Characterisation of an outbreak of acute liver injury after ingestion of plant-based food supplement.

BACKGROUND: In April 2022, French Lentil and Leek Crumble (FLLC), a new frozen food preparation manufactured by Daily Harvest™ (containing Tara flour) was offered as a natural high-protein meal product. Soon thereafter, widespread anecdotal reports of acute gastrointestinal symptoms with liver injury were reported, leading to its voluntary withdrawal in June 2022, after shipment of 28,000 preparations. AIMS: To summarise the clinical and laboratory features of 17 patients with FLLC associated liver injury from the Drug Induced Liver Injury Network (DILIN). METHODS: Patients with FLLC-associated liver injury were enrolled into a prospective protocol and followed for 6 months. Cases were adjudicated by expert opinion causality assessment with summary statistics for data analysis. RESULTS: Enrolled subjects had a mean age of 41 years, 82% were female with mean BMI of 24 kg/m2. All were Caucasian without underlying liver disease. In most cases, abdominal pain and nausea arose within hours of FLLC ingestion. Mean days from ingestion to identification of liver injury was 3.1 days (±2.8). On enrolment, 53% had jaundice, 47% nausea, 24% fever, 59% abdominal pain, 41% itching and 12% rash. The mean initial serum ALT was 475 U/L (±302), AST 315 U/L (±315), alkaline phosphatase 190 U/L (±76), with a total bilirubin of 2.6 mg/dL (±2). In this study, 63% presented with a hepatocellular pattern of liver injury, 6% cholestatic and 31% mixed as determined by the R value. In addition, 24% of patients were hospitalised, and there were no fatalities or liver transplants. Liver biopsy in one subject revealed acute hepatitis with mild ductular reaction, mild lymphocytic and eosinophilic portal inflammation, mild lobular inflammation, preserved bile ducts and absence of interface hepatitis, steatosis, granulomatous reaction or cholestasis. Phylogenetic analysis confirmed the presence of Tara spinosa, the source of Tara flour. CONCLUSIONS: Natural food products are increasingly ubiquitous and may unexpectedly cause significant illness. All clinicians should inquire whether patients are consuming natural food products or herbal supplements and consider them as a potential cause of liver injury.

Phytochemical Profiles and In Vitro Immunomodulatory Activities of Extracts Obtained from Limonium gmelinii Using Different Extraction Methods.

Limonium (L.) gmelinii is a valuable pharmacopoeial Kazakhstani plant. Several studies have reported on the various biological activities of the plant. The purpose of our research was to study and compare the extraction yields, immunomodulatory activities, and chemical compositions of extracts from the above-ground parts of L. gmelinii obtained via conventional extraction (CE; Extract 1) and ultrasound-assisted extraction (UAE; Extract 2). The extracts were characterized by a considerable number of polyphenols and flavonoids: 378.1 ± 4.5 and 382.2 ± 3.3 GAE mg/g, and 90.22 ± 2.8 and 94.61 ± 1.9 QE mg/g in Extract 1 and Extract 2, respectively. Extract 2 had a slightly higher extraction yield (33.5 ± 2.4%) than Extract 1 (30.2 ± 1.6%). Liquid Chromatography-Diode-Array Detection-Electrospray Ionization-Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS) revealed the presence of 54 biologically active compounds in both extracts. It was shown that the studied extracts stimulate the secretion of TNF-α and IL-6 by intact mouse peritoneal macrophages and splenic lymphocytes, whilst they have an inhibitory effect on the secretion of these cytokines by activated immune cells. Both extracts demonstrated similar patterns of stimulation and inhibition in a splenocyte proliferation assay. Altogether, the L. gmelinii extracts obtained via CE and UAE might be suggested as effective immunomodulatory agents. The application of UAE for this purpose seems to be more efficient with a view of obtaining of a highly potent extract in a much shorter time.

Phytochemical profiling of three Amaranthus species using LC-MS/MS metabolomic approach and chemometric tools.

