A comparative metabolomics analysis of Açaí (Euterpe oleracea Mart.) fruit, food powder, and botanical dietary supplement extracts.

INTRODUCTION: Euterpe oleracea Mart. (açaí) is a botanical of interest to many who seek functional foods that provide antioxidant and anti-inflammatory properties. Cancer patients are increasingly taking botanical dietary supplements containing açaí to complement their conventional therapeutics, which may lead to serious adverse events. Before testing our açaí extracts in vitro for botanical-drug interactions, the goal is to chemically characterize our extracts for compounds whose biological activity in açaí is unknown. OBJECTIVE: The objective of this work was to develop a chemical fingerprinting method for untargeted characterization of açaí samples from a variety of sources, including food products and botanical dietary supplement capsules, made with multiple extraction solvents. METHODS: An optimized LC-MS method was generated for in-depth untargeted fingerprinting of chemical constituents in açaí extracts. Statistical analysis models were used to describe relationships between the açaí extracts based on molecular features found in both positive and negative mode ESI. RESULTS: In an attempt to elucidate the differences in metabolites among açaí extracts from different cultivars, we identified or tentatively identified 173 metabolites from the 16 extracts made from 6 different sources. Of these compounds, there are 138 reported in açaí for the first time. Statistical models showed similar yet distinct differences between the extracts tested based on the polarity of compounds present and the origin of the source material. CONCLUSION: A high-resolution mass spectrometry method was generated that allowed us to greatly characterize 16 complex extracts made from different sources of açaí with different extraction solvent polarities.

Bioactive Natural Products Identification Using Automation of Molecular Networking Software.

Secondary metabolites from natural sources are promising starting points for discovering and developing drug prototypes and new drugs, as many current treatments for numerous diseases are directly or indirectly related to such compounds. Recent advances in bioinformatics tools and molecular networking methods have made it possible to identify novel bioactive compounds. In this study, a workflow combining network-based methods for identifying bioactive compounds found in natural products was streamlined by innovating an automated bioinformatics software. The workflow relies on Global Natural Product Social Molecular Networking (GNPS), a web-based mass spectrometry ecosystem that aims to be an open-access knowledge base for community-wide organization and sharing of raw, processed, or annotated fragmentation mass spectrometry data. By combining computational tools including MZmine2, GNPS, and Cytoscape, the integrated dashboard quickly creates bioactive molecular networks with minimal user intervention and reduces the processing time of the original workflow by over 80%. This newly automated workflow quickens the process of discovering bioactive compounds from natural products. This study uses extracts from Psidium guajava leaves to demonstrate the application of our automated software.

United States Pharmacopeia comprehensive safety review of Styphnolobium japonicum flower and flower bud.

The dried flower and flower bud of Styphnolobium japonicum (L.) Schott (Japanese Sophora flower and Japanese Sophora flower bud, respectively) have long been used as herbal medicines in Asia. Today, they are marketed as dietary supplements in the United States for their anti-oxidative properties and as a source of flavonoids, including rutin and quercetin. This review focused on the safety of S. japonicum flower and flower bud as dietary supplement ingredients. No serious adverse events or toxicity were reported in the clinical or experimental animal studies we reviewed. Although some studies indicated that rutin or quercetin may have potential for drug interactions, none were identified for S. japonicum flower or flower bud. S. japonicum flower and flower bud are not known to have been associated with serious health risks when appropriately consumed in dietary supplements and have been admitted to the U.S. Pharmacopeial Convention monograph development process. However, pregnant and breastfeeding women should seek the advice of a healthcare professional because no data are available on their use by these special populations.

Natural Products with Potential for Nonhormonal Male Contraception.

Despite increased access to contraception over the last 60 years, unplanned pregnancies continue to contribute to economic disparities and overpopulation. Additionally, the burden of family planning falls primarily on women, as a reliable pharmaceutical male contraceptive has yet to be developed. The objective of this literature-based systematic review was to identify compounds for future study from natural sources with potential nonhormonal male contraceptive activity. After the exclusion of extracts and compounds with known hormonal mechanisms, 26 unique compounds were identified from natural species. The plant source, compound class, structure, target, mechanism of action, safety/toxicity profile, and in vitro, in vivo, and human studies for each compound were evaluated and discussed. ß-Caryophyllene, embelin, oleanolic acid, triptonide, and N-butyldeoxynojirimycin (NB-DNJ) were selected as the five most promising compounds for future study using prespecified criteria such as number of studies, efficacy and safety profile, reversibility, and previous use in humans for any indication. In order to move forward with development of a male contraceptive from a natural source, additional studies are needed to determine the predicted safety and efficacy for in vivo and human clinical trials.

