Intestinal epithelial damage due to herbal compounds - an in vitro study.

Intestinal epithelial injury from herbal products has rarely been reported, despite the gut being the first point of contact for oral preparations. These products often consist of multiple herbs, thereby potentially exposing consumers to higher levels of reactive phytochemicals than predicted due to pharmacokinetic interactions. The phytochemical coumarin, found in many herbal products, may be taken in combination with herbal medicines containing astragalosides and atractylenolides, purported cytochrome P450 (CYP) modulators. As herbal use increases, the need to predict interactions in multiple at-risk organ systems is becoming critical. Hence, to determine whether certain herbal preparations containing coumarin may cause damage to the intestinal epithelium, Caco2 cells were exposed to common phytochemicals. Coumarin, astragaloside IV (AST-IV) or atractylenolide I (ATR-I) solutions were exposed to Caco2 cultures in increasing concentrations, individually or combined. Coumarin produced a significant concentration-dependant fall in cell viability that was potentiated when CYP enzymes were induced with rifampicin and incubated with CYP3A4 inhibitor econazole, suggesting a role for other CYP enzymes generating toxic metabolites. ATR-I alone produced no toxicity in uninduced cells but showed significant toxicity in rifampicin-induced cells. ATR-I had no effect on coumarin-induced toxicity. AST-IV was nontoxic alone but produced significant toxicity when combined with nontoxic concentrations of coumarin. The combination of coumarin, ATR-I and AST-IV was significantly toxic, but no synergistic interaction was seen. This investigation was conducted to determine the likelihood for intestinal-based interactions, with the results demonstrating coumarin is potentially toxic to intestinal epithelium, and combinations with other phytochemicals can potentiate this toxicity.

The Extracellular Molecular Chaperone Clusterin Inhibits Amyloid Fibril Formation and Suppresses Cytotoxicity Associated with Semen-Derived Enhancer of Virus Infection (SEVI).

Clusterin is a glycoprotein present at high concentrations in many extracellular fluids, including semen. Its increased expression accompanies disorders associated with extracellular amyloid fibril accumulation such as Alzheimer's disease. Clusterin is an extracellular molecular chaperone which prevents the misfolding and amorphous and amyloid fibrillar aggregation of a wide variety of unfolding proteins. In semen, amyloid fibrils formed from a 39-amino acid fragment of prostatic acid phosphatase, termed Semen-derived Enhancer of Virus Infection (SEVI), potentiate HIV infectivity. In this study, clusterin potently inhibited the in vitro formation of SEVI fibrils, along with dissociating them. Furthermore, clusterin reduced the toxicity of SEVI to pheochromocytoma-12 cells. In semen, clusterin may play an important role in preventing SEVI amyloid fibril formation, in dissociating SEVI fibrils and in mitigating their enhancement of HIV infection.

Could herbal soup be a potentially unrecognized cause of hepatotoxicity at autopsy?

Unexpected hepatic failure with liver necrosis is sometimes encountered during a forensic autopsy. Determining the etiology may sometimes be difficult, although increasingly herbal medicines are being implicated. To determine whether such effects might also be caused by foodstuffs, the following in vitro study was undertaken. Four formulations of traditional herbal soup advertised as bak kut teh were prepared and added to cultures of liver carcinoma cells (HepG2). Cell viability was assessed using an MTT colorimetric assay at 48 h demonstrating that all formulations had significant toxicity prior to dilution (p < 0.05). Formulation #1 showed 21% cell death (p = 0.023), Formulation #2 30% (p = 0.009), and Formulation #3 41% (p < 0.0001). Formulations #1-3 showed no significant toxicity once diluted (p > 0.05). Formulation 4 showed approximately 83% cell death before dilution (p < 0.0001) and persistent toxicity even with dilutions at 1:10 (15% ± 3.7, p = 0.023) and 1:1000 (14% ± 3.8, p = 0.024). This study has shown that herbal foodstuffs such as bak kut teh may be responsible for variable degrees of in vitro hepatotoxicity, thus extending the range of herbal products that may be potentially injurious to the liver. If unexpected liver damage is encountered at autopsy, information on possible recent ingestion of herbal food preparations should be sought, as routine toxicology screening will not identify the active components. Liver damage may therefore be caused not only by herbal medicines but possibly by herbal products contained in food.

