Goldenseal (Hydrastis canadensis L.) Extracts Inhibit the Growth of Fungal Isolates Associated with American Ginseng (Panax quinquefolius L.).

American ginseng, a highly valuable crop in North America, is susceptible to various diseases caused by fungal pathogens, including Alternaria spp., Fusarium spp., and Pestalotiopsis spp. The development of alternative control strategies that use botanicals to control fungal pathogens in American ginseng is desired as it provides multiple benefits. In this study, we isolated and identified three fungal isolates, Alternaria panax, Fusarium sporotrichioides, and Pestalotiopsis nanjingensis, from diseased American ginseng plants. Ethanolic and aqueous extracts from the roots and leaves of goldenseal were prepared, and the major alkaloid constituents were assessed via liquid chromatography-mass spectrometry (LC-MS). Next, the antifungal effects of goldenseal extracts were tested against these three fungal pathogens. Goldenseal root ethanolic extracts exhibited the most potent inhibition against fungal growth, while goldenseal root aqueous extracts and leaf ethanolic extracts showed only moderate inhibition. At 2% (m/v) concentration, goldenseal root ethanolic extracts showed an inhibition rate of 86.0%, 94.9%, and 39.1% against A. panax, F. sporotrichioides, and P. nanjingensis, respectively. The effect of goldenseal root ethanolic extracts on the mycelial morphology of fungal isolates was studied via scanning electron microscopy (SEM). The mycelia of the pathogens treated with the goldenseal root ethanolic extract displayed considerable morphological alterations. This study suggests that goldenseal extracts have the potential to be used as a botanical fungicide to control plant fungal diseases caused by A. panax, F. sporotrichioides, or P. nanjingensis.

Neuroactivity screening of botanical extracts using microelectrode array (MEA) recordings.

Toxicity testing of botanicals is challenging because of their chemical complexity and variability. Since botanicals may affect many different modes of action involved in neuronal function, we used microelectrode array (MEA) recordings of primary rat cortical cultures to screen 16 different botanical extracts for their effects on cell viability and neuronal network function in vitro. Our results demonstrate that extract materials (50 µg/mL) derived from goldenseal, milk thistle, tripterygium, and yohimbe decrease mitochondrial activity following 7 days exposure, indicative of cytotoxicity. Importantly, most botanical extracts alter neuronal network function following acute exposure. Extract materials (50 µg/mL) derived from aristolochia, ephedra, green tea, milk thistle, tripterygium, and usnea inhibit neuronal activity. Extracts of kava, kratom and yohimbe are particularly potent and induce a profound inhibition of neuronal activity at the low dose of 5 µg/mL. Extracts of blue cohosh, goldenseal and oleander cause intensification of the bursts. Aconite extract (5 µg/mL) evokes a clear hyperexcitation with a marked increase in the number of spikes and (network) bursts. The distinct activity patterns suggest that botanical extracts have diverse modes of action. Our combined data also highlight the applicability of MEA recordings for hazard identification and potency ranking of botanicals.

Usefulness of Hydrastis for the prevention of encrustation of long-term indwelling catheters in persons with neurogenic bladder dysfunction: a case series.

INTRODUCTION: Virtually every person with a spinal cord injury (SCI) suffers from a neurogenic lower urinary tract dysfunction (NLUTD). In the long term, about 15% of persons with SCI depend on indwelling (suprapubic or transurethral) catheters for bladder management. About 50% of these patients suffer from catheter encrustation and blockage, which may become a vital threat for persons with SCI, as it can lead to septicemia or autonomic dysreflexia. Until today, no prophylaxis of catheter encrustations with an evidence-based proof of efficacy exists. CASE PRESENTATION: The homeopathic remedy Hydrastis, made from the goldenseal root, is used for the treatment of thick, mucous urine sediment. In four patients with tetraplegia (three female, one male) who managed NLUTD by suprapubic catheters, recurrent encrustations and catheter blockage occurred despite irrigation and medical treatment. Surgical urinary diversion was envisioned. Applying Hydrastis C30 once weekly as a long-term medication, in three of the four patients, catheter obstructions ceased, with a follow-up for at least 1 year. One patient is awaiting ileal conduit surgery. DISCUSSION: According to the results of our case series, the application of Hydrastis seems to be beneficial in the prevention of encrustations of indwelling catheters in patients with SCI. As the treatment was effective and well tolerated, the problem is frequent, and effective solutions are scarce, a prospective trial seems justified.

