemically undefined and can be used for treatment of diseases like influenza. There are several classes of biotherapics and, among these, there are some called "living biotherapics" or "Roberto Costa?s Biotherapics". This study aimed to compare the cellular and biochemical effects of biotherapics prepared from intact influenza virus diluted in water and the one obtained from the same viral sample inactivated by ethanol 70% (v / v), both in the potencies of 12x and 30x. Transmission electron microscopy (TEM) analyses were performed on both preparations to assess the integrity of viral particles, which showed that ethanol 70% (v/v) induced a complete denaturation of viral particles. In contrast, the integrity of virus particles was preserved when water was used as the biotherapic solvent. Cellular and biochemical alterations induced by the preparations on MDCK cells were analyzed and compared with those induced by respective controls (water 30x-treated and untreated cells). Cellular viability analyzed by MTT method showed statistically significant differences (p <0.05) in MDCK cells treated with intact biotherapic for 5 (3 stimuli) and 30 (18 stimuli) days in comparison with untreated control. TEM analysis did not show significant cellular changes when the different experimental groups were compared. The enzymatic activity of phosphofructokinase 1 (PFK), an important enzyme in the glycolytic pathway, presented a statistically significant increase (p <0.05) after 30 days of treatment when compared to control groups. The results obtained suggest that inactivation of viral sample with ethanol 70% induces lysis and disruption of viral particles. In addition, preliminary results indicated that treatment with intact biotherapic seems to induce higher variations on MDCK cells responses when compared to inactivated-biotherapic-treated cells. Further analyses are ongoing, including scanning electron microscopy and quantification of the number of mitosis, in order to elucidate the mechanisms involved with biochemical and cellular responses induced by theses biotherapics.(AU)

Tratamento com bioterápico Candida albicans influencia a adesão de fungos a células Ma104 in vitro / Treatment with Candida albicans biotherapic influences in vitro fungal adhesion to Ma-104 cells

Oral candidiasis is an opportunist fungal infection in humans, mainly caused by Candida albicans. It occurs when the host presents an imbalance in the immune system and Candida spp., normally found in human flora, become able to develop the infection [1]. This disease is very common in HIV patients, and in all individuals that present immunossupression, such as patients treated with chemotherapy. Considering this scenario, the development of new medicines to treat oral candidiasis is mandatory.These results showed that the biotherapic did not present any citotoxicity, but was able to modify the morphological aspects of Ma-104 cells. Additionally, the interaction between host cells and ethilogic agent is directly influenced by biotherapic treatment, suggesting a promising antifungal potential of this medicine.(AU)