Several Amaranthus vegetables (Amaranthaceae) have been recognized as valuable sources of minerals, vitamins, proteins, and phytonutrients, with health-promoting characteristics. In this study, three edible Amaranthus species, namely A. hybridus (AH), A. blitum (AB), and A. caudatus (AC), were chemically characterized using non-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. Further, multivariate chemometric analyses were conducted, including principal component analysis (PCA) and correlation-covariance plot (C-C plot). As a result, forty-one diverse compounds were identified, which varied in distribution and abundance across the investigated species. Amino acids and flavonoid glycosides were the most prevalent metabolites. Other identified compounds comprised nucleoside, chlorogenic acids, hydroxy cinnamoyl amides, and triterpenoid saponins. The most discriminant metabolites were flavonoid glycosides and hydroxy cinnamoyl amides, giving each species a chemotaxonomic identity. Advancing the chemotaxonomy of Amaranthaceae, adenosine nucleoside and N-coumaroyl-ʟ-tryptophan were first reported from this family. Isorhamnetin and tricin glycosides were uniquely identified in AC, offering useful chemotaxonomic markers for this species. Notably, AB and AH profiles shared most metabolites, yet with varying abundance. These include adenosine, nicotiflorin, dicaffeoylquinic acids, and N-trans-feruloyl-4-O-methyldopamine. However, N-coumaroyl-ʟ-tryptophan and kaempferol dirhamnoside were exclusively found in AB, separating it from AH. In conclusion, the applied analytical techniques established molecular fingerprints for the included species, identified specific biomarkers, and investigated their interconnections.

Evaluation of the Herb-Drug Interaction (HDI) Potential of Zingiber officinale and Its Major Phytoconstituents.

Ginger is currently one of the most popular herbs commonly added to diverse foods, beverages, and dietary supplements. We evaluated the ability of a well-characterized ginger extract, and several of its phytoconstituents, to activate select nuclear receptors as well as modulate the activity of various cytochrome P450s and ATP-binding cassette (ABC) transporters because phytochemical-mediated modulation of these proteins underlies many clinically relevant herb-drug interactions (HDI). Our results revealed ginger extract activated the aryl hydrocarbon receptor (AhR) in AhR-reporter cells and pregnane X receptor (PXR) in intestinal and hepatic cells. Among the phytochemicals investigated, (S)-6-gingerol, dehydro-6-gingerdione, and (6S,8S)-6-gingerdiol activated AhR, while 6-shogaol, 6-paradol, and dehydro-6-gingerdione activated PXR. Enzyme assays showed that ginger extract and its phytochemicals dramatically inhibited the catalytic activity of CYP3A4, 2C9, 1A2, and 2B6, and efflux transport capabilities of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). Dissolution studies with ginger extract conducted in biorelevant simulated intestinal fluid yielded (S)-6-gingerol and 6-shogaol concentrations that could conceivably exceed cytochrome P450 (CYP) IC50 values when consumed in recommended doses. In summary, overconsumption of ginger may disturb the normal homeostasis of CYPs and ABC transporters, which in turn, may elevate the risk for HDIs when consumed concomitantly with conventional medications.

Elderberry Extracts: Characterization of the Polyphenolic Chemical Composition, Quality Consistency, Safety, Adulteration, and Attenuation of Oxidative Stress- and Inflammation-Induced Health Disorders.

Elderberry is highly reputed for its health-improving effects. Multiple pieces of evidence indicate that the consumption of berries is linked to enhancing human health and preventing or delaying the onset of chronic medical conditions. Compared with other fruit, elderberry is a very rich source of anthocyanins (approximately 80% of the polyphenol content). These polyphenols are the principals that essentially contribute to the high antioxidant and anti-inflammatory capacities and the health benefits of elderberry fruit extract. These health effects include attenuation of cardiovascular, neurodegenerative, and inflammatory disorders, as well as anti-diabetic, anticancer, antiviral, and immuno-stimulatory effects. Sales of elderberry supplements skyrocketed to $320 million over the year 2020, according to an American Botanical Council (ABC) report, which is attributable to the purported immune-enhancing effects of elderberry. In the current review, the chemical composition of the polyphenolic content of the European elderberry (Sambucus nigra) and the American elderberry (Sambucus canadensis), as well as the analytical techniques employed to analyze, characterize, and ascertain the chemical consistency will be addressed. Further, the factors that influence the consistency of the polyphenolic chemical composition, and hence, the consistency of the health benefits of elderberry extracts will be presented. Additionally, adulteration and safety as factors contributing to consistency will be covered. The role of elderberry in enhancing human health alone with the pharmacological basis, the cellular pathways, and the molecular mechanisms underlying the observed health benefits of elderberry fruit extracts will be also reviewed.