Assessing Transporter-Mediated Natural Product-Drug Interactions Via In vitro-In Vivo Extrapolation: Clinical Evaluation With a Probe Cocktail.

The botanical natural product goldenseal can precipitate clinical drug interactions by inhibiting cytochrome P450 (CYP) 3A and CYP2D6. Besides P-glycoprotein, effects of goldenseal on other clinically relevant transporters remain unknown. Established transporter-expressing cell systems were used to determine the inhibitory effects of a goldenseal extract, standardized to the major alkaloid berberine, on transporter activity. Using recommended basic models, the extract was predicted to inhibit the efflux transporter BCRP and uptake transporters OATP1B1/3. Using a cocktail approach, effects of the goldenseal product on BCRP, OATP1B1/3, OATs, OCTs, MATEs, and CYP3A were next evaluated in 16 healthy volunteers. As expected, goldenseal increased the area under the plasma concentration-time curve (AUC0-inf ) of midazolam (CYP3A; positive control), with a geometric mean ratio (GMR) (90% confidence interval (CI)) of 1.43 (1.35-1.53). However, goldenseal had no effects on the pharmacokinetics of rosuvastatin (BCRP and OATP1B1/3) and furosemide (OAT1/3); decreased metformin (OCT1/2, MATE1/2-K) AUC0-inf (GMR, 0.77 (0.71-0.83)); and had no effect on metformin half-life and renal clearance. Results indicated that goldenseal altered intestinal permeability, transport, and/or other processes involved in metformin absorption, which may have unfavorable effects on glucose control. Inconsistencies between model predictions and pharmacokinetic outcomes prompt further refinement of current basic models to include differential transporter expression in relevant organs and intestinal degradation/metabolism of the precipitant(s). Such refinement should improve in vitro-in vivo prediction accuracy, contributing to a standard approach for studying transporter-mediated natural product-drug interactions.

Effects of Hydrastis Canadensis, Commiphora Habessinica, Phytolacca Americana, and Echinacea Purpurea on Bacterial Growth.

CONTEXT: With the rise of antibiotic resistance, new strategies are needed to treat minor bacterial infections so that conventional antibiotics may be reserved for more serious conditions. One herbal formula, known as the HMPE formula, is often prescribed for minor infections. It includes Hydrastis canadensis (H. canadensis), Commiphora habessinica (C. habessinica), Phytolacca americana (P. americana), and Echinacea purpurea (E. purpurea). These herbs offer promise as treatments that may inhibit bacterial growth and stimulate the immune system. OBJECTIVE: To investigate the antibacterial effects of the HMPE formula and its constituent herbs against two organisms, Staphylococcus epidermidis and Escherichia coli. DESIGN: The research team performed an in-vitro study. SETTING: The study occurred at the Helfgott Research Institute at the National University of Natural Medicine in Portland, OR, USA. INTERVENTION: The study tested HMPE and each of its ingredients alone for antibacterial properties. OUTCOME MEASURES: The outcome measure was a disc diffusion assay. Sterile paper discs were impregnated with 15 µl of E. purpurea, H. canadensis, C. habessinica , or P. americana as herbal tinctures; with the complete HMPE formula; or with 65% ethanol as the negative control, and dried at room temperature for 40 minutes. Commercially prepared 10 µg ampicillin discs were used as a positive control. RESULTS: H. Canadensis and, to a lesser extent, the complete HMPE formula significantly inhibited the growth of the gram-positive bacteria Staphylococcus epidermidis, but not the gram-negative bacteria Escherichia coli. C. habessinica, P. americana, and E. purpurea alone did not inhibit growth of either bacterial strain. CONCLUSIONS: The results demonstrated that H. canadensis had antibacterial activity against S. epidermidis, but the HMPE formula was not active against S. epidermidis, when a zone of inhibition threshold of 12 millimeters (mm) was used to determine antibiotic activity. Because the HMPE formula was shown to be less effective than H. canadensis alone, the formula might benefit from an increased percentage of H. canadensis.