Safety of Cranberry: Evaluation of Evidence of Kidney Stone Formation and Botanical Drug-Interactions.

Cranberry is a popular ingredient in dietary supplements in the U. S. and is commonly used for preventing urinary tract infections. Because of its popularity in dietary supplements, the U. S. Pharmacopeial Convention has developed quality standards for cranberry ingredients. The purpose of this review was to determine if there are safety issues that should preclude the admission of cranberry ingredients from the development of U. S. Pharmacopeial Convention quality standards. Based on the totality of the data, the U. S. Pharmacopeial Convention concluded that cranberry ingredients are not known to be associated with serious risks to human health when consumed properly in dietary supplements and therefore were admitted for standard development. Although published clinical and animal data indicated that cranberry is not associated with serious adverse effects, interactions with warfarin and kidney stone formation were identified as potential risks. Studies have reported contradictory data regarding the role of cranberry in kidney stone formation, with some reports suggesting cranberry is associated with a reduced risk of kidney stones. Interactions with warfarin were not associated with moderate intakes of cranberry juice (240 - 480 mL). Some reports suggested that the potential for warfarin interactions requires excessive intakes of cranberry juice (1 - 2 L/day) or cranberry extracts (3000 mg/day). Cases of warfarin interactions with cranberry have mostly involved patients with serious illnesses and/or individuals taking concomitant medications. Based on these findings, the U. S. Pharmacopeial Convention concluded that the use of cautionary labeling statements regarding interactions with warfarin or kidney stone formation is not necessary in the development of quality standards for cranberry ingredients.

Slow-Binding Inhibition of Mycobacterium tuberculosis Shikimate Kinase by Manzamine Alkaloids.

Tuberculosis represents a significant public health crisis. There is an urgent need for novel molecular scaffolds against this pathogen. We screened a small library of marine-derived compounds against shikimate kinase from Mycobacterium tuberculosis ( MtSK), a promising target for antitubercular drug development. Six manzamines previously shown to be active against M. tuberculosis were characterized as MtSK inhibitors: manzamine A (1), 8-hydroxymanzamine A (2), manzamine E (3), manzamine F (4), 6-deoxymanzamine X (5), and 6-cyclohexamidomanzamine A (6). All six showed mixed noncompetitive inhibition of MtSK. The lowest KI values were obtained for 6 across all MtSK-substrate complexes. Time-dependent analyses revealed two-step, slow-binding inhibition. The behavior of 1 was typical; initial formation of an enzyme-inhibitor complex (EI) obeyed an apparent KI of ∼30 µM with forward ( k5) and reverse ( k6) rate constants for isomerization to an EI* complex of 0.18 and 0.08 min-1, respectively. In contrast, 6 showed a lower KI for the initial encounter complex (∼1.5 µM), substantially faster isomerization to EI* ( k5 = 0.91 min-1), and slower back conversion of EI* to EI ( k6 = 0.04 min-1). Thus, the overall inhibition constants, KI*, for 1 and 6 were 10 and 0.06 µM, respectively. These findings were consistent with docking predictions of a favorable binding mode and a second, less tightly bound pose for 6 at MtSK. Our results suggest that manzamines, in particular 6, constitute a new scaffold from which drug candidates with novel mechanisms of action could be designed for the treatment of tuberculosis by targeting MtSK.

Trends in botanical dietary supplement use among US adults by cancer status: The National Health and Nutrition Examination Survey, 1999 to 2014.

