Toxins in Botanical Drugs and Plant-derived Food and Feed - from Science to Regulation: A Workshop Review.

In September 2022, the 3rd International Workshop on pyrrolizidine alkaloids (PAs) and related phytotoxins was held on-line, entitled 'Toxins in botanical drugs and plant-derived food and feed - from science to regulation'. The workshop focused on new findings about the occurrence, exposure, toxicity, and risk assessment of PAs. In addition, new scientific results related to the risk assessment of alkenylbenzenes, a distinct class of herbal constituents, were presented. The presence of PAs and alkenylbenzenes in plant-derived food, feed, and herbal medicines has raised health concerns with respect to their acute and chronic toxicity but mainly related to the genotoxic and carcinogenic properties of several congeners. The compounds are natural constituents of a variety of plant families and species widely used in medicinal, food, and feed products. Their individual occurrence, levels, and toxic properties, together with the broad range of congeners present in nature, represent a striking challenge to modern toxicology. This review tries to provide an overview of the current knowledge on these compounds and indicates needs and perspectives for future research.

Antiviral activity of the "Virus Blocking Factor" (VBF) derived i.a. from Pelargonium extract and Sambucus juice against different human-pathogenic cold viruses in vitro.

THE AIM: The in-vitro antiviral activity of the "Virus Blocking Factor" (VBF), a combination of Pelargonium extract and Sambucus juice with addition of Betaglucan 1,3 / 1,6, Zincum gluconium, Acidum ascorbicum, was studied against human pathogenic viruses: Influenza A H1N1 (FluA H1N1), Rhinovirus B subtype 14 (HRV14), Respiratory Syncytial Virus (RSV), Parainfluenzavirus subtype 3 (Para 3), and Adenovirus C subtype 5 (Adeno 5). METHOD: Antiviral activity was assessed using plaque-reduction assays after adding the test substance post infection of the MDCK, HeLa and HEp-2 cells with the viruses. Ribavirin Virazol and - in case of Adenovirus an internal laboratory standard - were used as positive controls. Cytotoxic effects of VBF and VBF Control onto the virus permissive MDCK, HeLa and HEp-2 cells were examined. Non-toxic concentrations of VBF were determined by the Methylthiazoletetrazolium test (MTT-Test). RESULTS AND CONCLUSIONS: In all antiviral studies VBF showed (2.1%) a dose-dependent antiviral activity against FluA H1N1 and HRV14 at non-toxic concentrations. A very strong effect was demonstrated in concentrations of 2.5% and 1.25% where replication of H1N1 and HRV14 was nearly completely blocked. Dose-dependent antiviral activity was detectable against RSV in a concentration range of 1.25% to 0.63% of the test item. Due to toxic side effects of a 2.5% concentration at least a "minor effect" of about 30% (1.25% solution) against Para 3 infected HEp-2 cells could be determined. Concerning Adeno 5 not any antiviral activity could be demonstrated in all studies with all tested substance concentrations of VBF. VBF Control did not show any cytotoxicity and antiviral effects. Further research is needed to elucidate clinical effect of VBF.

Antiviral activity of the "Virus Blocking Factor" (VBF) derived i.a. from Pelargonium extract and Sambucus juice against different human-pathogenic cold viruses in vitro.

THE AIM: The in-vitro antiviral activity of the "Virus Blocking Factor" (VBF), a combination of Pelargonium extract and Sambucus juice with addition of Betaglucan 1,3 / 1,6, Zincum gluconium, Acidum ascorbicum, was studied against human pathogenic viruses: Influenza A H1N1 (FluA H1N1), Rhinovirus B subtype 14 (HRV14), Respiratory Syncytial Virus (RSV), Parainfluenzavirus subtype 3 (Para 3), and Adenovirus C subtype 5 (Adeno 5). METHOD: Antiviral activity was assessed using plaque-reduction assays after adding the test substance post infection of the MDCK, HeLa and HEp-2 cells with the viruses. Ribavirin Virazol and - in case of Adenovirus an internal laboratory standard - were used as positive controls. Cytotoxic effects of VBF and VBF Control onto the virus permissive MDCK, HeLa and HEp-2 cells were examined. Non-toxic concentrations of VBF were determined by the Methylthiazoletetrazolium test (MTT-Test). RESULTS AND CONCLUSIONS: In all antiviral studies VBF showed (2.1%) a dose-dependent antiviral activity against FluA H1N1 and HRV14 at non-toxic concentrations. A very strong effect was demonstrated in concentrations of 2.5% and 1.25% where replication of H1N1 and HRV14 was nearly completely blocked. Dose-dependent antiviral activity was detectable against RSV in a concentration range of 1.25% to 0.63% of the test item. Due to toxic side effects of a 2.5% concentration at least a "minor effect" of about 30% (1.25% solution) against Para 3 infected HEp-2 cells could be determined. Concerning Adeno 5 not any antiviral activity could be demonstrated in all studies with all tested substance concentrations of VBF. VBF Control did not show any cytotoxicity and antiviral effects. Further research is needed to elucidate clinical effect of VBF.