Goldenseal (Hydrastis canadensis L.) and its active constituents: A critical review of their efficacy and toxicological issues.

Goldenseal (Hydrastis canadensis L.) is a medicinal plant widely used in various traditional systems of medicine and as a food supplement. It has been traditionally used by Native Americans as a coloring agent and as medicinal remedy for common diseases and conditions like wounds, digestive disorders, ulcers, skin and eye ailments, and cancer. Over the years, goldenseal has become a popular food supplement in the USA and other regions. The rhizome of this plant has been used for the treatment of a variety of diseases including, gastrointestinal disorders, ulcers, muscular debility, nervous prostration, constipation, skin and eye infections, cancer, among others. Berberine is one of the most bioactive alkaloid that has been identified in different parts of goldenseal. The goldenseal extract containing berberine showed numerous therapeutic effects such as antimicrobial, anti-inflammatory, hypolipidemic, hypoglycemic, antioxidant, neuroprotective (anti-Alzheimer's disease), cardioprotective, and gastrointestinal protective. Various research finding suggest the health promoting effects of goldenseal components and their extracts. However, few studies have also suggested the possible neurotoxic, hepatotoxic and phototoxic activities of goldenseal extract and its alkaloids. Thus, large randomized, double-blind clinical studies need to be conducted on goldenseal supplements and their main alkaloids to provide more evidence on the mechanisms responsible for the pharmaceutical activity, clinical efficacy and safety of these products. Thus, it is very important to review the scientific information about goldenseal to understand about the current scenario.

Chemical Evaluation of the Effects of Storage Conditions on the Botanical Goldenseal using Marker-based and Metabolomics Approaches.

Hydrastis canadensis, commonly known as goldenseal, is a botanical native to the southeastern United States that has been used for the treatment of infection. The activity of goldenseal is often attributed to the presence of alkaloids (cyclic, nitrogen-containing compounds) present within its roots. Chemical components of botanical supplements like goldenseal may face degradation if not stored properly. The purpose of the research was to analyze the stability of known and unknown metabolites of H. canadensis during exposure to different storage conditions using mass spectrometry. Three abundant metabolites of H. canadensis, berberine, canadine, and hydrastine, were chosen for targeted analysis, and the stability of unknown metabolites was evaluated using untargeted metabolomics. The analysis and evaluation of H. canadensis samples were performed utilizing LC-MS and Principal Component Analysis (PCA). The research project focused on identifying the chemical changes in the metabolite content of H. canadensis under different temperature conditions (40°C ± 5°C, 20°C ± 5°C , and 4°C ± 5°C), different light:dark (hr:hr) cycles (16:8, 12:12, and 0:24), and different sample conditions (powdered roots versus whole roots) over a six month period. The results of this 6-month study revealed that the storage conditions evaluated had no significant effects on the chemical composition of H. canadensis roots. Hence, as long as H. canadensis roots are stored within the storage conditions tested in the study, no significant changes in chemical compositions of metabolites are expected.

In Vitro Assessment of Homeopathic Potencies of Hydrastis canadensis on Hormone-Dependent and Independent Breast Cancer.