BACKGROUND: Patients with cancer may use botanical dietary supplements (BDS) in an attempt to manage the side effects of chemotherapy, yet evidence about BDS use among patients with cancer is limited. The authors examined trends in BDS use among US adults according to cancer status and patient characteristics. METHODS: A serial, cross-sectional study was conducted using data from the National Health and Nutrition Examination Survey from 1999 through 2014 (n = 43,644). Self-reported cancer diagnosis history and any BDS use in the preceding 30 days were determined. The prevalence of BDS use was calculated in each cycle for respondents with and without cancer, both overall and by patient characteristics. Simple linear regression models were applied to test for trends in BDS use at a 2-sided P value < .05. Multiple logistic regression models were performed to identify the patient factors associated with BDS use. The results were weighted to represent national estimates. RESULTS: The prevalence of BDS use was greater among participants who had cancer compared with participants who did not have cancer, but trends remained stable during 1999 through 2014 for both groups. Trends in BDS use declined in patients with cancer who were older (Ptrend = .047), had a low annual family income (Ptrend = .028), and had a lower education level (Ptrend = .004). Among the respondents without cancer, trends in BDS use declined in those who were middle-aged (Ptrend = .025), non-Hispanic whites (Ptrend = .025), those with a lower education level (Ptrend = .011), and those who were not receiving prescription medication (Ptrend = .036). Patient age, sex, race/ethnicity, income, education, and health conditions were associated with BDS use. CONCLUSIONS: The overall use of BDS remained stable during 1999 through 2014 for US adults with and without cancer, but it varied by individual characteristics. Cancer 2018;124:1207-15. © 2017 American Cancer Society.

Mechanism of irreversible inhibition of Mycobacterium tuberculosis shikimate kinase by ilimaquinone.

Ilimaquinone (IQ), a marine sponge metabolite, has been considered as a potential therapeutic agent for various diseases due to its broad range of biological activities. We show that IQ irreversibly inactivates Mycobacterium tuberculosis shikimate kinase (MtSK) through covalent modification of the protein. Inactivation occurred with an apparent second-order rate constant of about 60 M-1 s-1. Following reaction with IQ, LC-MS analyses of intact MtSK revealed covalent modification of MtSK by IQ, with the concomitant loss of a methoxy group, suggesting a Michael-addition mechanism. Evaluation of tryptic fragments of IQ-derivatized MtSK by MS/MS demonstrated that Ser and Thr residues were most frequently modified with lesser involvement of Lys and Tyr. In or near the MtSK active site, three residues of the P-loop (K15, S16, and T17) as well as S77, T111, and S44 showed evidence of IQ-dependent derivatization. Accordingly, inclusion of ATP in IQ reactions with MtSK partially protected the enzyme from inactivation and limited IQ-based derivatization of K15 and S16. Additionally, molecular docking models for MtSK-IQ were generated for IQ-derivatized S77 and T111. In the latter, ATP was observed to sterically clash with the IQ moiety. Out of three other enzymes evaluated, lactate dehydrogenase was derivatized and inactivated by IQ, but pyruvate kinase and catalase-peroxidase (KatG) were unaffected. Together, these data suggest that IQ is promiscuous (though not entirely indiscriminant) in its reactivity. As such, the potential of IQ as a lead in the development of antitubercular agents directed against MtSK or other targets is questionable.

A multifaceted approach to identify non-specific enzyme inhibition: Application to Mycobacterium tuberculosis shikimate kinase.

Single dose high-throughput screening (HTS) followed by dose-response evaluations is a common strategy for the identification of initial hits for further development. Early identification and exclusion of false positives is a cost-saving and essential step in early drug discovery. One of the mechanisms of false positive compounds is the formation of aggregates in assays. This study evaluates the mechanism(s) of inhibition of a set of 14 compounds identified previously as actives in Mycobacterium tuberculosis (Mt) cell culture screening and in vitro actives in Mt shikimate kinase (MtSK) assay. Aggregation of hit compounds was characterized using multiple experimental methods, LC-MS, 1HNMR, dynamic light scattering (DLS), transmission electron microscopy (TEM), and visual inspection after centrifugation for orthogonal confirmation. Our results suggest that the investigated compounds containing oxadiazole-amide and aminobenzothiazole moieties are false positive hits and non-specific inhibitors of MtSK through aggregate formation.

A Review of the Toxicity of Compounds Found in Herbal Dietary Supplements.