Bioactive fraction from Plumeria obtusa L. attenuates LPS-induced acute lung injury in mice and inflammation in RAW 264.7 macrophages: LC/QToF-MS and molecular docking.

In this study, the anti-inflammatory effects of the methanolic extract (TE) of Plumeria obtusa L. (aerial parts) and its fractions were evaluated in vitro, and active fraction was evaluated in vivo. Among tested extracts, dichloromethane fraction (DCM-F) exhibited the strongest inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) in RAW 264.7 macrophages. The effect of DCM-F on LPS-induced acute lung injury (ALI) in mice was studied. The animals were divided into five groups (n = 7) randomly; Gp I: negative control, GP II: positive control (LPS group), GP III: standard (dexamethasone, 2 mg/kg b.wt), GP IV and V: DCM-F (100 mg/kg), and DEM-F (200 mg/kg), respectively. DCM-F at a dose of 200 mg/kg suppressed the ability of LPS to increase the levels of nitric oxide synthase (iNOS), NO, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), as measured by ELISA. In addition, the expression of cyclooxygenase-2 (COX-2) was reduced (determined by immunohistochemistry) and the level of malondialdehyde (MDA) was decreased while that of catalase was restored to the normal values. Furthermore, the histopathological scores of inflammation induced by LPS were reduced. Twenty-two compounds were tentatively identified in DCM-F using LC/ESI-QToF with iridoids, phenolic derivatives and flavonoids as major constituents. Identified compounds were subjected to two different molecular docking processes against iNOS and prostaglandin E synthase-1 target receptors. Notably, protoplumericin A and 13-O-coumaroyl plumeride were the most promising members compared to the co-crystallized inhibitor in each case. These findings suggested that DCM-F attenuates the LPS-induced ALI in experimental animals through its anti-inflammatory and antioxidant potential.

Cytotoxic and chemomodulatory effects of Phyllanthus niruri in MCF-7 and MCF-7ADR breast cancer cells.

The members of the genus Phyllanthus have long been used in the treatment of a broad spectrum of diseases. They exhibited antiproliferative activity against various human cancer cell lines. Breast cancer is the most diagnosed cancer and a leading cause of cancer death among women. Doxorubicin (DOX) is an anticancer agent used to treat breast cancer despite its significant cardiotoxicity along with resistance development. Therefore, this study was designed to assess the potential cytotoxicity of P. niruri extracts (and fractions) alone and in combination with DOX against naïve (MCF-7) and doxorubicin-resistant breast cancer cell lines (MCF-7ADR). The methylene chloride fraction (CH2Cl2) showed the most cytotoxic activity among all tested fractions. Interestingly, the CH2Cl2-fraction was more cytotoxic against MCF-7ADR than MCF-7 at 100 µg/mL. At sub-cytotoxic concentrations, this fraction enhanced the cytotoxic effect of DOX against the both cell lines under investigation (IC50 values of 0.054 µg/mL and 0.14 µg/mL vs. 0.2 µg/mL for DOX alone against MCF-7) and (1.2 µg/mL and 0.23 µg/mL vs. 9.9 µg/mL for DOX alone against MCF-7ADR), respectively. Further, TLC fractionation showed that B2 subfraction in equitoxic combination with DOX exerted a powerful synergism (IC50 values of 0.03 µg/mL vs. 9.9 µg/mL for DOX alone) within MCF-7ADR. Untargeted metabolite profiling of the crude methanolic extract (MeOH) and CH2Cl2 fraction exhibiting potential cytotoxicity was conducted using liquid chromatography diode array detector-quadrupole time-of-flight mass spectrometry (LC-DAD-QTOF). Further studies are needed to separate the active compounds from the CH2Cl2 fraction and elucidate their mechanism(s) of action.

Breast cancer cells that preferentially metastasize to lung or bone are more glycolytic, synthesize serine at greater rates, and consume less ATP and NADPH than parent MDA-MB-231 cells.