Candelabra aloe (Aloe arborescens) in the therapy and prophylaxis of upper respiratory tract infections: traditional use and recent research results.

Aloe arborescens (Candelabra Aloe) has been used in the treatment of upper respiratory tract infections in Central and Eastern European countries for many decades. Originally introduced to support the healing and recovery in cornea transplant patients, aqueous A. arborescens extracts soon became popular in the treatment of upper respiratory tract infections with a focus on toddlers and children. Recent preclinical and clinical data show that immunomodulatory, antiinflammatory, and antiviral effects contribute to its therapeutic efficacy. Based on its well documented, longstanding traditional use and its excellent safety and tolerability, A. arborescens may be considered a valuable addition to the spectrum of herbal medicinal products for the treatment and prophylaxis of upper respiratory tract infections, in particular common cold, in adults and children.

Genotoxicity of selected pyrrolizidine alkaloids in human hepatoma cell lines HepG2 and Huh6.

INTRODUCTION: Pyrrolizidine alkaloids (PAs) are found in many plant species as secondary metabolites which affect humans via contaminated food sources, herbal medicines and dietary supplements. Hundreds of compounds belonging to PAs have been identified. PAs undergo hepatic metabolism, after which they can induce hepatotoxicity and carcinogenicity. Many aspects of their mechanism of carcinogenicity are still unclear and it is important for human risk assessment to investigate this class of compounds further. MATERIAL AND METHODS: Human hepatoma cells HepG2 were used to investigate the genotoxicity of different chemical structural classes of PAs, namely europine, lycopsamine, retrorsine, riddelliine, seneciphylline, echimidine and lasiocarpine, in the cytokinesis-block micronucleus (CBMN) assay. The different ester type PAs europine, seneciphylline, and lasiocarpine were also tested in human hepatoma Huh6 cells. Six different PAs were investigated in a crosslink comet assay in HepG2 cells. RESULTS: The maximal increase of micronucleus formation was for all PAs in the range of 1.64-2.0 fold. The lowest concentrations at which significant induction of micronuclei were found were 3.2 µM for lasiocarpine and riddelliine, 32 µM for retrorsine and echimidine, and 100 µM for seneciphylline, europine and lycopsamine. Significant induction of micronuclei by lasiocarpine, seneciphylline, and europine were achieved in Huh6 cells at similar concentrations. Reduced tail formation after hydrogen peroxide treatment was found in the crosslink comet assay for all diester type PAs, while an equimolar concentration of the monoesters europine and lycopsamine did not significantly reduce DNA migration. CONCLUSION: The widely available human hepatoma cell lines HepG2 and Huh6 were suitable for the assessment of PA-induced genotoxicity. Selected PAs confirmed previously published potency rankings in the micronucleus assay. In HepG2 cells, the crosslinking activity was related to the ester type, which is a first report of PA mediated effects in the comet assay.

Patterns and Trends of Herbal Medicine Use among Patients with Gynecologic Cancer.

Background More and more information about complementary and integrative medicine is becoming available, especially among cancer patients. However, little is known about the use of herbal medicine by patients with gynecologic cancers. This study aimed to assess the use of herbal products by gynecologic cancer patients compared with healthy controls. Methods This cross-sectional study was conducted at the Department for Gynecology and Obstetrics of Erlangen University Hospital and included 201 patients with gynecologic cancer and 212 healthy controls. Use of herbal medicines was evaluated using a standardized questionnaire. Medical information on cancer patients was collected from hospital records. Group comparisons were done using a logistic regression model. Risk ratios were assessed using a Poisson regression model. Results Gynecologic cancer patients used herbal medicine significantly less often than healthy persons. 69% of gynecologic cancer patients and 81% of healthy participants reported using herbal products. 40% of cancer patients and 56% of healthy persons reported using plants for medicinal purposes. Motives of cancer patients for using herbal medicine included treatment of cancer-related symptoms. The major source of information for both groups was family and friends. Conclusions Although herbal medicine was used less by patients with gynecologic cancer, herbal products were used by both cancer patients and healthy individuals. To provide cancer patients with optimal therapy, oncologists should be informed about the herbal products used by their patients as this will allow them to take their patients' self-medication with herbal medicine into account. Counseling by oncologists on the use of herbal medicine should be encouraged.