Use of herbal dietary supplements by the public is common and has been happening for centuries. In the United States, the Food and Drug Administration has a limited scope of regulation over marketed herbal dietary supplements, which may contain toxic botanical compounds that pose a public health risk. While the Food and Drug Administration has made efforts to prohibit the sale of unsafe herbal dietary supplements, numerous reports have proliferated of adverse events due to these supplements. This literature review investigates bioactive plant compounds commonly used in herbal dietary supplements and their relative toxicities. Using primarily the National Library of Medicine journal database and SciFinder for current reports, 47 toxic compounds in 55 species from 46 plant families were found to demonstrate harmful effects due to hepatic, cardiovascular, central nervous system, and digestive system toxicity. This review further contributes a novel and comprehensive view of toxicity across the botanical dietary market, and investigates the toxicity of the top ten botanical dietary supplements purchased in the United States of America to gauge the exposure risk of toxicity to the public. The criteria of measuring toxicity in this review (plant compound, family, quantity, and toxicity effects) across the entire market in the United States, with special attention to those supplements whose exposure to the consumer is maximal, provides a unique contribution to the investigation of botanical supplements.

Therapeutic Perspectives on Chia Seed and Its Oil: A Review.

The attraction of novel foods proceeds alongside epidemic cardiovascular disease, diabetes, obesity, and related risk factors. Dieticians have identified chia (Salvia hispanica) as a product with a catalog of potential health benefits relating to these detriments. Chia is currently consumed not only as seeds, but also as oil, which brings about similar effects. Chia seeds and chia seed oil are used mainly as a food commodity and the oil is also used popularly as a dietary ingredient used in various dietary supplements available in the U. S. market. Chia seed is rich in α-linolenic acid, the biological precursor to eicosapentaenoic acid, a polyunsaturated fatty acid, and docosahexaenoic acid. Because the body cannot synthesize α-linolenic acid, chia has a newfound and instrumental role in diet. However, the inconclusive nature of the scientific community's understanding of its safety warrants further research and appropriate testing. The focus of this work is to summarize dietary health benefits of S. hispanica seed and oil to acknowledge concerns of adverse events from its ingestion, to assess current research in the field, and to highlight the importance of quality compendial standards to support safe use. To achieve this end, a large-scale literature search was partaken on the two well-known databases, PubMed and SciFinder. Hundreds of articles detailing such benefits as decreased blood glucose, decreased waist circumference and weight in overweight adults, and improvements in pruritic skin and endurance in distance runners have been recorded. These benefits must be considered within the appropriate circumstances.

Identification and quantification of thymol metabolites in plasma, liver and duodenal wall of broiler chickens using UHPLC-ESI-QTOF-MS.

In the present study, we aimed to develop a method for thymol sulfate and thymol glucuronide determination in plasma, liver and duodenal wall of broiler chickens after feeding with a Thymus vulgaris essential oil at the different concentrations (0.01, 0.05 and 0.1% w/w). UHPLC coupled with accurate-mass QTOF-MS was used for identification and quantification of thymol metabolites. Novel Waters Oasis Prime HLB solid-phase extraction cartridges were applied to sample clean-up with extraction recoveries ranged from 85 to 92%. The presence of thymol glucuronide was confirmed by MS software according to molecular formula, score, mass error and double bond equivalent. In terms of validation, calibration curves of thymol sulfate were constructed in matrix samples with linearity from 3.91 to 250.0 ng/mL and correlation coefficients were within the range of 0.9979-0.9995. Limits of detection were 0.97, 0.29 and 0.63 ng/mL and limits of quantification were 3.23, 0.97 and 2.09 ng/mL for plasma, liver and duodenal wall, respectively. Intra-day and inter-day precision expressed as relative standard deviation were <4.35%. To highlight, thymol metabolites were directly detected for the first time in liver and duodenal wall and this method was shown to be successfully applicable for investigation of thymol metabolism in chickens after thyme essential oil ingestion.

Chemical Adulterants in Herbal Medicinal Products: A Review.

Many herbal medicinal products have been found to contain synthetic prescription drugs as chemical adulterants. This has become evident by the number of toxicity cases and adverse reactions reported in which casualties were reported via analytical techniques that detected the presence of chemical adulterants in them, which could be responsible for their toxicity. The adulteration of herbal medicinal products with synthetic drugs continues to be a serious problem for regulatory agencies. This review provides up to date information on cases of toxicity, major chemical adulterants in herbal medicinal products, and current analytical techniques used for their detection.

LC-MS-Based Quality Assessment of a Traditional Chinese Medicine YANG XIN Formulation.

