Isolation of bitter acids from hops (Humulus lupulus L.) using countercurrent chromatography

Commercially available hops (Humulus lupulus L.) bitter acid extracts contain a mixture of three major congeners (co-, n-, and ad-) in addition to cis/trans diastereomers for each congener. Individual isomerized α-acids were obtained by the consecutive application of two separate countercurrent chromatography methods. First, individual isomerized α-acid congeners as a mixture of cis/trans diastereomers were obtained using a solvent system consisting of hexane and aqueous buffer. The second purification, capable of separating cis/trans diastereomers, was accomplished using a quaternary solvent system; an alternative procedure using β-cyclodextrin followed by countercurrent chromatography was also investigated. The NaBH(4) reduction of the purified isomerized α-acid compounds followed by countercurrent chromatography purification resulted in individual ρ iso α-acids (>95%). Similarly, catalytic hydrogenation of the purified isomerized α-acid compounds followed by countercurrent chromatography purification produced individual tetrahydro isomerized α-acids (>95%). Reported herein is a widely applicable approach that focuses on three critical variables--solvent system composition, pH, and buffer-to-sample ratio--that enable the efficient purification of individual bitter acids (≥95%) from commercially available hops extracts.     

Metabolism of xanthohumol and isoxanthohumol, prenylated flavonoids from hops (Humulus lupulus L.), by human liver microsomes

The female flowers of hops (Humulus lupulus L.) used to flavor beer contain the prenylated flavonoids xanthohumol (XN) and isoxanthohumol (IX). IX is moderately estrogenic in vitro and XN has pharmacological properties that might make it useful as a cancer chemopreventive agent. The metabolism of these dietary flavonoids was investigated in vitro using human liver microsomes. Hydroxylation of a prenyl methyl group was the primary route of oxidative metabolism forming either cis or trans hydroxylated metabolites of IX but only the trans isomer of XN. The double bond on the prenyl group of both compounds formed an epoxide which was opened by an intramolecular reaction with the neighboring hydroxyl group. The potent phytoestrogen 8-prenylnaringenin (8-PN) was detected as a demethylation product of IX. However, the analogous demethylation reaction was not observed for XN. Since XN can be converted to IX through acid-catalyzed cyclization in the stomach, XN might contribute to the in vivo levels of estrogenic 8-PN following consumption of hops extracts.     

A new formylated chalcone from Humulus lupulus with protective effect on HUVECs injury by angiotensin II

One new formylated chalcone, 3′- formyl xanthohumol (1) was isolated from the EtOAc-soluble partition of the cones of Humulus lupulus, along with two other known chalcones, namely dehydrocycloxanthohumol (2) and xanthohumol (3). The structure of compound 1 was elucidated on the basis of its 1D, 2D NMR and MS data. The structures of the known compounds were identified by comparison of their spectroscopic data with those reported by the literatures. The isolates were tested for their protective effects on human umbilical vein endothelial cells (HUVECs) injured by angiotensin II (Ang II), all the three compounds protected against the cell injury at the concentration of 20 μΜ, and compound 3 showed the most potent activity by improving cell viability from 53.9 to 74.9%.     

Capillary electrophoresis in association with chemometrics approach for bitterness hop (Humulus lupulus L.) classification

The precursor compounds related to the bitterness of beer are called α‐acids. These compounds are extracted from the hop, which is an important ingredient in the brewing process. These compounds were analyzed by capillary electrophoresis. The electrophoretic method used 160 mmol/L of ammonium carbonate (pH 9) as BGE (background electrolyte), a voltage of +20 kV in a capillary with 50 μm of internal diameter and with a 62.5 cm of total length (54 cm effective). The samples were injected in hydrodynamic mode applying a pressure of 25 mbar for 5 s and the analytes were detected at 230 nm. A hydromethanolic extraction during 3 h was considered as the optimum condition for the sample preparation using MeOH/H₂O 80:20 v/v as the extract solution. From the optimized conditions the electropherograms were evaluated for their use as input for chemometric modeling. Preprocessing investigation for electrophoretic data taking into account the alignment, denoising and baseline correction, and variable selection were considered before the chemometric modeling using principal component analysis (PCA). The electrophoretic data were systematically evaluated to find the optimum conditions to modeling. A PCA analysis for all tests was carried out using different preprocessing methods and, an explained variance higher than 90% was achieved in all of them. The optimized chemometric method worked with aligned and meancentered data. From this approach, a simple and efficient method to classify hop samples with high and low α‐acids content without the use of analytical standards was established from a simple electrophoretic analysis.     

