Lucas R. Chadwick

Resveratrol and red wine extracts inhibit the growth of CagA+ strains of Helicobacter pylori in vitro

TO THE EDITOR: In 1994, Helicobacter pylori was classified as a group I carcinogen and a definite cause of gastric cancer in humans by the International Agency for Research on Cancer (1). Since then, H. pylori has been epidemiologically linked to adenocarcinoma of the distal stomach (2, 3), and a recent study has also found a positive association between H. pylori infection and colorectal adenomas (4). CagA is the strain-specific H. pylori gene that has been linked to the development of premalignant and malignant histological lesions (5). Thus, susceptibility of cagA+ H. pylori strains is of note because, as compared with cagA– strains, infections caused by cagA+ strains significantly increase the risk for developing severe gastric inflammation, atrophic gastritis, and noncardia gastric adenocarcinoma (5). Previously, we have demonstrated that resveratrol, a stilbene from red wine, inhibited the growth of 15 clinical strains of H. pylori in vitro and suggested that the anti–H. pylori activity of resveratrol may play a role in its chemopreventative effects (6). In this investigation, the antibacterial activities of two red wine extracts (Pinot Noir) and resveratrol were assessed against five cagA+ H. pylori strains: accession numbers M23-3, GTD7-13, G1-1, SS1 (Sydney Strain cagA+), and the ATCC 43504 (Rockville, MD) possessing the cagA+ gene and expressing vacuolating cytotoxin. The H. pylori cagA+ strains were obtained from Drs. Richard Peek and Dawn Israel, Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN. Susceptibility testing was performed with the agar dilution procedure, and all other methods were as previously described (6). Red wine extracts (Pinot Noir) were prepared, one by concentrating and drying under reduced pressure, and the second by separating the alcohol-soluble and -insoluble components. The second extract was prepared by dissolving 1500 ml of red wine in 3 L of methanol, concentrating under reduced pressure, filtering, and collecting both the filtrate (alcohol soluble) and the particulate matter (alcohol insoluble). The filtrate was termed a methanol-soluble extract, and the particulate matter was termed a methanol-insoluble extract. The methanol-soluble extract was not active in our assay at concentrations up to 500 μg/ml. However, both the methanol-insoluble and the concentrated red wine extracts were active against all cagA+ HP strains, with minimum inhibitory concentrations (MIC) of 25 μg/ml and 50 μg/ml, respectively (range of 25–50 μg/ml). Resveratrol was also active against all five strains, with an MIC of 12.5 μg/ml (range of 6.25–25 μg/ml). The control drug, amoxicillin, had an MIC range of 0.0039 to 0.25 μg/ml. These data demonstrate that both red wine and resveratrol inhibit the growth of H. pylori cagA+ strains in vitro and further support their role as chemopreventive agents.

Identification of human hepatic cytochrome P450 enzymes involved in the metabolism of 8-prenylnaringenin and isoxanthohumol from hops (Humulus lupulus L.).

The female flowers of hops (Humulus lupulus L.) are used in the brewing of beer and are under investigation for use in dietary supplements for the management of menopausal symptoms in women. Hop extracts contain the weakly estrogenic compound isoxanthohumol (IX), proestrogenic xanthohumol, and the potent estrogen 8-prenylnaringenin (8PN). Because IX can be metabolized in the human liver to form 8PN, the specific cytochrome P450 (P450) enzymes responsible for this O-demethylation reaction were identified. In addition, the enzymes that convert IX and 8PN to their most abundant metabolites were identified because these metabolic pathways might also affect the estrogenicity of hop preparations. Specifically, the P450 enzymes that catalyze the oxidation of the prenyl side chains of IX and 8PN into trans- or cis-alcohols were investigated. Human liver microsomes and monoclonal antibodies that inhibit specific P450 enzymes were used in combination with liquid chromatography/mass spectrometry to identify the enzymes responsible for these transformations. CYP2C19 was found to catalyze the formation of both cis- and trans-alcohols of the prenyl side chain of 8PN with Km values of 14.8 ± 3.2 and 16.6 ± 4.6 μM, respectively. CYP2C8 converted 8PN regioselectively to the trans-alcohol of the prenyl group with a Km of 3.7 ± 0.9 μM. Finally, CYP1A2 was found to catalyze the O-demethylation of IX to generate 8PN, with a Km value of 17.8 ± 3.7 μM. These results suggest that the estrogenicity of hop constituents in vivo will depend in part on metabolic conversion that may show individual variation.

In vivo estrogenic comparisons of Trifolium pratense (red clover) Humulus lupulus (hops), and the pure compounds isoxanthohumol and 8-prenylnaringenin.