Gene expression signatures associated with breast cancer metastases suggest that metabolic re-wiring is important for metastatic growth in lungs, bones, and other organs. However, since pathway fluxes depend on additional factors such as ATP demand, allosteric effects, and post-translational modification, flux analysis is necessary to conclusively establish phenotypes. In this study, the metabolic phenotypes of breast cancer cell lines with low (T47D) or high (MDA-MB-231) metastatic potential, as well as lung (LM)- and bone (BoM)-homing lines derived from MDA-MB-231 cells, were assessed by 13C metabolite labeling from [1,2-13C] glucose or [5-13C] glutamine and the rates of nutrient and oxygen consumption and lactate production. MDA-MB-231 and T47D cells produced 55 and 63%, respectively, of ATP from oxidative phosphorylation, whereas LM and BoM cells were more glycolytic, deriving only 20-25% of their ATP from mitochondria. ATP demand by BoM and LM cells was approximately half the rate of the parent cells. Of the anabolic fluxes assessed, nucleotide synthesis was the major ATP consumer for all cell lines. Glycolytic NADH production by LM cells exceeded the rate at which it could be oxidized by mitochondria, suggesting that the malate-aspartate shuttle was not involved in re-oxidation of these reducing equivalents. Serine synthesis was undetectable in MDA-MB-231 cells, whereas 3-5% of glucose was shunted to serine by LM and BoM lines. Proliferation rates of T47D, BoM, and LM lines tightly correlated with their respiration-normalized NADPH production rates. In contrast, MDA-MB-231 cells produced NADPH and GSH at higher rates, suggesting this line is more oxidatively stressed. Approximately half to two-thirds of NADPH produced by T47D, MDA-MB-231, and BoM cells was from the oxidative PPP, whereas the majority in LM cells was from the folate cycle. All four cell lines used the non-oxidative PPP to produce pentose phosphates, although this was most prominent for LM cells. Taken together, the metabolic phenotypes of LM and BoM lines differed from the parent line and from each other, supporting the metabolic re-wiring hypothesis as a feature of metastasis to lung and bone.

Advances in the Chemistry, Analysis and Adulteration of Anthocyanin Rich-Berries and Fruits: 2000-2022.

Anthocyanins are reported to exhibit a wide variety of remedial qualities against many human disorders, including antioxidative stress, anti-inflammatory activity, amelioration of cardiovascular diseases, improvement of cognitive decline, and are touted to protect against neurodegenerative disorders. Anthocyanins are water soluble naturally occurring polyphenols containing sugar moiety and are found abundantly in colored fruits/berries. Various chromatographic (HPLC/HPTLC) and spectroscopic (IR, NMR) techniques as standalone or in hyphenated forms such as LC-MS/LC-NMR are routinely used to gauge the chemical composition and ensure the overall quality of anthocyanins in berries, fruits, and finished products. The major emphasis of the current review is to compile and disseminate various analytical methodologies on characterization, quantification, and chemical profiling of the whole array of anthocyanins in berries, and fruits within the last two decades. In addition, the factors affecting the stability of anthocyanins, including pH, light exposure, solvents, metal ions, and the presence of other substances, such as enzymes and proteins, were addressed. Several sources of anthocyanins, including berries and fruit with their botanical identity and respective yields of anthocyanins, were covered. In addition to chemical characterization, economically motivated adulteration of anthocyanin-rich fruits and berries due to increasing consumer demand will also be the subject of discussion. Finally, the health benefits and the medicinal utilities of anthocyanins were briefly discussed. A literature search was performed using electronic databases from PubMed, Science Direct, SciFinder, and Google Scholar, and the search was conducted covering the period from January 2000 to November 2022.

Synthesis and Inhibitory Activity of Machaeridiol-Based Novel Anti-MRSA and Anti-VRE Compounds and Their Profiling for Cancer-Related Signaling Pathways.