BACKGROUND: Breast cancer is the second leading cause of cancer-related deaths in women. Conventional treatment such as chemotherapy, hormonal therapy and radiotherapy has decreased the mortality rate among cancer patients but has also revealed long-term side effects. Drug resistance and toxicity to normal cells compound the problems associated with the use of modern medicines. Hence, complementary or alternative treatment options are being explored. The current study, using different homeopathic potencies of Hydrastis canadensis, was conducted to distinguish between any effects they might have on hormone-dependent and independent breast cancer. MATERIALS AND METHODS: The cytotoxic effect of homeopathic medicine Hydrastis on hormone-dependent (MCF 7) and hormone-independent (MDA-MB-468) breast cancer cells was assessed using viability and colony-forming assays after 48 or 72 hours of treatment. Flow cytometry-based Annexin V-PI (propidium iodide), caspase 3 and cell cycle analysis was performed following treatment of cells with mother tincture or various potencies of Hydrastis (1C, 2C, 30C, 200C). RESULTS: Different potencies of Hydrastis displayed selective cytotoxic effects against MCF 7 cells, but only marginal effects against MDA-MB-468. The maximum cytotoxicity was established in the case of 1C following 72 hours of treatment. Treatment of breast cancer cells revealed an increase in the G0/G1 cell population, along with an increase in the caspase 3 levels and induction of apoptosis. CONCLUSION: Hydrastis may have a selective cytotoxic effect against hormone-dependent breast cancer MCF 7 cells, leading to cell cycle arrest in the G0/G1 phase, which could be the plausible reason for the induction of apoptosis. The results need to be validated in vivo.

Modulation of Major Human Liver Microsomal Cytochromes P450 by Component Alkaloids of Goldenseal: Time-Dependent Inhibition and Allosteric Effects.

Botanical and other natural products (NPs) are often coconsumed with prescription medications, presenting a risk for cytochrome P450 (P450)-mediated NP-drug interactions. The NP goldenseal (Hydrastis canadensis) has exhibited antimicrobial activities in vitro attributed to isoquinoline alkaloids contained in the plant, primarily berberine, (-)-ß-hydrastine, and to a lesser extent, hydrastinine. These alkaloids contain methylenedioxyphenyl rings, structural alerts with potential to inactivate P450s through formation of metabolic intermediate complexes. Time-dependent inhibition experiments were conducted to evaluate their ability to inhibit major P450 activities in human liver microsomes by using a cocktail of isozyme-specific substrate probes. Berberine inhibited CYP2D6 (dextromethorphan O-demethylation; K I = 2.7 µM, kinact = 0.065 minute-1) and CYP3A4/5 (midazolam 1'-hydroxylation; K I = 14.8 µM, kinact = 0.019 minute-1); (-)-ß-hydrastine inhibited CYP2C9 (diclofenac 4'-hydroxylation; K I = 49 µM, kinact = 0.036 minute-1), CYP2D6 (K I > 250 µM, kinact > 0.06 minute-1), and CYP3A4/5 (K I = 28 µM, kinact = 0.056 minute-1); and hydrastinine inhibited CYP2D6 (K I = 37 µM, kinact = 0.049 minute-1) activity. Berberine additionally exhibited allosteric effects on midazolam hydroxylation, showing both positive and negative heterotropic cooperativity. Experiments with recombinant isozymes showed that berberine activated midazolam 1'-hydroxylation by CYP3A5, lowering K m(app), but showed mixed inhibition and negative cooperativity toward this reaction when catalyzed by CYP3A4. Berberine inactivated CYP3A4 at a much faster rate than CYP3A5 and was a noncompetitive inhibitor of midazolam 4-hydroxylation by CYP3A4 but a strong mixed inhibitor of the CYP3A5 catalyzed reaction. These complex kinetics should be considered when extrapolating the risk for NP-drug interactions involving goldenseal. SIGNIFICANCE STATEMENT: Robust kinetic parameters were determined for the reversible and time-dependent inhibition of CYP2C9, CYP2D6, and CYP3A4/5 activities in human liver microsomes by major component isoquinoline alkaloids contained in the botanical natural product goldenseal. The alkaloid berberine also exhibited opposing, isozyme-specific allosteric effects on midazolam hydroxylation mediated by recombinant CYP3A4 (inhibition) and CYP3A5 (activation). These data will inform the development of a physiologically based pharmacokinetic model that can be used to predict potential clinically relevant goldenseal-drug interactions.

Identification of adulteration in botanical samples with untargeted metabolomics.