Implications for herbal polypharmacy: coumarin-induced hepatotoxicity increased through common herbal phytochemicals astragaloside IV and atractylenolide I.

Hepatotoxicity is a well-known adverse effect of many substances, with toxicity often resulting from interactions of drugs with other drug-like substances. With the increased availability of complementary and alternative medicines, including herbal medicines, the likelihood of adverse interactions between drugs and drug-like substances in herbs increases. However, the impact of potential herb-herb interactions is little understood. To assess the potential of two cytochrome P450 enzyme modulating phytochemicals common to many herbal medicines, atractylenolide I (ATR-I) and astragaloside IV (AST-IV), to interact with coumarin, another phytochemical common in many foods, a hepatocyte function model with a liver carcinoma cell line, HepG2, was exposed to these agents. To determine the effects of cytochrome P450 modulation by these phytochemicals certain cells were induced with rifampicin to induce cytochrome P450. Increasing concentrations of ATR-I combined with a fixed, nontoxic concentration of coumarin (200 µM), demonstrated significant additive interactions. 300 µM ATR-I produced a 31% reduction in cell viability (p < 0.01) with coumarin in rifampicin uninduced cells. In rifampicin-induced cells, ATR-I (100-300 µM) produced a significant reduction in cell viability (p < 0.01) with coumarin (200 µM). AST-IV with fixed coumarin (200 µM) showed 27% toxicity at 300 µM AST-IV in rifampicin uninduced cells (p < 0.05) and 30% toxicity in rifampicin induced cells (p < 0.05). However, when fixed coumarin and AST-IV were combined with increasing concentrations of ATR-I no further significant increase in toxicity was observed (p > 0.05). These results demonstrate the potential toxic interactive capabilities of common traditional Chinese herbal medicine phytochemicals and underline the potential importance of coumarin-mediated toxicity.

Flavonoid-statin interactions causing myopathy and the possible significance of OATP transport, CYP450 metabolism and mevalonate synthesis.

3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, statins, are a primary treatment for hyperlipidemic cardiovascular diseases which are a leading global cause of death. Statin therapy is life saving and discontinuation due to adverse events such as myotoxicity may lead to unfavourable outcomes. There is no known mechanism for statin-induced myotoxicity although it is theorized that it is due to inhibition of downstream products of the HMG-CoA pathway. It is known that drug-drug interactions with conventional medicines exacerbate the risk of statin-induced myotoxicity, though little attention has been paid to herb-drug interactions with complementary medicines. Flavonoids are a class of phytochemicals which can be purchased as high dose supplements. There is evidence that flavonoids can raise statin plasma levels, increasing the risk of statin-induced myopathy. This could be due to pharmacokinetic interactions involving hepatic cytochrome 450 (CYP450) metabolism and organic anion transporter (OATP) absorption. There is also the potential for flavonoids to directly and indirectly inhibit HMG-CoA reductase which could contraindicate statin-therapy. This review aims to discuss what is currently known about the potential for high dose flavonoids to interact with the hepatic CYP450 metabolism, OATP uptake of statins or their ability to interact with HMG-CoA reductase. Flavonoids of particular interest will be covered and the difficulties of examining herbal products will be discussed throughout.

Normal aging, motor neurone disease, and Alzheimer's disease are characterized by cortical changes in inflammatory cytokines.