Three unique 5,6-seco-hexahydrodibenzopyrans (seco-HHDBP) machaeridiols A−C, reported previously from Machaerium Pers., have displayed potent activities against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium, and E. faecalis (VRE). In order to enrich the pipeline of natural product-derived antimicrobial compounds, a series of novel machaeridiol-based analogs (1−17) were prepared by coupling stemofuran, pinosylvin, and resveratrol legends with monoterpene units R-(−)-α-phellandrene, (−)-p-mentha-2,8-diene-1-ol, and geraniol, and their inhibitory activities were profiled against MRSA ATCC 1708, VRE ATCC 700221, and cancer signaling pathways. Compounds 5 and 11 showed strong in vitro activities with MIC values of 2.5 µg/mL and 1.25 µg/mL against MRSA, respectively, and 2.50 µg/mL against VRE, while geranyl analog 14 was found to be moderately active (MIC 5 µg/mL). The reduction of the double bonds of the monoterpene unit of compound 5 resulted in 17, which had the same antibacterial potency (MIC 1.25 µg/mL and 2.50 µg/mL) as its parent, 5. Furthermore, a combination study between seco-HHDBP 17 and HHDBP machaeriol C displayed a synergistic effect with a fractional inhibitory concentrations (FIC) value of 0.5 against MRSA, showing a four-fold decrease in the MIC values of both 17 and machaeriol C, while no such effect was observed between vancomycin and 17. Compounds 11 and 17 were further tested in vivo against nosocomial MRSA at a single intranasal dose of 30 mg/kg in a murine model, and both compounds were not efficacious under these conditions. Finally, compounds 1−17 were profiled against a panel of luciferase genes that assessed the activity of complex cancer-related signaling pathways (i.e., transcription factors) using T98G glioblastoma multiforme cells. Among the compounds tested, the geranyl-substituted analog 14 exhibited strong inhibition against several signaling pathways, notably Smad, Myc, and Notch, with IC50 values of 2.17 µM, 1.86 µM, and 2.15 µM, respectively. In contrast, the anti-MRSA actives 5 and 17 were found to be inactive (IC50 > 20 µM) across the panel of these cancer-signaling pathways.

Computational Tools to Expedite the Identification of Potential PXR Modulators in Complex Natural Product Mixtures: A Case Study with Five Closely Related Licorice Species.

The genus Glycyrrhiza, comprising approximately 36 spp., possesses complex structural diversity and is documented to possess a wide spectrum of biological activities. Understanding and finding the mechanisms of efficacy or safety for a plant-based therapy is very challenging, yet it is crucial and necessary to understand the polypharmacology of traditional medicines. Licorice extract was shown to modulate the xenobiotic receptors, which might manifest as a potential route for natural product-induced drug interactions. However, different mechanisms could be involved in this phenomenon. Since the induced herb-drug interaction of licorice supplements via Pregnane X receptor (PXR) is understudied, we ventured out to analyze the potential modulators of PXR in complex mixtures such as whole extracts by applying computational mining tools. A total of 518 structures from five species of Glycyrrhiza: 183 (G. glabra), 180 (G. uralensis), 100 (G. inflata), 33 (G. echinata), and 22 (G. lepidota) were collected and post-processed to yield 387 unique compounds. Visual inspection of top candidates with favorable ligand-PXR interactions and the highest docking scores were identified. The in vitro testing revealed that glabridin (GG-14) is the most potent PXR activator among the tested compounds, followed by licoisoflavone A, licoisoflavanone, and glycycoumarin. A 200 ns molecular dynamics study with glabridin confirmed the stability of the glabridin-PXR complex, highlighting the importance of computational methods for rapid dereplication of potential xenobiotic modulators in a complex mixture instead of undertaking time-consuming classical biological testing of all compounds in a given botanical.

Streptomyces distallicus, a Potential Microbial Biolarvicide.