UNLABELLED The lack of a safe and reliable alternative to hormone therapy (HT) for treating menopausal symptoms underscores the need for alternative therapies. OBJECTIVE The purpose of this study was to assess the in vivo estrogenic effects of the botanical dietary supplements Trifolium pratense (red clover) and Humulus lupulus (hops), and two compounds obtained from H. lupulus, isoxanthohumol and 8-prenylnaringenin (8-PN) using the ovariectomized uterotrophic adult rat model. A H. lupulus extract and a 30% isoflavone extract of T. pratense were tested at three escalating doses as was one dose of isoxanthohumol for 21d. 8-Prenylnaringenin, the major estrogen in H. lupulus, was also tested at three relevant escalating doses. In order to determine the in vivo metabolism of 8-PN, the major phases I and II metabolites were also identified. The primary outcome measure, uterus weight gain, indicated that H. lupulus and T. pratense did not have an estrogenic effect on the uterus, and none of the secondary outcome measures were positive. In contrast, there was a clear dose response when 8-PN was evaluated where the middle and high doses of 8-PN were active. 8-Prenylnaringenin in rat plasma, liver, and mammary gland was measured and the major phases I and II 8-PN metabolites were detected. Our findings suggest that while both the H. lupulus and T. pratense extracts do not have an effect on the rat uterus, 8-PN at equivalent doses to those previously used in humans did have an effect, and may therefore have a deleterious effect in women.

Screening natural products for inhibitors of quinone reductase-2 using ultrafiltration LC-MS

Inhibitors of quinone reductase-2 (NQO2; QR-2) can have antimalarial activity and antitumor activities or can function as chemoprevention agents by preventing the metabolic activation of toxic quinones such as menadione. To expedite the search for new natural product inhibitors of QR-2, we developed a screening assay based on ultrafiltration liquid chromatography-mass spectrometry that is compatible with complex samples such as bacterial or botanical extracts. Human QR-2 was prepared recombinantly, and the known QR-2 inhibitor, resveratrol, was used as a positive control and as a competitive ligand to eliminate false positives. Ultrafiltration LC-MS screening of extracts of marine sediment bacteria resulted in the discovery of tetrangulol methyl ether as an inhibitor of QR-2. When applied to the screening of hop extracts from the botanical, Humulus lupulus L., xanthohumol and xanthohumol D were identified as ligands of QR-2. Inhibition of QR-2 by these ligands was confirmed using a functional enzyme assay. Furthermore, binding of xanthohumol and xanthohumol D to the active site of QR-2 was confirmed using X-ray crystallography. Ultrafiltration LC-MS was shown to be a useful assay for the discovery of inhibitors of QR-2 in complex matrixes such as extracts of bacteria and botanicals.

Purity−Activity Relationships of Natural Products: The Case of Anti-TB Active Ursolic Acid

The present study explores the variability of biological responses from the perspective of sample purity and introduces the concept of purity-activity relationships (PARs) in natural product research. The abundant plant triterpene ursolic acid (1) was selected as an exemplary natural product due to the overwhelming number yet inconsistent nature of its approximate 120 reported biological activities, which include anti-TB potential. Nine different samples of ursolic acid with purity certifications were obtained, and their purity was independently assessed by means of quantitative 1H NMR (qHNMR). Biological evaluation consisted of determining MICs against two strains of virulent Mycobacterium tuberculosis and IC50 values in Vero cells. Ab initio structure elucidation provided unequivocal structural confirmation and included an extensive 1H NMR spin system analysis, determination of nearly all J couplings and the complete NOE pattern, and led to the revision of earlier reports. As a net result, a sigmoid PAR profile of 1 was obtained, demonstrating the inverse correlation of purity and anti-TB bioactivity. The results imply that synergistic effects of 1 and its varying impurities are the likely cause of previously reported antimycobacterial potential. Generating PARs is a powerful extension of the routinely performed quantitative correlation of structure and activity ([Q]SAR). Advanced by the use of primary analytical methods such as qHNMR, PARs enable the elucidation of cases like 1 when increasing purity voids biological activity. This underlines the potential of PARs as a tool in drug discovery and synergy research and accentuates the need to routinely combine biological testing with purity assessment.

Essential Parameters for Structural Analysis and Dereplication by 1H NMR Spectroscopy

The present study demonstrates the importance of adequate precision when reporting the δ and J parameters of frequency domain 1H NMR (HNMR) data. Using a variety of structural classes (terpenoids, phenolics, alkaloids) from different taxa (plants, cyanobacteria), this study develops rationales that explain the importance of enhanced precision in NMR spectroscopic analysis and rationalizes the need for reporting Δδ and ΔJ values at the 0.1–1 ppb and 10 mHz level, respectively. Spectral simulations paired with iteration are shown to be essential tools for complete spectral interpretation, adequate precision, and unambiguous HNMR-driven dereplication and metabolomic analysis. The broader applicability of the recommendation relates to the physicochemical properties of hydrogen (1H) and its ubiquity in organic molecules, making HNMR spectra an integral component of structure elucidation and verification. Regardless of origin or molecular weight, the HNMR spectrum of a compound can be very complex and encode a wealth of structural information that is often obscured by limited spectral dispersion and the occurrence of higher order effects. This altogether limits spectral interpretation, confines decoding of the underlying spin parameters, and explains the major challenge associated with the translation of HNMR spectra into tabulated information. On the other hand, the reproducibility of the spectral data set of any (new) chemical entity is essential for its structure elucidation and subsequent dereplication. Handling and documenting HNMR data with adequate precision is critical for establishing unequivocal links between chemical structure, analytical data, metabolomes, and biological activity. Using the full potential of HNMR spectra will facilitate the general reproducibility for future studies of bioactive chemicals, especially of compounds obtained from the diversity of terrestrial and marine organisms.