Adulteration remains an issue in the dietary supplement industry, including botanical supplements. While it is common to employ a targeted analysis to detect known adulterants, this is difficult when little is known about the sample set. With this study, untargeted metabolomics using liquid chromatography coupled to ultraviolet-visible spectroscopy (LC-UV) or high-resolution mass spectrometry (LC-MS) was employed to detect adulteration in botanical dietary supplements. A training set was prepared by combining Hydrastis canadensis L. with a known adulterant, Coptis chinensis Franch., in ratios ranging from 5 to 95% adulteration. The metabolomics datasets were analyzed using both unsupervised (principal component analysis and composite score) and supervised (SIMCA) techniques. Palmatine, a known H. canadensis metabolite, was quantified as a targeted analysis comparison. While the targeted analysis was the most sensitive method tested in detecting adulteration, statistical analyses of the untargeted metabolomics datasets detected adulteration of the goldenseal samples, with SIMCA providing the greatest discriminating potential. Graphical abstract.

Botanical metabolite ions extraction from full electrospray ionization mass spectrometry using high-dimensional penalized regression.

INTRODUCTION: Mass spectrometric data analysis of complex biological mixtures can be a challenge due to its vast datasets. There is lack of data treatment pipelines to analyze chemical signals versus noise. These tasks, so far, have been up to the discretion of the analysts. OBJECTIVES: The aim of this work is to demonstrate an analytical workflow that would enhance the confidence in metabolomics before answering biological questions by serial dilution of botanical complex mixture and high-dimensional data analysis. Furthermore, we would like to provide an alternative approach to a univariate p-value cutoff from t-test for blank subtraction procedure between negative control and biological samples. METHODS: A serial dilution of complex mixture analysis under electrospray ionization was proposed to study firsthand chemical complexity of metabolomics. Advanced statistical models using high-dimensional penalized regression were employed to study both the concentration and ion intensity relationship and the ion-ion relationship per second of retention time sub dataset. The multivariate analysis was carried out with a tool built in-house, so called metabolite ions extraction and visualization, which was implemented in R environment. RESULTS: A test case of the medicinal plant goldenseal (Hydrastis canandensis L.), showed an increase in metabolome coverage of features deemed as "important" by a multivariate analysis compared to features deemed as "significant" by a univariate t-test. For an illustration, the data analysis workflow suggested an unexpected putative compound, 20-hydroxyecdysone. This suggestion was confirmed with MS/MS acquisition and literature search. CONCLUSION: The multivariate analytical workflow selects "true" metabolite ions signals and provides an alternative approach to a univariate p-value cutoff from t-test, thus enhancing the data analysis process of metabolomics.

Pharmacological effects of berberine on mood disorders.

Berberine, a natural isoquinoline alkaloid, is used in herbal medicine and has recently been shown to have efficacy in the treatment of mood disorders. Furthermore, berberine modulates neurotransmitters and their receptor systems within the central nervous system. However, the detailed mechanisms of its action remain unclear. This review summarizes the pharmacological effects of berberine on mood disorders. Therefore, it may be helpful for potential application in the treatment of mood disorders.

Quantification of natural products in herbal supplements: A combined NMR approach applied on goldenseal.

Authentication of natural products is of major relevance in the context of manufactured drugs or herbal supplements since such active products generate a lucrative market. The analytical method to identify and quantify valuable natural products is critical for quality control and product assignment of herbal supplements. In this framework, we propose to apply a recently developed quantitative 2D NMR approach called Q QUIPU (Quick QUantItative Perfected and pUre shifted) in combination with 1D 1H NMR capable to access the concentration of three major alkaloids, berberine, ß-hydrastine and canadine, in the root extract of goldenseal (Hydrastis canadensis), one of the 20 most popular herbal supplements used worldwide. We highlight the complementarity of 1D and 2D quantitative NMR to accurately assess the amount of alkaloids with different range of concentrations and stability within extracts. In particular, unstable natural products having non-overlapped signals like berberine could only be quantified by sensitive and fast 1D 1H, while overlapped signals of ß-hydrastine and low intense ones of canadine could only be quantified with the recent 2D Q QUIPU HSQC. Results obtained from this combined approach have led to a good accuracy (<10%) as compared with coupled UHPLC-MS/UV techniques. This quantitative NMR approach paves the way to numerous applications where the accurate quantification of targeted compounds in complex mixtures is required, for instance in agricultural, food and pharmaceuticals products.