The role of increased brain inflammation in the development of neurodegenerative diseases is unclear. Here, we have compared cytokine changes in normal aging, motor neurone disease (MND), and Alzheimer's disease (AD). After an initial analysis, six candidate cytokines, interleukin (IL)- 4, 5, 6, 10, macrophage inhibitory protein (MIP)-1α, and fibroblast growth factor (FGF)-2, showing greatest changes were assayed in postmortem frozen human superior frontal gyri (n = 12) of AD patients, aging and young adult controls along with the precentral gyrus (n = 12) of MND patients. Healthy aging was associated with decreased anti-inflammatory IL-10 and FGF-2 levels. AD prefrontal cortex was associated with increased levels of IL-4, IL-5, and FGF-2, with the largest increase seen for FGF-2. Notwithstanding differences in the specific frontal lobe gyrus sampled, MND patients' primary motor cortex (precentral gyrus) was associated with increased levels of IL-5, IL-6, IL-10, and FGF-2 compared to the aging prefrontal cortex (superior frontal gyrus). Immunocytochemistry showed that FGF-2 is expressed in neurons, astrocytes, and microglia in normal aging prefrontal cortex, AD prefrontal cortex, and MND motor cortex. We report that healthy aging and age-related neurodegenerative diseases have different cortical inflammatory signatures that are characterized by increased levels of anti-inflammatory cytokines and call into question the view that increased inflammation underlies the development of age-related neurodegenerative diseases.

Defining and unpacking the core concepts of pharmacology education.

Pharmacology education currently lacks a research-based consensus on which core concepts all graduates should know and understand, as well as a valid and reliable means to assess core conceptual learning. The Core Concepts in Pharmacology Expert Group (CC-PEG) from Australia and New Zealand recently identified a set of core concepts of pharmacology education as a first step toward developing a concept inventory-a valid and reliable tool to assess learner attainment of concepts. In the current study, CC-PEG used established methodologies to define each concept and then unpack its key components. Expert working groups of three to seven educators were formed to unpack concepts within specific conceptual groupings: what the body does to the drug (pharmacokinetics); what the drug does to the body (pharmacodynamics); and system integration and modification of drug-response. First, a one-sentence definition was developed for each core concept. Next, sub-concepts were established for each core concept. These twenty core concepts, along with their respective definitions and sub-concepts, can provide pharmacology educators with a resource to guide the development of new curricula and the evaluation of existing curricula. The unpacking and articulation of these core concepts will also inform the development of a pharmacology concept inventory. We anticipate that these resources will advance further collaboration across the international pharmacology education community to improve curricula, teaching, assessment, and learning.

The potential side effects of herbal preparations in domestic animals.

A recent series of deaths in previously healthy dogs in Victoria, Australia associated with the ingestion of raw meat contaminated by indospicine derived from native Australian plants of the Indigofera species draws attention to the potential that exists for herbal toxicity in domestic animals. Although the efficacy of herbal remedies generally remains unproven in domestic animals, herbal preparations are being increasingly used as supplements and treatments. Issues with incorrect ingredients, inadequate processing, faulty, incomplete or inaccurate product labelling, contamination with toxins, adulteration with undeclared pharmaceutical agents and herb-herb interactions are well recognized as causes of adverse effects in humans. However, apart from of the effects of noxious weed species, the literature on herbal toxicity in domestic animals is sparse. Thus, the forensic evaluation of cases of suspected poisoning in domestic animals should also encompass an accurate description the type and dose of any herbal preparations that may have been recently administered.

Identifying the core concepts of pharmacology education.

Pharmacology education currently lacks an agreed knowledge curriculum. Evidence from physics and biology education indicates that core concepts are useful and effective structures around which such a curriculum can be designed to facilitate student learning. Building on previous work, we developed a novel, criterion-based method to identify the core concepts of pharmacology education. Five novel criteria were developed, based on a literature search, to separate core concepts in pharmacology from topics and facts. Core concepts were agreed to be big ideas, enduring, difficult, applicable across contexts, and useful to solve problems. An exploratory survey of 33 pharmacology educators from Australia and New Zealand produced 109 terms, which were reduced to a working list of 26 concepts during an online workshop. Next, an expert group of 12 educators refined the working list to 19 concepts, by applying the five criteria and consolidating synonyms, and added three additional concepts that emerged during discussions. A confirmatory survey of a larger group resulted in 17 core concepts of pharmacology education. This list may be useful for educators to evaluate existing curricula, design new curricula, and to inform the development of a concept inventory to test attainment of the core concepts in pharmacology.

Paracetamol (acetaminophen) hepatotoxicity increases in the presence of an added herbal compound.