No clinically relevant CYP3A induction after St. John's wort with low hyperforin content in healthy volunteers.

OBJECTIVE: Induction of CYP3A by St. John's wort (SJW) products with high hyperforin content is well described. Since CYP3A induction is mediated by hyperforin in a concentration-dependent manner, and SJW preparations differ significantly in hyperforin content, the aim of the study was to evaluate the effect of an SJW powder with low hyperforin content on CYP3A function. METHODS: Twenty healthy male volunteers received an SJW powder with low hyperforin content for 2 weeks. Midazolam plasma concentration time profiles were characterized after a single oral dose of 7.5 mg midazolam on the day before and on the 14th day of SJW medication. RESULTS: Midazolam AUC(0-infinity) slightly decreased from 124.0 +/- 62.5 ng/ml.h at baseline to 105.6 +/- 53.2 ng/ml.h after SJW (P < 0.05), representing a mean 11.3% decrease (95% CI: -22.8 to 0.21). No significant change in midazolam C(max), t(1/2) and t(max) was observed. For all pharmacokinetic parameters, the 90% CI for the geometric mean ratio of treatment over baseline were within the no-effect boundaries of 0.70-1.43. CONCLUSION: Administration of an SJW product with low hyperforin content resulted in a mild induction of CYP3A not considered clinically relevant.

Herbal reference standards.

This review describes the current definitions and regulatory requirements that apply to reference standards that are used to analyse herbal products. It also describes and discusses the current use of reference substances and reference extracts in the European and United States pharmacopoeias.

Transition rates of selected metals determined in various types of teas (Camellia sinensis L. Kuntze) and herbal/fruit infusions.

Teas and raw materials used as ingredients of herbal and fruit infusions (HFI) were analysed by means of ICP-MS for their content of aluminium, arsenic, cadmium, copper, lead and mercury in the dry product and in the infusion. Samples of tea (Camellia sinensis L. Kuntze) were selected to include different origins, types (black, green), leaf grades (whole leaf, broken, fannings, dust) and manufacturing techniques (orthodox, "crush, tear, curl"). The selected HFI raw materials (chamomile, elderberries, fennel, hibiscus, mate, peppermint, rooibos and rose hip) cover the most important matrices (flower, fruit, seed, herb, leaf) and reflect the economic significance of these HFI materials in trade. Infusions were prepared under standardised conditions representing typical household brewing. Transition rates for the investigated metals vary significantly but are mostly well below 100%. We propose default transition rates for metals to avoid overestimation of exposure levels from tea/HFI consumption.

Effect of St John's wort dose and preparations on the pharmacokinetics of digoxin.

BACKGROUND AND OBJECTIVE: St John's wort preparations vary in composition, main constituents, formulation, and daily dose administered. The aim of the study was to evaluate the possible pharmacokinetic interaction of marketed St John's wort formulations and doses with digoxin. METHODS: A randomized, placebo-controlled, parallel-group study was performed in 96 healthy volunteers in 3 study parts. A 7-day loading phase with digoxin was followed by 14 days of comedication with placebo or one of 10 St John's wort products varying in dose and formulation. The pharmacokinetics of digoxin was determined before comedication and on day 14 of comedication. RESULTS: Comedication comprised traditionally used Hypericum products; 2 g powder without hyperforin, tea, juice, oil extract, and placebo had no significant interaction with digoxin nor did hyperforin-free extract (Ze 117) or low daily doses of hyperforin-containing Hypericum powder (1 g, 0.5 g). However, comedication with the high-dose hyperforin-rich extract LI 160 resulted in a reduction of digoxin area under the curve from time 0 to 24 hours (AUC(0-24)) of -24.8% (95% confidence interval [CI], -28.3 to -21.3), a reduction in digoxin maximal plasma concentration (C(max)) of -37% (95% CI, -42 to -32), and a reduction in digoxin plasma concentration at 24 hours after previous dosing (C(trough)) of -19% (95% CI, -27 to -11). Comedication with 4 g Hypericum powder with comparable hyperforin content resulted in a reduction in digoxin AUC(0-24) of -26.6% (95% CI, -37.3 to -15.9), a reduction in digoxin C(max) of -38% (95% CI, -48 to -18), and a reduction in digoxin C(trough) of -19% (95% CI, -27 to -10). Two grams of Hypericum powder with half the hyperforin content resulted in a less prominent reduction in AUC(0-24) of -17.7% (95% CI, -21.6 to -13.7), C(max) (-21%; 95% CI, -40 to -2), and C(trough) (-13%; 95% CI, -21 to -5). CONCLUSIONS: The interaction of St John's wort and digoxin varies within St John's wort preparations and doses and seems to be correlated with the dose, particularly of hyperforin.