Detection of adulteration in Hydrastis canadensis (goldenseal) dietary supplements via untargeted mass spectrometry-based metabolomics.

Current estimates report that approximately 25% of U.S. adults use dietary supplements for medicinal purposes. Yet, regulation and transparency within the dietary supplement industry remains a challenge, and economic incentives encourage adulteration or augmentation of botanical dietary supplement products. Undisclosed changes to the dietary supplement composition could impact safety and efficacy; thus, there is a continued need to monitor possible botanical adulteration or mis-identification. Goldenseal, Hydrastis canadensis L. (Ranunculaceae), is a well-known botanical used to combat bacterial infections and digestive problems and is widely available as a dietary supplement. The goal of this study was to evaluate potential adulteration in commercial botanical products using untargeted metabolomics, with H. canadensis supplements serving as a test case. An untargeted ultraperformance liquid chromatography-mass spectrometry (LC-MS) metabolomics analysis was performed on 35 H. canadensis commercial products. Visual inspection of the chemometric data via principal component analysis (PCA) revealed several products that were distinct from the main groupings of samples, and subsequent evaluation of contributing metabolites led to their confirmation of the outliers as originating from a non-goldenseal species or a mixture of plant materials. The obtained results demonstrate the potential for untargeted metabolomics to discriminate between multiple unknown products and predict possible adulteration.

FT-NIR characterization with chemometric analyses to differentiate goldenseal from common adulterants.

Goldenseal (Hydrastis canadensis L.) has been a popular herb since the 1970s, with a US market share of over $32 million in 2014. Wild goldenseal has been listed in the Convention on International Trade in Endangered Species for decades. Limits in supply and greed for profit have led to adulteration with similar but more accessible and inexpensive plant materials. Fourier transform near-infrared spectroscopy (FT-NIR) coupled with three different chemometric models, partial least squares (PLS) regression, soft independent modeling of class analogy (SIMCA), and moving window principal component analysis (MW-PCA) provide fast, simple, nondestructive approaches to differentiating pure goldenseal from 4 common pure adulterants (yellow dock, yellow root, coptis, Oregon grape). All three models successfully differentiated authentic goldenseal from adulterants. The models were t-tested for detection of goldenseal intentionally mixed with individual adulterants at 2% to 95% theoretical levels made computationally. The PLS model was unable to detect adulterants mixed with goldenseal at any level. The SIMCA model was the best for detection of yellow root and Oregon grape adulteration in goldenseal, as low as 10%. The MW-PCA model proved best for detection of yellow dock at ≥ 15% and coptis adulteration ≥5% in goldenseal. This study demonstrates that NIR spectroscopy coupled with chemometric analyses is a good tool for industry and investigators to implement for rapid detection of goldenseal adulteration in the marketplace, but also indicates that the specific approach to chemometric analysis must be evaluated and selected on a case-by-case basis in order to achieve useful sensitivity and specificity.

Variability in Potency Among Commercial Preparations of Berberine.

Berberine is an isoquinoline alkaloid plant extract that is widely available as a dietary supplement in the United States and has demonstrated efficacy in the treatment of type 2 diabetes mellitus and dyslipidemia. Because of its increased use and purported pharmacological properties, potential variations in product quality could pose a barrier to berberine's safety and effectiveness in clinical practice. Thus, this study evaluated the potency of dietary supplements containing berberine available in the U.S. commercial market. Fifteen unique dietary supplements containing berberine were purchased through U.S. dietary supplement vendors. For each product, berberine was extracted from 3 unique capsules and analyzed by ultra-high-performance liquid chromatography tandem mass spectrometry. Percentage content based on the product label claim was determined for each product. The average berberine content across the products was found to be 75% ± 25% of the product label claim, with product potency ranging from 33% to 100%. Nine of the 15 tested products (60%) failed to meet the potency standards of 90% to 110% of labeled content claim, as commonly required of pharmaceutical preparations by the U.S. Pharmacopeial Convention. Evaluation of the relationship between product cost and the measured potency failed to demonstrate an association between quality and cost. Variability in product quality may significantly contribute to inconsistencies in the safety and effectiveness of berberine. In addition, the quality of the berberine product cannot be inferred from its cost.

