Botanical Ingredient Forensics: Detection of Attempts to Deceive Commonly Used Analytical Methods for Authenticating Herbal Dietary and Food Ingredients and Supplements.

Botanical ingredients are used widely in phytomedicines, dietary/food supplements, functional foods, and cosmetics. Products containing botanical ingredients are popular among many consumers and, in the case of herbal medicines, health professionals worldwide. Government regulatory agencies have set standards (collectively referred to as current Good Manufacturing Practices, cGMPs) with which suppliers and manufacturers must comply. One of the basic requirements is the need to establish the proper identity of crude botanicals in whole, cut, or powdered form, as well as botanical extracts and essential oils. Despite the legal obligation to ensure their authenticity, published reports show that a portion of these botanical ingredients and products are adulterated. Most often, such adulteration is carried out for financial gain, where ingredients are intentionally substituted, diluted, or "fortified" with undisclosed lower-cost ingredients. While some of the adulteration is easily detected with simple laboratory assays, the adulterators frequently use sophisticated schemes to mimic the visual aspects and chemical composition of the labeled botanical ingredient in order to deceive the analytical methods that are used for authentication. This review surveys the commonly used approaches for botanical ingredient adulteration and discusses appropriate test methods for the detection of fraud based on publications by the ABC-AHP-NCNPR Botanical Adulterants Prevention Program, a large-scale international program to inform various stakeholders about ingredient and product adulteration. Botanical ingredients at risk of adulteration include, but are not limited to, the essential oils of lavender (Lavandula angustifolia, Lamiaceae), rose (Rosa damascena, Rosaceae), sandalwood (Santalum album, Santalaceae), and tea tree (Melaleuca alternifolia, Myrtaceae), plus the extracts of bilberry (Vaccinium myrtillus, Ericaceae) fruit, cranberry (Vaccinium macrocarpon, Ericaceae) fruit, elder (Sambucus nigra, Viburnaceae) berry, eleuthero (Eleutherococcus senticosus, Araliaceae) root, ginkgo (Ginkgo biloba, Ginkgoaceae) leaf, grape (Vitis vinifera, Vitaceae) seed, saw palmetto (Serenoa repens, Arecaceae) fruit, St. John's wort (Hypericum perforatum, Hypericaceae) herb, and turmeric (Curcuma longa, Zingiberaceae) root/rhizome, among numerous others.

Cinnamon - Differentiation of Four Species by Linking Classical Botany to an Automated Chromatographic Authentication System.

One of the world's oldest spices, cinnamon is also one of the most popular. Species of the genus Cinnamomum offer a variety of extractable oils with aroma and flavor characteristics of importance to the flavor industry, so differentiating cinnamon samples for culinary-based applications is very important. Cinnamon also has reported health benefits associated with specific phytochemical constituents, but its composition can vary greatly depending on species and source region. A substantial amount of the research reported on cinnamon does not provide thorough documentation of the source and taxonomic identification of the study material, a very common issue with studies of food and medicinal plants. In the interest of providing some clarity to the discussion of the health benefits and culinary attributes of the different cinnamon types in the marketplace, we offer the results of a long-term chemotaxonomic study of cinnamon samples sourced from different regions of the world and link those chemical data to classical taxonomic identification of the source plants. We provide details of the effective use of an automated chemotaxonomic analytical method to differentiate cinnamons from various geographic regions. Also included are chromatographic data for the polyphenolic/procyanidin fractions of each species, as cinnamon type-A procyanidins are often the purported source of biological activity in cinnamon and cinnamon extracts.

A matrix-focused structure-activity and binding site flexibility study of quinolinol inhibitors of botulinum neurotoxin serotype A.