Hibiscus sabdariffa L. - a phytochemical and pharmacological review.

Hibiscus sabdariffa L. (Hs, roselle; Malvaceae) has been used traditionally as a food, in herbal drinks, in hot and cold beverages, as a flavouring agent in the food industry and as a herbal medicine. In vitro and in vivo studies as well as some clinical trials provide some evidence mostly for phytochemically poorly characterised Hs extracts. Extracts showed antibacterial, anti-oxidant, nephro- and hepato-protective, renal/diuretic effect, effects on lipid metabolism (anti-cholesterol), anti-diabetic and anti-hypertensive effects among others. This might be linked to strong antioxidant activities, inhibition of α-glucosidase and α-amylase, inhibition of angiotensin-converting enzymes (ACE), and direct vaso-relaxant effect or calcium channel modulation. Phenolic acids (esp. protocatechuic acid), organic acid (hydroxycitric acid and hibiscus acid) and anthocyanins (delphinidin-3-sambubioside and cyanidin-3-sambubioside) are likely to contribute to the reported effects. More well designed controlled clinical trials are needed which use phytochemically characterised preparations. Hs has an excellent safety and tolerability record.

Intestinal anti-inflammatory activity of the Serpylli herba extract in experimental models of rodent colitis.

INTRODUCTION: Nowadays, there is an increasing interest for alternative options in the treatment of inflammatory bowel diseases (IBDs) that combine efficacy and an adequate safety profile. METHODS: The intestinal anti-inflammatory effects of Serpylli herba, the officinal drug in the European Pharmacopeia composed by the aerial parts of wild thyme (Thymus serpyllum), were evaluated in the trinitrobenzenesulfonic acid (TNBS)-induced rat colitis and dextran sodium sulfate (DSS)-induced mouse colitis, which are well characterized experimental models with some resemblance to human IBD. RESULTS: S. herba extract exerted an intestinal anti-inflammatory effect in both experimental models of colitis, as evidenced both histologically, since it facilitated the tissue recovery of the damaged colon, and biochemically as showed by the improvement of the different inflammatory markers evaluated, including myeloperoxidase activity, glutathione content, and leukotriene B4 levels as well as the expression of the inducible proteins iNOS and COX-2. This beneficial effect was associated with the reduction in the expression of different cytokines, like TNFα, IL-1ß, IFNγ, IL-6 and IL-17, the chemokine MCP-1, and the adhesion molecule ICAM-1, thus ameliorating the altered immune response associated with the colonic inflammation. CONCLUSION: S. herba extract displays an anti-inflammatory effect on different models of rodent colitis that could be attributed to its immunomodulatory properties.

Additive insulinogenic action of Opuntia ficus-indica cladode and fruit skin extract and leucine after exercise in healthy males.

BACKGROUND: Oral intake of a specific extract of Opuntia ficus-indica cladode and fruit skin (OpunDia™) (OFI) has been shown to increase serum insulin concentration while reducing blood glucose level for a given amount of glucose ingestion after an endurance exercise bout in healthy young volunteers. However, it is unknown whether OFI-induced insulin stimulation after exercise is of the same magnitude than the stimulation by other insulinogenic agents like leucine as well as whether OFI can interact with those agents. Therefore, the aims of the present study were: 1) to compare the degree of insulin stimulation by OFI with the effect of leucine administration; 2) to determine whether OFI and leucine have an additive action on insulin stimulation post-exercise. METHODS: Eleven subjects participated in a randomized double-blind cross-over study involving four experimental sessions. In each session the subjects successively underwent a 2-h oral glucose tolerance test (OGTT) after a 30-min cycling bout at ~70% VO2max. At t0 and t60 during the OGTT, subjects ingested 75 g glucose and capsules containing either 1) a placebo; 2) 1000 mg OFI; 3) 3 g leucine; 4) 1000 mg OFI + 3 g leucine. Blood samples were collected before and at 30-min intervals during the OGTT for determination of blood glucose and serum insulin. RESULTS: Whereas no effect of leucine was measured, OFI reduced blood glucose at t90 by ~7% and the area under the glucose curve by ~15% and increased serum insulin concentration at t90 by ~35% compared to placebo (P<0.05). From t60 to the end of the OGTT, serum insulin concentration was higher in OFI+leucine than in placebo which resulted in a higher area under the insulin curve (+40%, P<0.05). CONCLUSION: Carbohydrate-induced insulin stimulation post-exercise can be further increased by the combination of OFI with leucine. OFI and leucine could be interesting ingredients to include together in recovery drinks to resynthesize muscle glycogen faster post-exercise. Still, it needs to be confirmed that such nutritional strategy effectively stimulates post-exercise muscle glycogen resynthesis.