Dynamic Residual Complexity of Natural Products by qHNMR: Solution Stability of Desmethylxanthohumol

The use of chromatographic assays to assess the residual complexity of materials that are purified from natural sources by chromatographic means is, in a sense, a case of the fox watching the henhouse. Beside their static residual complexity, which is intrinsic to their metabolic origin, biologically active natural materials can also be involved in chemical reactions that lead to dynamic residual complexity. The present study examines the dynamics of the hop prenylphenol, desmethylxanthohumol (DMX), by means of quantitative (1)H-NMR (qHNMR) in a setting that mimics IN VITRO and physiological conditions. The experiments provide a comprehensive, time-resolved, and mechanistic picture of the spontaneous isomerization of DMX into congeneric flavanones, including their (1)H/(2)D isotopomers. Formation of the potent phytoestrogen, 8-prenylnaringenin (8PN), suggests that measurable estrogenic activity even of high-purity DMX is an artifact. Together with previously established qHNMR assays including purity activity relationships (PARs), dynamic qHNMR assays complement important steps of the post-isolation evaluation of natural products. Thus, qHNMR allows assessment of several unexpected effects that potentially break the assumed linkage between a single chemical entity (SCE) and biological endpoints.

ISOLATION OF ALKALOIDS FROM GOLDENSEAL (HYDRASTIS CANADENSIS RHIZOMES) USING pH-ZONE REFINING COUNTERCURRENT CHROMATOGRAPHY

Goldenseal (the rhizomes of Hydrastis canadensis) has a long history of use in North American folk medicine and today is one of the top-selling herbal dietary supplements in the United States. The alkaloids present in the plant have been shown to be responsible for a broad range of biological activities, and the purpose of this work was to isolate preparative quantities of alkaloids present in Goldenseal for subsequent biological evaluation. Berberine chloride, canadaline, canadine, β-hydrastine, and isocorypalmine were separated from a methanolic extract of Goldenseal by a combination of solvent/solvent partition, pH-zone refining countercurrent chromatographic, and recrystallization techniques.

CCC Sample Cutting for Isolation of Prenylated Phenolics from Hops

Abstract One of the most potent estrogenic substances from the plant kingdom is the prenylated flavanone, 8‐prenylnaringenin (8PN), present in hops (Humulus lupulus L., Cannabaceae) in low (<10 ppm) concentrations. The prenylated chalcone desmethylxanthohumol (DMX), which is also present in this plant material, serves as a precursor to 8PN. A method for the preparative isolation of these and related bioactive phytochemicals has been developed that involves the use of multiple chromatographic techniques including complementary countercurrent chromatography (CCC) solvent systems. This paper describes the isolation of 8PN and DMX employing “sample‐cutting CCC”. The Hexanes‐EtOAc‐MeOH‐H2O (HEMWat) solvent systems found to be effective for isolation of prenylated phenolics from hops range from HEMWat 8‐2‐8‐2 for diprenylxanthohumol and 6,8‐diprenylnaringenin, to HEMWat 6‐4‐6‐4 for xanthohumol (XH), 6PN and 8PN, to HEMWat 5‐5‐5‐5 for DMX, hydroxylated derivatives of XH, and humulinones.

Extra-column volume in CCC

Abstract Countercurrent chromatography (CCC) is used by natural products and medicinal chemists for fractionation of extracts of flora and fauna to screen for pharmacological activity, and in isolating and purifying gram quantities of individual natural and synthetic compounds. The large extra‐column volume in CCC apparatus, particularly in the coil‐planet centrifuge designs employing an anti‐twisting flow line assembly, may lead to errors in the calculation of the distribution ratios of eluting fractions. The effect of extra‐column volume is to delay the time required for a peak to reach the detector. This delay is equal to the transit time of the extra‐column volume plus half of the mobile‐phase portion of the sample volume. The effect is negligible for columns of large volume (300 mL) but becomes more significant for small columns (15 mL) and for large sample volumes. Manual correction for the effect is very tedious, but results obtained using a generally applicable spreadsheet are presented and discussed. A second spreadsheet is also indicated wherein peak retention times, along with sample and extra‐column volume, are entered for calculation of distribution constants, which are summarized on a single page convenient for entry in a notebook.