Our initial discovery of 8-hydroxyquinoline inhibitors of BoNT/A and separation/testing of enantiomers of one of the more active leads indicated considerable flexibility in the binding site. We designed a limited study to investigate this flexibility and probe structure-activity relationships; utilizing the Betti reaction, a 36 compound matrix of quinolinol BoNT/A LC inhibitors was developed using three 8-hydroxyquinolines, three heteroaromatic amines, and four substituted benzaldehydes. This study has revealed some of the most effective quinolinol-based BoNT/A inhibitors to date, with 7 compounds displaying IC50 values ⩽1µM and 11 effective at ⩽2µM in an ex vivo assay.

Fungal bis-Naphthopyrones as Inhibitors of Botulinum Neurotoxin Serotype A.

An in silico screen of the NIH Molecular Library Small Molecule Repository (MLSMR) of ∼350000 compounds and confirmatory bioassays led to identification of chaetochromin A (1) as an inhibitor of botulinum neurotoxin serotype A (BoNT A). Subsequent acquisition and testing of analogues of 1 uncovered two compounds, talaroderxines A (2) and B (3), with improved activity. These are the first fungal metabolites reported to exhibit BoNT/A inhibitory activity.

Isoflavonoids and coumarins from Glycyrrhiza uralensis: antibacterial activity against oral pathogens and conversion of isoflavans into isoflavan-quinones during purification.

Phytochemical investigation of a supercritical fluid extract of Glycyrrhiza uralensis has led to the isolation of 20 known isoflavonoids and coumarins, and glycycarpan (7), a new pterocarpan. The presence of two isoflavan-quinones, licoriquinone A (8) and licoriquinone B (9), in a fraction subjected to gel filtration on Sephadex LH-20 is due to suspected metal-catalyzed oxidative degradation of licoricidin (1) and licorisoflavan A (2). The major compounds in the extract, as well as 8, were evaluated for their ability to inhibit the growth of several major oral pathogens. Compounds 1 and 2 showed the most potent antibacterial activities, causing a marked growth inhibition of the cariogenic species Streptococcus mutans and Streptococcus sobrinus at 10 µg/mL and the periodontopathogenic species Porphyromonas gingivalis (at 5 µg/mL) and Prevotella intermedia (at 5 µg/mL for 1 and 2.5 µg/mL for 2). Only 1 moderately inhibited growth of Fusobacterium nucleatum at the highest concentration tested (10 µg/mL).

Separation of Betti Reaction Product Enantiomers: Absolute Configuration and Inhibition of Botulinum Neurotoxin A.

The racemic product of the Betti reaction of 5-chloro-8-hydroxyquinoline, benzaldehyde and 2-aminopyridine was separated by chiral HPLC to determine which enantiomer inhibited botulinum neurotoxin serotype A. When the enantiomers unexpectedly proved to have comparable activity, the absolute structures of (+)-(R)-1 and (-)-(S)-1 were determined by comparison of calculated and observed circular dichroism spectra. Molecular modeling studies were undertaken in an effort to understand the observed bioactivity and revealed different ensembles of binding modes, with roughly equal binding energies, for the two enantiomers.

Chemical modification of the insecticidal briantheins X and Y.

Manipulation of the allylic chloride functionality in briantheins X and Y provided 10 new analogues of these gorgonian diterpenes as part of a continuing study of structure-activity relationships in this family of insecticidal compounds. Modified Finkelstein reaction conditions led not only to halogen substitution products but also to rearrangement, dehydrohalogenation, and dehydration products. None of the new compounds showed superior insecticidal activity to brianthein X or Y, although most did result in lower weight gains versus controls.

Cellular inhibition of checkpoint kinase 2 (Chk2) and potentiation of camptothecins and radiation by the novel Chk2 inhibitor PV1019 [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide].

Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

Cytotoxic and HIF-1alpha inhibitory compounds from Crossosoma bigelovii.

Cytotoxicity-guided fractionation of an organic solvent extract of the plant Crossosoma bigelovii led to the discovery of a new strophanthidin glycoside (1) and two new 2-methylchromone glycosides (2 and 3). Also isolated were the known chromones eugenin and noreugenin, the indole alkaloid ajmalicine, the dibenzylbutane lignan secoisolariciresinol, the dibenzylbutyrolactone lignan matairesinol, and the furanone 5-tetradec-5-enyldihydrofuran-2-one. Further investigation into the biological properties of strophanthidin glycosides revealed a connection between inhibition of HIF-1 activation and the glycosylation of the genin. This work is the first published study of the bioactive phytochemicals of the family Crossosomataceae.