Hepatotoxicity from paracetamol/acetaminophen has occasionally been reported at lower than expected doses. As herbal preparations may interact with pharmaceutical drugs the following in vitro study was undertaken to determine whether the toxic effects of paracetamol on liver cell growth in culture would be exacerbated by the addition of psoralen, a furanocoumarin compound that is present in Psoralea corylifolia, a common Chinese herb. The following study utilising a liver carcinoma cell line (HepG2) showed that Psoralea corylifolia was significantly toxic from 0.3 mg/ml to 5 mg/ml (p < 0.05), whereas paracetamol was not toxic below 50 mM (p = 0.0026). Interactions between previously non-toxic levels of 0.1 mg/ml of Psoralea corylifolia and increasing concentrations of paracetamol (0-50 mM), however, were observed, with a significant increase in toxicity compared to paracetamol alone (30% cell death vs. 72% cell death with Psoralea corylifolia). A significant synergistic interaction was observed at 40 mM paracetamol with 0.1 mg/ml of Psoralea (p = 0.038). This study has, therefore, shown significantly increased hepatotoxicity in cell cultures exposed to paracetamol when herbal compounds containing furanocoumarins were added. Fulminant acute liver failure occurring after the ingestion of low doses of paracetamol may not, therefore, always be due to an occult idiosyncratic response to paracetamol, but instead possibly to the combined effects of paracetamol and herbal preparations. Given the widespread use of both paracetamol and herbal preparations this possibility should be considered in cases of unexplained hepatic necrosis and liver failure that present for medicolegal investigation.

Combined Liquid Chromatography-mass Spectrometry and Next-generation DNA Sequencing Detection of Adulterants and Contaminants in Analgesic and Anti-inflammatory Herbal Medicines.

INTRODUCTION: Methods for assessing the quality of herbal medicine preparations have advanced significantly in recent years in conjunction with increases in herbal medicine use and reports of adulteration and contamination. OBJECTIVE: This study examined the quality of analgesic and anti-inflammatory herbal medicine preparations available on the Australian market by detecting the presence of listed ingredients, adulterants and contaminants. METHODS: Forty-nine analgesic and anti-inflammatory herbal medicine preparations were randomly sourced from Australian capital cities. They were audited using a dual approach of liquid chromatography-mass spectrometry (LC-MS) combined with next-generation DNA sequencing. Once screened, a comparison of listed ingredients with verified ingredients was conducted to determine the accuracy of labelling, and the extent of adulteration and contamination. RESULTS: Twenty-six of 49 (53%) herbal medicines were adulterated or contaminated with undeclared ingredients. LC-MS revealed the presence of pharmaceutical adulterants including atropine and ephedrine. DNA sequencing uncovered concerning levels of herbal substitution, adulteration and contamination, including the use of fillers (alfalfa, wheat and soy), as well as pharmacologically relevant species (Centella asiatica, Panax ginseng, Bupleurum and Passiflora). Pig/boar and bird DNA was found in some preparations, inferring substandard manufacturing practices. Of the 26 contaminated samples, 19 (73%) were manufactured in Australia, and 7 (27%) were imported from other countries (6 from China, 1 from New Zealand). In 23 of 49 (47%) herbal medicine samples, no biological ingredients were detected at all. These were predominantly pain and anti-inflammatory preparations such as glucosamine and eicosapentaenoic and docosahexaenoic acids found in krill and fish oils, so DNA would not be expected to survive the manufacturing process. CONCLUSION: The high level of contamination and substitution of herbal medicine preparations sourced from Australian dispensaries supports the need for more stringent pharmacovigilance measures in Australia and abroad.

Hypersensitive adverse drug reactions to glucosamine and chondroitin preparations in Australia between 2000 and 2011.