Quality control of plant food supplements.

It is essential to guarantee the safety of unprocessed plants and food supplements if consumers' health is to be protected. Although botanicals and their preparations are regulated at EU level, at least in part, there is still considerable discretion at national level, and Member States may choose to classify a product either as a food supplement or as a drug. Accurate data concerning the finished products and the plant used as the starting point are of major importance if risks and safety are to be properly assessed, but in addition standardized criteria for herbal preparation must be laid down and respected by researchers and manufacturers. Physiologically active as well as potentially toxic constituents need to be identified, and suitable analytical methods for their measurement specified, particularly in view of the increasing incidence of economically motivated adulteration of herbal raw materials and extracts. It remains the duty of food operators to keep up with the scientific literature and to provide sufficient information to enable the adaptation of specifications, sampling schemes and analytical methods to a fast-changing environment.

Extracts of Valeriana officinalis L. s.l. show anxiolytic and antidepressant effects but neither sedative nor myorelaxant properties.

Extracts of Valeriana officinalis L. s.l. are used for treating mild sleep disorders and nervous tension. Despite intensive research efforts, the pharmacological actions accounting for the clinical efficacy of valerian remain unclear. Thus, it was the aim of this study to evaluate CNS-related effects of different valerian extracts using behavioral paradigms (mice and rats). Following oral administration two commercially available preparations (extraction solvents: 45% methanol m/m and 70% ethanol v/v), a 35% ethanolic v/v extract and a refined extract derived from it (patented special extract phytofin Valerian 368) were tested for sedative (locomotor activity, ether-induced anaesthesia) and anxiolytic (elevated plus maze) activity. Using the forced swimming and the horizontal wire test the latter two extracts were additionally tested for antidepressant and myorelaxant properties. Up to maximum dosages of 500 or 1000 mg/kg bw none of the valerian extracts displayed sedative effects. Neither spontaneous activity was reduced nor the duration of ether-induced narcosis was prolonged. In contrast, results obtained in the elevated plus maze test revealed a pronounced anxiolytic effect of the 45% methanolic and 35% ethanolic extract as well as of phyotofin Valerian 368 in a dose range of 100-500 mg/kg bw. Additionally and different from its primary extract (35% ethanolic extract) phytofin Valerian 368 showed antidepressant activity in the forced swimming test after subacute treatment. Myorelaxant effects were not observed in dosages up to 1000 mg/kg bw. Due to these findings it is proposed that not sedative but anxiolytic and antidepressant activity, which was elaborated particularly in the special extract phytofin Valerian 368, considerably contribute to the sleep-enhancing properties of valerian.

The extent of induction of CYP3A by St. John's wort varies among products and is linked to hyperforin dose.

OBJECTIVE: Induction of CYP3A by St. John's wort (SJW) extracts with high hyperforin (HYF) content is well described. Since SJW products vary in the amount of HYF and other main constituents, the aim of the study was to evaluate the effect on CYP3A function of SJW preparations with a range from very low to high HYF content. METHODS: Forty-two male, healthy volunteers were randomized into six parallel SJW medication groups with varying composition especially with regard to HYF content. Midazolam plasma concentration profiles were characterized after a single oral dose of 7.5 mg midazolam on the day before and on the 14th day of SJW medication. RESULTS: All SJW preparations tested resulted in a decrease in midazolam AUC, although the extent of the effect differed. The extract LI 160 (HYF 41 mg/day) decreased midazolam AUC0-12h by 79.4% (95% CI -88.6; -70.1), which was significantly greater than the effect by any other medication (p<0.05). SJW powder tablets 2.7 g/day (HYF 12 mg/day) resulted in a midazolam AUC0-12h decrease of 47.9% (95% CI -59.7;-36.2), while 2.7 g/day SJW powder tablets that were almost devoid of HYF (0.13 mg/day) reduced midazolam AUC0-12h by only 21.1% (95% CI -33.9; -8.3). Considering all six SJW medications tested, the extent of midazolam AUC decrease correlated significantly with increasing HYF dose (r=-0.765, p<0.001), but not with hypericin dose (r=-0.067; p=0.673). CONCLUSION: The extent of induction of CYP3A varies among St. John's wort products and depends on hyperforin dose.