Inhibitors of the NF-kappaB activation pathway from Cryptocarya rugulosa.

The nuclear factor-kappaB (NF-kappaB) signaling pathway is constitutively active in many types of cancers and is a potential therapeutic target. Using a cell-based assay for stability of inhibitor of kappa B (IkappaB), a critical regulator of NF-kappaB activity, we found that an organic solvent extract of the plant Cryptocarya rugulosa inhibited constitutive NF-kappaB activity in human lymphoma cell lines. The active components were identified as rugulactone, a new alpha-pyrone (1), and the known cryptocaryone (2). Rugulactone was the more active compound, exhibiting up to 5-fold induction of IkappaB at 25 microg/mL; maximal activity was observed with 10 h exposure of test cells to 1 or 2.

Induction of apoptosis in human cancer cells by candidaspongiolide, a novel sponge polyketide.

BACKGROUND: Candidaspongiolide (CAN), a novel polyketide from a marine sponge, is the active component of a mixture that was found to be potently cytotoxic in the National Cancer Institute's 60-cell-line screen. METHODS: Effects of CAN on U251 glioma and HCT116 colorectal cancer cells and on normal fibroblasts were assessed using radiolabeling studies to measure protein synthesis, clonogenic assays to measure cell survival, flow cytometry of annexin V- and propidium iodide-stained cells to measure apoptosis, and western blots in the presence or absence of specific inhibitors to assess accumulation and phosphorylation of potential downstream target proteins. RESULTS: CAN inhibited protein synthesis and potently induced apoptosis in both U251 and HCT116 cells, the latter in part by a caspase 12-dependent pathway. For example, 25%-30% of U251 or HCT116 cells became apoptotic after 24 hours of treatment with 100 nM CAN. CAN also rapidly induced sustained phosphorylation of eukaryotic translation initiation factor-2 (eIF2)-alpha at Ser51 and of the translation elongation factor eEF2 at Thr56, which could contribute to its dose-dependent inhibition of protein synthesis. Stable expression of dominant-negative eIF2alpha was sufficient to prevent CAN-induced eIF2alpha phosphorylation and induction of apoptosis but insufficient to prevent inhibition of protein synthesis. CAN induction of eIF2alpha phosphorylation did not occur by a classic endoplasmic reticulum stress pathway. However, an inhibitor of and small-interfering RNAs to the double-stranded RNA-dependent protein kinase PKR prevented CAN-mediated eIF2alpha phosphorylation and apoptosis, respectively. Although CAN inhibited protein synthesis in both cancer cells and normal human fibroblasts, it induced eIF2alpha phosphorylation and apoptosis only in cancer cells. CONCLUSIONS: CAN triggers PKR/eIF2alpha/caspase 12-dependent apoptosis and inhibits protein synthesis in cancer cells but only inhibits protein synthesis in normal cells.

Arylstibonic acids: novel inhibitors and activators of human topoisomerase IB.

Human topoisomerase IB (hTopo) forms a covalent phosphotyrosyl linkage with the DNA backbone, and controls genomic DNA topology by relaxing DNA supercoils during the processes of DNA replication, transcription, chromosome condensation and decondensation. The essential role of hTopo in these processes has made it a preeminent anticancer drug target. We have screened a small library of arylstibonic acids for their effects on plasmid supercoil relaxation catalyzed by hTopo. Despite the similar structures of the library compounds, some compounds were found to be effective competitive inhibitors, and others, nonessential activators. Some arylstibonic acids show selectivity in their action against hTopo and the related enzyme from poxvirus (vTopo). Structure-activity relationships and structural modeling suggest that competitive inhibition may result from positioning of the negatively charged stibonic acid and carboxylate groups of the inhibitors into DNA phosphate binding pockets on hTopo. The hTopo activators act by a surprising allosteric mechanism without interfering with DNA binding or binding of the widely used hTopo poison camptothecin. Arylstibonic acid competitive inhibitors may become useful small molecules for elucidating the cellular functions of hTopo.