PURPOSE OF THE STUDY: This study investigates spontaneous adverse drug reactions (ADRs) to glucosamine and chondroitin in the Australian population between 2000 and 2011, with a primary focus on hypersensitivity reactions. STUDY DESIGN: Case reports of ADR to glucosamine and chondroitin sent to the Therapeutic Goods Administration between 2000 and 2011 were obtained and analysed. The demographic information and severity of the ADR were recorded for individual ADR cases. These reactions were classified according to the Brown et al grading system for generalised hypersensitivity reactions. This included mild hypersensitivity reactions (generalised erythema, urticaria and angioedema) through to moderate hypersensitivity reactions (wheeze, nausea, vomiting, dizziness (presyncope), diaphoresis, chest or throat tightness and abdominal pain), and more severe reactions (hypotension, confusion and collapse). RESULTS: In this study of 366 ADRs to glucosamine and chondroitin preparations, 71.85% of cases (n=263) were found to have hypersensitivity reactions. Of these 263 cases, 92 cases were classified as mild (eg, pruritus, urticaria and lip oedema), 128 cases classified as moderate (such as dyspnoea, nausea and abdominal pain), and 43 cases classified as severe (including amnesia, gait disturbance, somnolence and hypotension). It is not clear whether the patients involved had a known shellfish allergy or underlying atopy. CONCLUSION: Results of this investigation support the need for clear labelling on glucosamine and chondroitin preparations to raise awareness of possible adverse events for those predisposed to allergy or atopy in response to shellfish.

Synthesis and biological properties of tetranuclear ruthenium complexes containing the bis[4(4'-methyl-2,2'-bipyridyl)]-1,7-heptane ligand.

Linear and non-linear tetranuclear ruthenium(ii) complexes containing the bridging ligand bis[4(4'-methyl-2,2'-bipyridyl)]-1,7-heptane have been synthesised and their biological properties examined. The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) of the ruthenium(ii) complexes were determined against six strains of bacteria: Gram-positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA); and the Gram-negative Escherichia coli (E. coli) strains MG1655, APEC, UPEC and Pseudomonas aeruginosa (P. aeruginosa). The results showed that both tetranuclear complexes had significant antimicrobial activity, with the non-linear (branched) species (Rubb7-TNL) having slightly higher activity than the corresponding linear analogue (Rubb7-TL). The corresponding toxicity against three eukaryotic cell lines - BHK (baby hamster kidney), Caco-2 (heterogeneous human epithelial colorectal adenocarcinoma) and Hep-G2 (liver carcinoma) - have also been determined. Interestingly, both Rubb7-TNL and Rubb7-TL were as toxic to the eukaryotic cells as they were to the bacteria, a rarity for kinetically-inert cationic polypyridylruthenium(ii) complexes, and exhibited lower IC50 values than cisplatin over 24-, 48- or 72-hour incubation times. Fluorescence spectroscopy was used to study the binding of the ruthenium complexes with human serum albumin (HSA). Rubb7-TNL and Rubb7-TL exhibited strong HSA binding, with equilibrium binding constants in the order of 107 M-1. Confocal microscopy was used to examine the cellular localisation of Rubb7-TNL in BHK cells. The results indicated that the ruthenium complex localised in the nucleolus. Significant accumulation was also observed in the cytoplasm, but not in the mitochondria. Taken together, the results of this study suggest that Rubb7-TNL is an unlikely candidate as an antimicrobial agent, but may have potential as an anticancer drug.

Toxicological screening and DNA sequencing detects contamination and adulteration in regulated herbal medicines and supplements for diet, weight loss and cardiovascular health.

Use of herbal medicines and supplements by consumers to prevent or treat disease, particularly chronic conditions continues to grow, leading to increased awareness of the minimal regulation standards in many countries. Fraudulent, adulterated and contaminated herbal and traditional medicines and dietary supplements are a risk to consumer health, with adverse effects and events including overdose, drug-herb interactions and hospitalisation. The scope of the risk has been difficult to determine, prompting calls for new approaches, such as the combination of DNA metabarcoding and mass spectrometry used in this study. Here we show that nearly 50% of products tested had contamination issues, in terms of DNA, chemical composition or both. Two samples were clear cases of pharmaceutical adulteration, including a combination of paracetamol and chlorpheniramine in one product and trace amounts of buclizine, a drug no longer in use in Australia, in another. Other issues include the undeclared presence of stimulants such as caffeine, synephrine or ephedrine. DNA data highlighted potential allergy concerns (nuts, wheat), presence of potential toxins (Neem oil) and animal ingredients (reindeer, frog, shrew), and possible substitution of bird cartilage in place of shark. Only 21% of the tested products were able to have at least one ingredient corroborated by DNA sequencing. This study demonstrates that, despite current monitoring approaches, contaminated and adulterated products are still reaching the consumer. We suggest that a better solution is stronger pre-market evaluation, using techniques such as that outlined in this study.