YANG XIN is a traditional Chinese medicine formulation used for nervous fatigue and consists of a proprietary blend of concentrated extracts from 18 plant ingredients. The 18 constituent plant ingredients, YANG XIN capsules, and formulations 2014-005_1 A and 1B were extracted by consecutive 24-hour macerations with dichloromethane followed by methanol. Metabolite separation was carried out through LC-MS in 40 minutes. Data acquisitions for qualitative and quantitative analyses of the samples were collected under (±) ESI modes and (+) APCI mode using full spectrum scan analysis.A total of 18 analytical markers were identified by LC-MS for YANG XIN formulations based on accurate mass measurements, molecular formula, double bond equivalent, MFG score, and error (ppm) of the measurement. Aditionally, a comparison of the data with previously reported results for the compounds, followed by identity confirmation with standard compounds, was performed. Seventeen analytical markers representing 17 plant ingredients in the different YANG XIN formulations were quantified for the first time. The YANG XIN capsules and the 2014-005_1B formulation were similar to each other and different from the 2014-005_1 A formulation based on the fact that both YANG XIN capsules and the 2014-005_1B formulation contain the same analytical markers. This method provides good linearity (r(2) > 0.9945), intraday precision (R. S. D. < 3.9 %), interday precision (R. S. D. < 5.6 %), accuracy (99.2-101 %), recovery (145.7 %), limit of detection (0.0011-0.0732 µg/mL), and limit of quantitation (0.0038-0.2441 µg/mL).

Development of a functional assay to detect inhibitors of Plasmodium falciparum glutathione reductase utilizing liquid chromatography-mass spectrometry.

Plasmodium falciparum (Pf) like most other organisms, has a sophisticated antioxidant system, part of which includes glutathione reductase (GR). GR works by recycling toxic glutathione disulfide to glutathione, thereby reducing reactive oxygen species and making a form of glutathione (GSH) the parasite can use. Inhibition of this enzyme in Pf impedes parasite growth. In addition, it has been confirmed that PfGR is not identical to human GR. Thus, PfGR is an excellent target for antimalarial drug development. A functional assay utilizing liquid chromatography-mass spectrometry was developed to specifically identify and evaluate inhibitors of PfGR. Using recombinant PfGR enzyme and 1,4-naphthoquinone (1) as a reference compound and 4-nitrobenzothiadiazole (2) and methylene blue (3) as additional compounds, we quantified the concentration of GSH produced compared with a control to determine the inhibitory effect of these compounds. Our results coincide with that presented in literature: compounds 1-3 inhibit PfGR with IC50 values of 2.71, 8.38, and 19.23 µm, respectively. Good precision for this assay was exhibited by low values of intraday and interday coefficient of variation (3.1 and 2.4%, respectively). Thus, this assay can be used to screen for other potential inhibitors of PfGR quickly and accurately.

Preliminary LC-MS Based Screening for Inhibitors of Plasmodium falciparum Thioredoxin Reductase (PfTrxR) among a Set of Antimalarials from the Malaria Box.

Plasmodium falciparum thioredoxin reductase (PfTrxR) has been identified as a potential drug target to combat growing antimalarial drug resistance. Medicines for Malaria Venture (MMV) has pre-screened and identified a set of 400 antimalarial compounds called the Malaria Box. From those, we have evaluated their mechanisms of action through inhibition of PfTrxR and found new active chemical scaffolds. Five compounds with significant PfTrxR inhibitory activity, with IC50 values ranging from 0.9-7.5 µM against the target enzyme, were found out of the Malaria Box.

Development of an ESI-LC-MS-based assay for kinetic evaluation of Mycobacterium tuberculosis shikimate kinase activity and inhibition.

A simple and reliable liquid chromatography-mass spectrometry (LC-MS) assay has been developed and validated for the kinetic characterization and evaluation of inhibitors of shikimate kinase from Mycobacterium tuberculosis (MtSK), a potential target for the development of novel antitubercular drugs. This assay is based on the direct determination of the reaction product shikimate-3-phosphate (S3P) using electrospray ionization (ESI) and a quadrupole time-of-flight (Q-TOF) detector. A comparative analysis of the kinetic parameters of MtSK obtained by the LC-MS assay with those obtained by a conventional UV-assay was performed. Kinetic parameters determined by LC-MS were in excellent agreement with those obtained from the UV assay, demonstrating the accuracy, and reliability of this method. The validated assay was successfully applied to the kinetic characterization of a known inhibitor of shikimate kinase; inhibition constants and mode of inhibition were accurately delineated with LC-MS.