Infected mosquitos from the genus Aedes have become one of the world's most influential contributors to human morbidity and death. To explore new biopesticides with activity against Aedes aegypti, Streptomyces distallicus, a species related to the subspecies group of Streptomyces netropsis, was investigated. Six metabolites, aureothin, allo-aureothin, deoxyaureothin, 4',7-dihydroxy isoflavone, 2-methyl-5-(3-indolyl)oxazole, and 2-ethyl-5-(3-indolyl)oxazole were isolated, and chemical structures, were elucidated based on one- and two-dimensional NMR spectroscopy analyses and HRMS. The A. aegypti larvicidal activity of these compounds was evaluated. Only two isomeric compounds, aureothin and allo-aureothin, showed larvicidal activity against A. aegypti with LC50 values of 1.5 and 3.1 ppm for 24 h post-treatment, respectively, and 3.8 and 7.4 ppm for 48 h post-treatment, respectively. The crude extract of S. distallicus also demonstrated potent larvicidal activity with LC50 values of 1.46 and 1.2 ppm for 24 and 48 h post-treatment, respectively. Deoxyaureothin, a furan ring reduced form of aureothin, showed no activity against A. aegypti. The hybrid imported fire ants activity of aureothin was also evaluated, but it did not show any activity at the highest dose of 62.5 µg/g. Described here is the first report on a bioassay-directed investigation of the secondary metabolites of S. distallicus and biological evaluation of isolated compounds aureothin and its isomer and intermediates as potential microbial larvicides. S. distallicus and crude extracts thereof are a promising source of potential microbial biolarvicides.

Comparative analysis of five Salvia species using LC-DAD-QToF.

Several Salvia species, commonly known as sage plants, are an integral part of various culinary and folklore preparations for the perceived wide range of effects from organoleptic to psychological. As a result, many of these species are an integral part of botanical drug applications, highlighting the need for accurate identification and quality control for consumer's safety. Five closely related Salvia species (S. officinalis, S. miltiorrhiza, S. divinorum, S. mellifera, and S. apiana) within a same botanical family were analyzed and differentiated using LC-QToF. Accurate mass measurement (<5 ppm) of protonated and deprotonated molecules together with resulting fragments and product ions allowed unequivocal or tentative identification of more than 180 compounds either by comparison with reference standards or literature data. The leaf part were identified based on various phenolic acids, flavonoids as well as di- and tri-terpenoids. Polyphenolics, viz., salvianolic A/B and rosmarinic acids in S. officinalis, lipophilic diterpenoids, viz., tanshinones in S. miltiorrhiza, abietatriene diterpenes and triterpenoids (ursane-/olean-type) in S. mellifera, and S. apiana were identified as characteristic, significant components. In comparison, salvinorins and divinorins representing a class of neoclerodane diterpenoids were detected only in S. divinorum. The presented methodology can successfully be applied to qualitatively assess sage-based ingredients in various finished products and formulations.

Plant Extract of Limonium gmelinii Attenuates Oxidative Responses in Neurons, Astrocytes, and Cerebral Endothelial Cells In Vitro and Improves Motor Functions of Rats after Middle Cerebral Artery Occlusion.

There are numerous publications demonstrating that plant polyphenols can reduce oxidative stress and inflammatory processes in the brain. In the present study we have investigated the neuroprotective effect of plant extract isolated from the roots of L. gmelinii since it contains a rich source of polyphenols and other biologically active compounds. We have applied an oxidative and inflammatory model induced by NMDA, H2O2, and TNF-α in human primary neurons and astrocytes, and mouse cerebral endothelial cell (CECs) line in vitro. The levels of ROS generation, NADPH oxidase activation, P-selectin expression, and activity of ERK1/2 were evaluated by quantitative immunofluorescence analysis, confocal microscopy, and MAPK assay. In vivo, sensorimotor functions in rats with middle cerebral artery occlusion (MCAO) were assessed. In neurons NMDA induced overproduction of ROS, in astrocytes TNF-α initiated ROS generation, NADPH oxidase activation, and phosphorylation of ERK1/2. In CECs, the exposure by TNF-α induced oxidative stress and triggered the accumulation of P-selectin on the surface of the cells. In turn, pre-treatment of the cells with the extract of L. gmelinii suppressed oxidative stress in all cell types and pro-inflammatory responses in astrocytes and CECs. In vivo, the treatment with L. gmelinii extract improved motor activity in rats with MCAO.

Quantitative determination and characterization of polyphenols from Cissus quadrangularis L. and dietary supplements using UHPLC-PDA-MS, LC-QToF and HPTLC.