Biochemometrics to Identify Synergists and Additives from Botanical Medicines: A Case Study with Hydrastis canadensis (Goldenseal).

A critical challenge in the study of botanical natural products is the difficulty of identifying multiple compounds that may contribute additively, synergistically, or antagonistically to biological activity. Herein, it is demonstrated how combining untargeted metabolomics with synergy-directed fractionation can be effective toward accomplishing this goal. To demonstrate this approach, an extract of the botanical goldenseal ( Hydrastis canadensis) was fractionated and tested for its ability to enhance the antimicrobial activity of the alkaloid berberine (4) against the pathogenic bacterium Staphylococcus aureus. Bioassay data were combined with untargeted mass spectrometry-based metabolomics data sets (biochemometrics) to produce selectivity ratio (SR) plots, which visually show which extract components are most strongly associated with the biological effect. Using this approach, the new flavonoid 3,3'-dihydroxy-5,7,4'-trimethoxy-6,8- C-dimethylflavone (29) was identified, as were several flavonoids known to be active. When tested in combination with 4, 29 lowered the IC50 of 4 from 132.2 ± 1.1 µM to 91.5 ± 1.1 µM. In isolation, 29 did not demonstrate antimicrobial activity. The current study highlights the importance of fractionation when utilizing metabolomics for identifying bioactive components from botanical extracts and demonstrates the power of SR plots to help merge and interpret complex biological and chemical data sets.

Antimicrobial activity of conventional and plant-extract disinfectant solutions on microbial biofilms on a maxillofacial polymer surface.

STATEMENT OF PROBLEM: Dentists often note problems with infection in patients with maxillofacial prostheses. Conventional disinfection protocols are not always effective and may alter the properties of the polymer used in the prosthesis. Thus, the search for improved disinfection methods is important. PURPOSE: The purpose of this in vitro study was to evaluate and compare the antimicrobial activity of conventional disinfectant solutions (water and neutral soap and 4% chlorhexidine) and plant extracts (Cymbopogon nardus and Hydrastis canadensis) on specimens of maxillofacial silicone contaminated with Candida albicans and Staphylococcus aureus biofilms. MATERIAL AND METHODS: Seventy-two silicone (MDX4-4210) specimens were fabricated (5×2 mm) and sterilized. Thirty-six were contaminated with C albicans (10(6) cells/mL) and 36 with S aureus (10(8) cells/mL) to evaluate the antimicrobial activity of the cleaning protocols. After incubation (37°C/72 hours), the specimens were divided into 5 groups: not disinfected (positive control), soaking in saline solution for 10 minutes, soaking in 4% chlorhexidine for 10 minutes, soaking in C nardus for 10 minutes, soaking in H canadensis for 10 minutes, and washing by hand with water and neutral soap for 30 seconds. The viability of cells was evaluated by XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay and by scanning electron microscope analysis. The results were analyzed by ANOVA and the Tukey HSD test (α=.05). RESULTS: All disinfection solutions provided a statistically significant reduction in biofilm viability compared with the control group for both microorganisms (P<.05). Washing with water and neutral soap was significantly more effective in reducing biofilm viability than immersion in the disinfection solutions, with persistence of viable microorganisms between 1.05% for C albicans and 0.62% for S aureus after this cleaning protocol. Photomicrographs revealed that 4% chlorhexidine altered the surface of the polymer. CONCLUSIONS: Within the limitations of this in vitro study, it was concluded that the cleaning protocols with different disinfectant solutions produced a significant reduction in the viability of C albicans and S aureus biofilms on the silicone polymer. Washing with water and neutral soap was the most effective protocol against both microorganisms.

Ultra-highly diluted plant extracts of Hydrastis canadensis and Marsdenia condurango induce epigenetic modifications and alter gene expression profiles in HeLa cells in vitro.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: HDs triggered epigenetic modifications and alterations in microarray gene expression profiles of many genes associated with carcinogenesis in HeLa cells in vitro.