Separation and SAR study of HIF-1alpha inhibitory tubulosines from Alangium cf. longiflorum.

A crude organic solvent extract of Alangium cf. longiflorum exhibited potent inhibition of hypoxia-induced HIF-1 transcriptional activity in human U251 glioma cells. Dereplication and bioactivity-guided fractionation, including Sephadex LH-20 and chiral HPLC chromatographies, led to the isolation of tubulosine ( 1), 9-desmethyltubulosine ( 2), and isotubulosine ( 3). Structures were verified by complete (1)H and (13)C assignments using 1D- and 2D-NMR techniques. Tubulosine strongly inhibited HIF-1 transcriptional activity, isotubulosine was devoid of activity, and 9-desmethyltubulosine possessed 6-fold less potency than tubulosine.

Potent inhibition of human apurinic/apyrimidinic endonuclease 1 by arylstibonic acids.

Human apurinic/apyrimidinic endonuclease (Ape1) plays an important role by processing the >10,000 highly toxic abasic sites generated in the genome of each cell every day. Ape1 has recently emerged as a target for inhibition, in that its overexpression in tumors has been linked with poor response to both radiation and chemotherapy and lower overall patient survival. Inhibition of Ape1 using siRNA or the expression of a dominant-negative form of the protein has been shown to sensitize cells to DNA-damaging agents, including various chemotherapeutic agents. However, potent small-molecule inhibitors of Ape1 remain to be found. To this end, we screened Ape1 against the NCI Diversity Set of small molecules and discovered aromatic nitroso, carboxylate, sulfonamide, and arylstibonic acid compounds with micromolar affinities for the protein. A further screen of a 37-compound arylstibonic acid sublibrary identified ligands with IC(50) values in the range of 4 to 300 nM. The negatively charged stibonic acids act by a partial-mixed mode and probably serve as DNA phosphate mimics. These compounds provide a useful scaffold for development of chemotherapeutic agents against Ape1.

Identification of a Bis-guanylhydrazone [4,4'-Diacetyldiphenylurea-bis(guanylhydrazone); NSC 109555] as a novel chemotype for inhibition of Chk2 kinase.

Chk2 is a protein kinase involved in the ATM-dependent checkpoint pathway (http://discover.nci.nih.gov/mim). This pathway is activated by genomic instability and DNA damage and results in either cell cycle arrest, to allow DNA repair to occur, or cell death (apoptosis). Chk2 is activated by ATM-mediated phosphorylation and autophosphorylation and in turn phosphorylates its downstream targets (Cdc25A, Cdc25C, BRCA1, p53, Hdmx, E2F1, PP2A, and PML). Inhibition of Chk2 has been proposed to sensitize p53-deficient cells as well as protect normal tissue after exposure to DNA-damaging agents. We have developed a drug-screening program for specific Chk2 inhibitors using a fluorescence polarization assay, immobilized metal ion affinity-based fluorescence polarization (IMAP). This assay detects the degree of phosphorylation of a fluorescently linked substrate by Chk2. From a screen of over 100,000 compounds from the NCI Developmental Therapeutics Program, we identified a bis-guanylhydrazone [4,4'-diacetyldiphenylureabis(guanylhydrazone); NSC 109555] as a lead compound. In vitro data show the specific inhibition of Chk2 kinase activity by NSC 109555 using in vitro kinase assays and kinase-profiling experiments. NSC 109555 was shown to be a competitive inhibitor of Chk2 with respect to ATP, which was supported by docking of NSC 109555 into the ATP binding pocket of the Chk2 catalytic domain. The potency of NSC 109555 was comparable with that of other known Chk2 inhibitors, such as debromohymenialdisine and 2-arylbenzimidazole. These data define a novel chemotype for the development of potent and selective inhibitors of Chk2. This class of drugs may ultimately be useful in combination with current DNA-damaging agents used in the clinic.