Stem and leaf of Cissus quadrangularis L. (Vitaceae), indigenous to Asia and Africa, were used for medicinal and dietary purposes with limited information about the plant's phytochemistry. Stem and leaf samples were assessed for the simultaneous determination of polyphenolic compounds (catechin, epicatechin, quercetin-3-O-ß-glucopyranoside, kaempferol-3-O-ß-glucoside, quercetin-3-O-ß-rhamnoside, leachianol F, amurensin A, pallidol, resveratrol, and quadrangularin A), using UHPLC-PDA-MS. The validation data showed that the method is precise, specific, accurate, and linear over the range of 0.5-100 µg/mL. Reversed-phase ultra-high performance liquid chromatography (UHPLC) fingerprints of the crude methanolic stem and leaf extracts of C. quadrangularis were obtained at different wavelengths based on their λmax. Polyphenolics were characterized using both UHPLC-PDA-MS and LC-QToF analysis. From liquid chromatography quadrupole time of flight-electrospray ionization mass spectrometry (LC-QToF) spectra, over 40 components were structurally correlated, and confirmation was based on the fragmentation characteristics and also from the information available in the literature. In addition to the LC-QToF method, a simple, fast HPTLC method was developed as a visual aid for the rapid qualitative analytical tool to help establish the quality assessment of botanical raw materials and dietary supplements claiming to contain Cissus.

Modulation of CYP3A4 and CYP2C9 activity by Bulbine natalensis and its constituents: An assessment of HDI risk of B. natalensis containing supplements.

BACKGROUND: Bulbine natalensis is an African-folk medicinal plant used as a dietary supplement for enhancing sexual function and muscle strength in males by presumably boosting testosterone levels, but no scientific information is available about the possible herb-drug interaction (HDI) risk when bulbine-containing supplements are concomitantly taken with prescription drugs. PURPOSE: This study was aimed to investigate the HDI potential of B. natalensis in terms of the pregnane X receptor (PXR)-mediated induction of major drug-metabolizing cytochrome P450 enzyme isoforms (i.e., CYP3A4 and CYP2C9) as well as inhibition of their catalytic activity. RESULTS: We found that a methanolic extract of B. natalensis activated PXR (EC50 6.2 ± 0.6 µg/ml) in HepG2 cells resulting in increased mRNA expression of CYP3A4 (2.40 ± 0.01 fold) and CYP2C9 (3.37 ± 0.3 fold) at 30 µg/ml which was reflected in increased activites of the two enzymes. Among the constituents of B. natalensis, knipholone was the most potent PXR activator (EC50 0.3 ± 0.1 µM) followed by bulbine-knipholone (EC50 2.0 ± 0.5 µM), and 6'-methylknipholone (EC50 4.0 ± 0.5 µM). Knipholone was also the most effective in increasing the expression of CYP3A4 (8.47 ± 2.5 fold) and CYP2C9 (2.64 ± 0.3 fold) at 10 µM. Docking studies further confirmed the unique structural features associated with knipholones for their superior inductive potentials in the activation of PXR compared to other anthraquinones. In a CYP inhibition assay, the methanolic extract as well as the anthraquinones strongly inhibited the catalytic activity of CYP2C9 while, inhibition of CYP3A4 was weak. CONCLUSIONS: These results suggest that consumption of B. natalensis may pose a potential risk for HDI if taken with conventional medications that are substrates of CYP3A4 and CYP2C9 and may contribute to unanticipated adverse reactions or therapeutic failures. Further studies are warranted to validate these findings and establish their clinical relevancy.

Ashwagandha-induced liver injury: A case series from Iceland and the US Drug-Induced Liver Injury Network.