Plasmodium falciparum Thioredoxin Reductase (PfTrxR) and Its Role as a Target for New Antimalarial Discovery.

The growing resistance to current antimalarial drugs is a major concern for global public health. The pressing need for new antimalarials has led to an increase in research focused on the Plasmodium parasites that cause human malaria. Thioredoxin reductase (TrxR), an enzyme needed to maintain redox equilibrium in Plasmodium species, is a promising target for new antimalarials. This review paper provides an overview of the structure and function of TrxR, discusses similarities and differences between the thioredoxin reductases (TrxRs) of different Plasmodium species and the human forms of the enzyme, gives an overview of modeling Plasmodium infections in animals, and suggests the role of Trx functions in antimalarial drug resistance. TrxR of Plasmodium falciparum is a central focus of this paper since it is the only Plasmodium TrxR that has been crystallized and P. falciparum is the species that causes most malaria cases. It is anticipated that the information summarized here will give insight and stimulate new directions in which research might be most beneficial.

(1) H-NMR quantification of major saccharides in açaí raw materials: a comparison of the internal standard methodology with the absolute intensity qNMR method.

INTRODUCTION: While the use of internal standard methodology for qNMR is a proven and reliable form of quantification, simplified alternative approaches are needed. Agilent's absolute intensity qNMR utility software is a valuable alternative that has not yet been subjected to validation in the peer-reviewed literature. OBJECTIVE: To provide validation of Agilent's absolute intensity qNMR method with a specific application to natural product quantification by measuring saccharide content in açaí materials. METHODS: In order to validate the method, calibration test samples of ibuprofen were prepared in DMSO-d6 at nine different concentrations and measured with (1) H-NMR. A minimum of 40 spectra were collected for each sample, and the absolute intensity utility was used for quantification. The same methodology was then applied to the açaí materials, creating triplicates for each of the materials and using 3-(trimethylsilyl)-1-propanesulphonic acid sodium salt in water-d2 as both the solvent and internal standard. (1) H-NMR spectra were collected, and the amounts of glucose, sucrose and fructose were determined using both the internal standard approach and the absolute intensity qNMR method. RESULTS: Applying the absolute intensity utility to the ibuprofen samples demonstrated a linear response (R(2) = 0.99943). For the açaí investigations, results obtained from the absolute intensity method were comparable to those obtained from the internal standard approach, with percentage differences ranging from 0.5-6.2%. CONCLUSION: This study demonstrates the accuracy, precision and reliability of Agilent's absolute intensity qNMR method. In addition, practical information is provided for assessing the saccharide contents of açaí materials.

Standardization of açaí extracts for in-vitro assays based on anthocyanin quantitation.

Euterpe oleracea Mart., commonly known as açaí, is a fruit that grows on a palm tree native to the Amazon region. Quantitation of bioactive constituents is a crucial preliminary step before utilizing extracts for biological assays so they may be normalized and administered according to a specific constituent concentration. Açaí has four main anthocyanin analytes: cyanidin 3-glucoside, cyanidin 3-sambubioside, cyanidin 3-rutinoside, and peonidin 3-rutinoside. This is the first comparison of açaí anthocyanin profiles between fresh fruits, processed powders, and botanical dietary supplement capsules. The materials examined shared a similar anthocyanin profile, with cyanidin 3-rutinoside being the most abundant (0.380 ± 0.006 - 15.1 ± 0.01 mg/g), followed by cyanidin 3-glucoside (0.0988 ± 0.0031 - 8.95 ± 0.01 mg/g). Among the botanical dietary supplement capsules, the two formulations varied greatly in anthocyanin concentration despite both being aqueous extracts (0.650 ± 0.011 - 0.924 ± 0.010 mg/g versus 1.23 ± 0.01 - 1.27 ± 0.02 mg/g). Previous LC-MS methods range from 35-120 min per injection, while we report a 10 min quantitative method for analysis of anthocyanins in various açaí materials that is fast, reproducible, and accurate. The method produced is useful to assure the quality, efficacy and safety of food and dietary supplement materials containing açaí.