Ultra-highly diluted plant extracts of Hydrastis canadensis and Marsdenia condurango induce epigenetic modifications and alter gene expression profiles in HeLa cells in vitro / 中西医结合学报

<p><b>OBJECTIVE</b>Methylation-specific epigenetic process and gene expression profiles of HeLa cells treated with ultra-high dilutions (HDs) of two plant extracts, Hydrastis canadensis (HC-30) and Marsdenia condurango (Condu-30), diluted 1060 times, were analyzed against placebo 30C (Pl-30) for alterations in gene profiles linked to epigenetic modifications.</p><p><b>METHODS</b>Separate groups of cells were subjected to treatment of Condu-30, HC-30, and Pl-30 prepared by serial dilutions and succussions. Global microarray data recorded on Affymetrix platform, using 25-mer probes were provided by iLifeDiscoveries, India. Slides were scanned with 3000 7G microarray scanner and raw data sets were extracted from Cel (raw intensity) files. Analyses of global microarray data profile, differential gene expression, fold change and clusters were made using GeneSpring GX12.5 software and standard normalization procedure. Before microarray study, concentration of RNA (ng/μL), RIN value and rRNA ratio for all the samples were analysed by Agilant Bioanalyzer 2100. Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative RT-PCR were done for analyzing SMAD-4 expression. Fluorescence-activated cell sorting study was further made to elucidate fate of cells at divisional stages. Methylation-specific restriction enzyme assay was conducted for ascertaining methylation status of DNA at specific sites.</p><p><b>RESULTS</b>HDs of HC-30 and Condu-30 differentially altered methylation in specific regions of DNA and expression profiles of certain genes linked to carcinogenesis, as compared to Pl-30. Two separate cut sites were found in genomic DNA of untreated and placebo-treated HeLa cells when digested with McrBC, compared to a single cut observed in Condu-30-treated genomic DNA. SMAD-4 gene expression validated the expression pattern observed in microarray profile. Methylation-specific restriction enzyme assay elucidated differential epigenetic modifications in drug-treated and control cells.</p><p><b>CONCLUSION</b>HDs triggered epigenetic modifications and alterations in microarray gene expression profiles of many genes associated with carcinogenesis in HeLa cells in vitro.</p>

Evidence in support of gene regulatory hypothesis: Gene expression profiling manifests homeopathy effect as more than placebo

Background: Use of ultra-high diluted remedies in homeopathy and their claimed efficacy in curing diseases has been challenged time and again by non-believers despite many evidence-based positive results published in favor of their efficacy in curing/ameliorating disease symptoms. Aims: To test the ability of ultra-high diluted homeopathic remedies beyond Avogadro’s limit, if any, in manifesting gene modulating effects in controlled in vitro experimental model. Methods: Since cancer cells manifest aberrant epigenetic gene expressions, we conducted global microarray gene expression profiling of HeLa cells (an established epigenetic model of HPV18 positive cell line) treated with two different potentized homeopathic remedies, namely, Condurango 30c and Hydrastis canadensis 30C (used in the treatment of cancer), as compared to that of placebo (succussed alcohol 30c). Results: Data revealed distinctly different expression patterns of over 100 genes as a consequence of treatment with both homeopathc remedies compared to placebo. Conclusion: Results indicate that action of the potentized drugs was “more than placebo” and these ultra-highly diluted drugs acted primarily through modulation of gene expression.

Introdução: O uso de medicamentos homeopáticos ultra-diluídos e a sua presumível eficácia na cura de doenças têm sido reiteradamente contestados por céticos, apesar dos muitos resultados positivos, baseados em evidências, demonstrando a sua eficácia na cura/melhora de sintomas patológicos. Objetivos: Testar a possível capacidade de medicamentos homeopáticos diluídos além do limite de Avogadro para apresentar efeitos de modulação genética em modelo experimental in vitro. Métodos: Sendo que as células neoplásicas manifestam expressões epigenéticas aberrantes, investigamos através de micro-arranjo o perfil global de expressão genética de células HeLa (modelo epigenético já estabelecido de linhas celulares HPV18 positivas) tratadas com 2 medicamentos homeopáticos dinamizados, a saber, Condurango 30c e Hydrastis canadensis 30c (utilizados no tratamento de neoplasias) comparados com um placebo (álcool sucussionado 30c). Resultados: Os dados indicaram diferentes perfis de expressão em mais de 100 genes nas células tratadas com os medicamentos homeopáticos por comparação ao placebo. Conclusão: Os resultados indicam que o efeito dos medicamentos dinamizados foi “mais do que placebo” e que essa ação foi principalmente mediada por modulação da expressão genética.

Effect of homeopathic medicines on transplanted tumors in mice.

Ultra low doses used in homeopathic medicines are reported to have healing potential for various diseases but their action remains controversial. In this study we have investigated the antitumour and antimetastatic activity of selected homeopathic medicines against transplanted tumours in mice. It was found that Ruta graveolens 200c and Hydrastis canadensis 200c significantly increased the lifespan of Ehrlich Ascites Carcinoma and Dalton's Lymphoma Ascites induced tumour-bearing animals by 49.7%, and 69.4% respectively. Moreover there was 95.6% and 95.8% reduction of solid tumour volume in Ruta 200c and Hydrastis 200c treated animals on the 31st day after tumour inoculation. Hydrastis 1M given orally significantly inhibited the growth of developed solid tumours produced by DLA cells and increased the lifespan of tumour bearing animals. Some 9 out of 15 animals with developed tumors were completely tumour free after treatment with Hydrastis 1M. Significant anti-metastatic activity was also found in B16F-10 melanoma-bearing animals treated with Thuja1M, Hydrastis 1M and Lycopodium1M. This was evident from the inhibition of lung tumour nodule formation, morphological and histopathological analysis of lung and decreased levels of gamma-GT in serum, a cellular marker of proliferation. These findings support that homeopathic preparations of Ruta and Hydrastis have significant antitumour activity. The mechanism of action of these medicines is not known at present.

Extraction and HPLC analysis of alkaloids in goldenseal / 生物工程学报

An ambient extraction of goldenseal root powder followed by HPLC analysis of the alkaloids on a Zorbax Rapid Resolution Eclipse XDB-C18 column provides an accurate method for the determination of key alkaloids in goldenseal, including berberine and hydrastine. The extraction and HPLC analysis can be applied to several other alkaloids, including canadine, hydrastinine, and palmatine, and may be applicable to other berberine-containing plant roots. The Rapid Resolution Eclipse XDB-C18 column is used for an isocratic separation with high resolution of all componentsin under 15 minutes.