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.

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.

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.

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.

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.

Caffeine toxicity in forensic practice: possible effects and under-appreciated sources.

Caffeine is considered a very safe stimulant and is widely consumed in a variety of forms, from pure caffeine to beverages and foods. Typically, death is only seen when gram quantities of caffeine are consumed, usually in suicide attempts. Even in this scenario, death is rare. However, there are special populations that need to be considered in forensic presentations, who may be at greater risk. These include poor metabolizers, people with liver disease, and people with cardiac conditions, who can die as a result of caffeine intake at levels well below what is ordinarily considered toxic. Also, caffeine intake may be hidden. For example, herbal medicines with substantial caffeine content may not disclose these concentrations on their product label. The role of caffeine in medicolegal deaths is yet to be defined, however, herbal medicines and herbal weight loss supplements may represent an underappreciated source of caffeine in this context.

Combined DNA, toxicological and heavy metal analyses provides an auditing toolkit to improve pharmacovigilance of traditional Chinese medicine (TCM).

Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

A comparison of patterns of spontaneous adverse drug reaction reporting with St. John's Wort and fluoxetine during the period 2000-2013.

Herbal medicines are perceived to be safe by the general public and medical practitioners, despite abundant evidence from clinical trials and case reports that show herbal preparations can have significant adverse effects. The overall impact of adverse events to herbal medicines in Australia is currently unknown. Post marketing surveillance of medications through spontaneous adverse drug reaction (ADR) reports to the Therapeutic Goods Administration (TGA) is one way to estimate this risk. The patterns of spontaneously reported ADRs provide insight to herbal dangers, especially when compared with patterns of a mechanistically similar conventional drug. The study compared the pattern of spontaneously reported ADRs to St. John's Wort (Hypericum perforatum), a common herbal treatment for depression which contains selective serotonin reuptake inhibitors (SSRI), to fluoxetine, a commonly prescribed synthetic SSRI antidepressant. Spontaneous ADR reports sent to the TGA between 2000-2013 for St. John's Wort (n = 84) and fluoxetine (n = 447) were obtained and analysed. The demographic information, types of interaction, severity of the ADR, and the body systems affected (using the Anatomical Therapeutic Chemical classification system) were recorded for individual ADR cases. The majority of spontaneously reported ADRs for St. John's Wort and fluoxetine were concerning females aged 26-50 years (28.6%, 22.8%). The organ systems affected by ADRs to St John's Wort and fluoxetine have a similar profile, with the majority of cases affecting the central nervous system (45.2%, 61.7%). This result demonstrates that herbal preparations can result in ADRs similar to those of prescription medications.

Lethal hepatocellular necrosis associated with herbal polypharmacy in a patient with chronic hepatitis B infection.

Following a short treatment for irritable bowel with the following herbs: Astragalus propinquus, Codonopsis pilosula, Paeonia sp., Atractylodes macrocephala, Pueraria sp., Poria cocos, Dioscorea opposita, Patriniae, Psoralea corylifolia, Alpinia katsumadai, Glycyrrhiza uralensis and Dolomiaea souliei sp. a 43-year-old woman developed acute severe liver failure requiring liver transplantation. Histopathological examination of the liver showed massive hepatic necrosis in keeping with drug/chemical toxicity. Surgery was followed by multiorgan failure and death. While numerous studies have evaluated the effect of polypharmacy, the study of multiple concurrent herb use is only just emerging, despite the popularity of herbal medicine use in the western world. As this case demonstrates that fulminant hepatic failure and death may be caused by the concomitant use of a number of herbal products, the possibility of untoward effects from herbal polypharmacy must be increasingly considered in the evaluation of medicolegal cases.

Forensic problems with the composition and content of herbal medicines.