Candidaspongiolides, distinctive analogues of tedanolide from sponges of the genus Candidaspongia.

Fractionation of cytotoxic extracts of specimens of a newly described sponge genus, Candidaspongia, has yielded the candidaspongiolides (3), a complex mixture of acyl esters of a macrolide related to tedanolide. The general structure of the candidaspongiolides was determined by analyses of various 2D NMR and MS data sets. The acyl ester components were identified by GC-MS analysis of the derived fatty acid methyl esters. The mixture could be selectively converted to the deacylated macrolide core (4) by enzymolysis with immobilized porcine lipase, with the structure of the candidaspongiolide core then secured by NMR and MS analysis. The candidaspongiolide mixture was potently cytotoxic, exhibiting a mean panel 50% growth inhibition (GI50) of 14 ng/mL in the National Cancer Institute's 60-cell-line in vitro antitumor screen.

Structural assignment of poecillastrins B and C, macrolide lactams from the deep-water Caribbean sponge Poecillastra species.

Two new chondropsin-type macrolide lactams, poecillastrins B (1) and C (2), were isolated from aqueous extracts of the marine sponge Poecillastra sp. These trace metabolites were isolated in low yield (400-600 microg), and their structures were determined primarily by analysis of NMR data acquired using a cyrogenically cooled probe. High-quality 1D and 2D NMR data sets allowed complete assignment of the spectroscopic data and defined the new structures as 35-membered ring analogues of poecillastrin A (3). Compounds 1 and 2 showed potent cytotoxic activity against a human melanoma tumor cell line (LOX) with an IC50 value of less than 1 microg/mL.

Cell type-specific, topoisomerase II-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation by NSC 644221.

PURPOSE: The discovery and development of small-molecule inhibitors of hypoxia-inducible factor-1 (HIF-1) is an attractive, yet challenging, strategy for the development of new cancer therapeutic agents. Here, we report on a novel tricyclic carboxamide inhibitor of HIF-1alpha, NSC 644221. EXPERIMENTAL DESIGN: We investigated the mechanism by which the novel compound NSC 644221 inhibited HIF-1alpha. RESULTS: NSC 644221 inhibited HIF-1-dependent, but not constitutive, luciferase expression in U251-HRE and U251-pGL3 cells, respectively, as well as hypoxic induction of vascular endothelial growth factor mRNA expression in U251 cells. HIF-1alpha, but not HIF-1beta, protein expression was inhibited by NSC 644221 in a time- and dose-dependent fashion. Interestingly, NSC 644221 was unable to inhibit HIF-1alpha protein accumulation in the presence of the proteasome inhibitors MG132 or PS341, yet it did not directly affect the degradation of HIF-1alpha as shown by experiments done in the presence of cyclohexamide or pulse-chase labeling using [35S]methionine. In contrast, NSC 644221 decreased the rate of HIF-1alpha translation relative to untreated controls. Silencing of topoisomerase (topo) IIalpha, but not topo I, by specific small interfering RNA completely blocked the ability of NSC 644221 to inhibit HIF-1alpha. The data presented show that topo II is required for the inhibition of HIF-1alpha by NSC 644221. Furthermore, although NSC 644221 induced p21 expression, gammaH2A.X, and G2-M arrest in the majority of cell lines tested, it only inhibited HIF-1alpha in a distinct subset of cells, raising the possibility of pathway-specific "resistance" to HIF-1 inhibition in cancer cells. CONCLUSIONS: NSC 644221 is a novel HIF-1 inhibitor with potential for use as both an analytic tool and a therapeutic agent. Our data provide a strong rationale for pursuing the preclinical development of NSC 644221 as a HIF-1 inhibitor.