BACKGROUND & AIMS: Ashwagandha (Withania somnifera) is widely used in Indian Ayurvedic medicine. Several dietary supplements containing ashwagandha are marketed in the US and Europe, but only one case of drug-induced liver injury (DILI) due to ashwagandha has been published. The aim of this case series was to describe the clinical phenotype of suspected ashwagandha-induced liver injury. METHODS: Five cases of liver injury attributed to ashwagandha-containing supplements were identified; three were collected in Iceland during 2017-2018 and two from the Drug-Induced Liver Injury Network (DILIN) in 2016. Other causes for liver injury were excluded. Causality was assessed using the DILIN structured expert opinion causality approach. RESULTS: Among the five patients, three were males; mean age was 43 years (range 21-62). All patients developed jaundice and symptoms such as nausea, lethargy, pruritus and abdominal discomfort after a latency of 2-12 weeks. Liver injury was cholestatic or mixed (R ratios 1.4-3.3). Pruritus and hyperbilirubinaemia were prolonged (5-20 weeks). No patient developed hepatic failure. Liver tests normalized within 1-5 months in four patients. One patient was lost to follow-up. One biopsy was performed, showing acute cholestatic hepatitis. Chemical analysis confirmed ashwagandha in available supplements; no other toxic compounds were identified. No patient was taking potentially hepatotoxic prescription medications, although four were consuming additional supplements, and in one case, rhodiola was a possible causative agent along with ashwagandha. CONCLUSIONS: These cases illustrate the hepatotoxic potential of ashwagandha. Liver injury is typically cholestatic or mixed with severe jaundice and pruritus, but self-limited with liver tests normalizing in 1-5 months.

Severe and protracted cholestasis in 44 young men taking bodybuilding supplements: assessment of genetic, clinical and chemical risk factors.

BACKGROUND: Bodybuilding supplements can cause a profound cholestatic syndrome. AIM: To describe the drug-Induced liver injury network's experience with liver injury due to bodybuilding supplements. METHODS: Liver injury pattern, severity and outcomes, potential genetic associations, and exposure to anabolic steroids by product analysis were analysed in prospectively enrolled subjects with bodybuilding supplement-induced liver injury with causality scores of probable or higher. RESULTS: Forty-four males (mean age 33 years) developed liver injury with a median latency of 73 days. Forty-one per cent presented with hepatocellular pattern of liver injury as defined by the R > 5 ([Fold elevation of ALT] ÷ [Fold elevation of Alk Phos] (mean, range = 6.4, 0.5-31.4, n = 42) despite all presenting with clinical features of cholestatic liver injury (100% with jaundice and 84% with pruritus). Liver biopsy (59% of subjects) demonstrated a mild hepatitis and profound cholestasis in most without bile duct injury, loss or fibrosis. Seventy-one per cent were hospitalised, and none died or required liver transplantation. In some, chemical analysis revealed anabolic steroid controlled substances not listed on the label. No enrichment of genetic variants associated with cholestatic syndromes was found, although mutations in ABCB11 (present in up to 20%) were significantly different than in ethnically matched controls. CONCLUSIONS: Patients with bodybuilding supplements liver injury uniformly presented with cholestatic injury, which slowly resolved. The ingested products often contained anabolic steroids not identified on the label, and no enrichment in genetic variants was found, indicating a need for additional studies.

Piper nigrum Oil - Determination of Selected Terpenes for Quality Evaluation.

The growing demand and commercial value of black pepper (Piper nigrum) has resulted in considerable interest in developing suitable and cost-effective methods for chemical characterization and quality evaluation purposes. In the current study, an extensive set of oil samples (n = 23) that were extracted by steam distillation from black pepper seeds was investigated to compare the chemical profiles of samples originating from nine major producing countries, as well as to identify potential chemical markers for quality evaluation. The twenty-two most abundant volatile compounds, mainly terpenes, in these oils were determined by conventional GC/MS analysis. Principal component analysis with this set of data revealed distinct clusters for samples that originated from China and Malaysia. Relatively low concentrations of sabinene (< 0.2%) and high concentrations of 3-carene (10.9 - 21.1%) were observed in these samples, respectively, compared to oil samples from other countries. The enantiomeric distributions of key terpene markers, viz., ß-pinene, sabinene, limonene, and terpinen-4-ol, were determined by chiral GC/MS analysis. Interestingly, for these four monoterpenes, levo-isomers were found to be predominant, emphasizing the highly conserved enzymatic processes occurring in P. nigrum. Moreover, consistent enantiomeric ratios ((-) isomer/(+) isomer) of 92.2 ± 3.0% for ß-pinene, 94.8 ± 2.8% for sabinene, 60.7 ± 1.1% for limonene, and 78.3 ± 1.3% for terpinen-4-ol were observed, independent of geographical location. These results demonstrate the potential of using stereospecific compositions as chiral signatures for establishing the authenticity and quality of black pepper oil.