Exploring the Application of the DSA-TOF, a Direct, High-resolution Time-of-Flight Mass Spectrometry Technique for the Screening of Potential Adulterated and Contaminated Herbal Medicines.

Global consumption of complementary and alternative medicines, including herbal medicines, has increased substantially, and recent reports of adulteration demonstrate the need for high throughput and extensive pharmacovigilance to ensure product safety and quality. Three different standard reference materials and five previously analyzed herbal medicines have been used as a proof of concept for the application of adulteration/contamination screening using a Direct Sample Analysis (DSA) ion source with TOF MS on the Perkin Elmer AxION 2 TOF. This technique offers the advantages of minimum sample preparation, rapid analysis, and mass accuracies of 5 ppm. The DSA TOF analysis correlates well with the previous analysis on the initial sample set (which found undeclared herbal ingredients), with the added advantage of detecting previously untargeted compounds, including species-specific flavonoids and alkaloids. The rapid analysis using the DSA-TOF facilitates screening for hundreds of compounds in minutes with minimal sample preparation, generating a comprehensive profile for each sample. Graphical Abstract.

Analysis of spontaneous adverse drug reactions to echinacea, valerian, black cohosh and ginkgo in Australia from 2000 to 2015.

OBJECTIVE: Assessing adverse drug reactions (ADRs) is a proven method to estimate the safety of medicines. The ADRs to herbal medicines in Australia (and by inference, the safety of herbal medicines in Australia) remain unknown. This study examines spontaneous ADR cases to four of the most popular herbs in Australia from 2000 to 2015: echinacea (Echinacea purpurea), valerian (Valeriana officinalis), black cohosh (Actaea racemosa) and ginkgo (Ginkgo biloba). METHODS: ADRs of echinacea, valerian, black cohosh and ginkgo reported to the Australian Therapeutic Goods Administration (TGA) between 2000 and 2015 were obtained from the TGA database. Data were collated and analysed according to age, sex, severity, type of ADR and body system affected. Statistics were calculated using GraphPad Prism software. RESULTS: Most ADRs were mild or moderate. However, every herbal medicine was associated with life-threatening ADRs. In each life-threatening case, the herbal medicine was taken concomitantly with prescription medications. Black cohosh was associated with a significant number of severe ADRs (30.3% of the total), with 39.4% of these ADRs being associated with abnormal hepatic function, hepatitis or hepatotoxicity. CONCLUSION: This study highlights the lack of public awareness with regard to herb-drug interactions, since most of the severe ADRs involved a herb-drug interaction.

Control of temporal shape of nanosecond long lasers using feedback loops.

We present developments in the control of the temporal pulse shape of nanosecond lasers. An active feedback loop between a regenerative amplified laser's output and input was controlled in order to obtain the desired pulse shape. We used several algorithms to achieve this and the differences caused by the target pulse shape and duration are compared. It is found that the algorithm based on the ratio of the target pulse shape and measured pulse profile provides the most robust solution. The methods proposed here can be used to obtain any pulse shape with minimal knowledge of the laser amplification system.

Evidence for the efficacy and safety of herbal weight loss preparations.

Rising rates of obesity across the globe have been associated with an increase in the use of herbal preparations for weight control. However, the mechanisms of action for these substances are often not known, as is the potential for interaction with other herbal preparations or prescription pharmaceutical drugs. To investigate the reported efficacy and safety of herbal weight loss preparations, we conducted a review of the literature focusing on herbs that are most commonly used in weight loss preparations, specifically, Garcinia cambogia, Camellia sinensis, Hoodia gordonii, Citrus aurantium and Coleus forskohlii. There was no clear evidence that the above herbal preparations would cause sustained long-term weight loss in humans in the long term. Serious illness and even death have occasionally resulted from the use of herbal weight loss preparations. Few clinical trials have been undertaken to evaluate the efficacy and/or safety of herbal weight loss preparations. In addition, potential issues of herb-herb and herb-drug interactions are often not considered. Regulation of these products is much less rigorous than for prescription medications, despite documented cases of associated hepatotoxicity.