A survey of herbal medicines available for internet and over-the-counter purchase in South Australia, Australia, was conducted looking specifically at those used for 'arthritis', 'cold and flu', 'gastrointestinal', 'stress' and 'premenstrual syndrome'. 121 products consisted of 29 in the 'arthritis' category, 33 in 'cold and flu', 19 in 'gastrointestinal' 30 in 'stress' and 10 in 'premenstrual syndrome'. Twenty two (18%) of 121 products were not registered with the Australian Register of Therapeutic Goods (ARTG), despite this being a legal requirement for their sale. Of the registered products 59 (60%) of 99 had differing ingredient concentrations on the website compared to their ARTG listing. Only three of the 15 purchased products had ingredient concentrations which were consistent between the website, ARTG listing and product packaging. These findings demonstrate that it may not be possible to determine what herbal substance an individual has been exposed to prior to death and in what concentration, based on packaging from medications seized at the scene, or from examination of website data and the ARTG listing. These discrepancies may increase the problems that exist in attempting to determine what role herbal medicines may play in the mechanism of death in certain forensic cases.

Gallic acid is the major component of grape seed extract that inhibits amyloid fibril formation.

Many protein misfolding diseases, for example, Alzheimer's, Parkinson's and Huntington's, are characterised by the accumulation of protein aggregates in an amyloid fibrillar form. Natural products which inhibit fibril formation are a promising avenue to explore as therapeutics for the treatment of these diseases. In this study we have shown, using in vitro thioflavin T assays and transmission electron microscopy, that grape seed extract inhibits fibril formation of kappa-casein (κ-CN), a milk protein which forms amyloid fibrils spontaneously under physiological conditions. Among the components of grape seed extract, gallic acid was the most active component at inhibiting κ-CN fibril formation, by stabilizing κ-CN to prevent its aggregation. Concomitantly, gallic acid significantly reduced the toxicity of κ-CN to pheochromocytoma12 cells. Furthermore, gallic acid effectively inhibited fibril formation by the amyloid-beta peptide, the putative causative agent in Alzheimer's disease. It is concluded that the gallate moiety has the fibril-inhibitory activity.

Dietary polyphenol-derived protection against neurotoxic ß-amyloid protein: from molecular to clinical.

Polyphenolic compounds derived mainly from plant products have demonstrated neuroprotective properties in a number of experimental settings. Such protective effects have often been ascribed to antioxidant capacity, but specific augmentation of other cellular defences and direct interactions with neurotoxic proteins have also been demonstrated. With an emphasis on neurodegenerative conditions, such as Alzheimer's disease, we highlight recent findings on the neuroprotection ascribed to bioactive polyphenols capable of directly interfering with the Alzheimer's disease hallmark toxic ß-amyloid protein (Aß), thereby inhibiting fibril and aggregate formation. This includes compounds such as the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) and the phytoalexin resveratrol. Targeted studies on the biomolecular interactions between dietary polyphenolics and Aß have not only improved our understanding of the pathogenic role of ß-amyloid, but also offer fundamentally novel treatment options for Alzheimer's disease and potentially other amyloidoses.

Uptake, distribution, and speciation of selenoamino acids by human cancer cells: X-ray absorption and fluorescence methods.

Selenium compounds exhibit chemopreventative properties at supranutritional doses, but the efficacy of selenium supplementation in cancer prevention is dependent on the chemical speciation of the selenium supplement and its metabolites. The uptake, speciation, and distribution of the common selenoamino acid supplements, selenomethionine (SeMet) and Se-methylselenocysteine (MeSeCys), in A549 human lung cancer cells were investigated using X-ray absorption and fluorescence spectroscopies. X-ray absorption spectroscopy of bulk cell pellets treated with the selenoamino acids for 24 h showed that while selenium was found exclusively in carbon-bound forms in SeMet-treated cells, a diselenide component was identified in MeSeCys-treated cells in addition to the carbon-bound selenium species. X-ray fluorescence microscopy of single cells showed that selenium accumulated with sulfur in the perinuclear region of SeMet-treated cells after 24 h, but microprobe selenium X-ray absorption near-edge spectroscopy in this region indicated that selenium was carbon-bound rather than sulfur-bound. X-ray absorption and X-ray fluorescence studies both showed that the selenium content of MeSeCys-treated cells was much lower than that of SeMet-treated cells. Selenium was distributed homogeneously throughout the MeSeCys-treated cells.