Characterization of reduced iso‐α‐acids derived from hops (Humulus lupulus) by NMR

Reduced forms of iso‐α‐acids (isohumulones), used in modern beer brewing were separated and characterized by ¹H and ¹³C NMR spectroscopy. Components from mixtures of rho‐iso‐α‐acids, tetrahydro‐iso‐α‐acids, and hexahydro‐iso‐α‐acids were isolated using high‐performance liquid chromatography (HPLC) and analyzed by use of one‐ and two‐dimensional NMR experiments. The data presented assign the identities of the main peaks in the HPLC traces for the reduced iso‐α‐acids. Previous tentative assignments regarding the cis and trans configurations and the structures of the acyl residues of the reduced iso‐α‐acids were confirmed and extensive NMR assignments were made. Furthermore, the previously unknown stereochemistry in the C‐4 side‐chain of the rho‐ and hexahydro‐iso‐α‐acids was assigned. Copyright © 2003 John Wiley & Sons, Ltd.     

HPTLC Determination of Xanthohumol in Hops (Humulus lupulus L.) and Hop Products

JPC – Journal of Planar Chromatography – Modern TLC - A sensitive thin-layer chromatographic method has been established for quantification of xanthohumol in hops (Humulus lupulus L.)...     

Chemotaxonomic features associated with flavonoids of cannabinoid-free cannabis (Cannabis sativa subsp. sativa L.) in relation to hops (Humulus lupulus L.)

The major flavonoids present in the leaves and flowers of the cannabinoid-free cannabis (Cannabis sativa subsp. sativa L.) cultivars Felina and Futura are orientin (1), vitexin (2), luteolin-7-O-beta-D-glucuronide (3), and apigenin-7-O-beta-D-glucuronide (4), while prenylated flavonoids, to which the potent estrogenicity of hops (Humilus lupulus L.) is associated, are absent. The different composition of flavonoids has chemotaxonomic value.     

Characterization of varietal differences in essential oil components of hops (Humulus lupulus) by SFC-FTIR spectroscopy

A supercritical fluid chromatographic method combined with Fourier-transform infrared spectroscopy detection (SFC-FTIR) was developed for determination of varietal differences in essential oil constituents in hops (Humulus lupulus). Infrared spectra (IR) of the major constituents of essential oil of hops were taken as films deposited on AgCl discs and compared with those obtained after chromatographic separation in the IR flow-cell with supercritical carbon dioxide (scCO2). Spectra from AgCl discs were comparable to those in scCO2, but in scCO2 most of the bands appeared approximately 8-10 cm-1 to higher wave numbers. Open-tubular SFC-FTIR analysis of the essential oil of 4 different hop varieties was performed. The SFC-FTIR chromatograms showed differences in the location and relative intensity of the peaks depending on the variety, which was further confirmed by consideration of their FTIR spectra.     

Production of monoclonal antibodies against hop-derived (Humulus lupulus L.) prenylflavonoids and the development of immunoassays

Monoclonal antibodies against the hop-derived prenylated chalcone xanthohumol (X) and the prenylated flavonoids isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) were developed. Carboxylic acid haptens of X, IX and 8-PN were synthesized by linking a spacer to their C4′-OH group followed by subsequent coupling to bovine serum albumin (BSA) to form conjugates that were employed as immunogens in BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the established hybridoma cell lines proved, in cross-reactivity studies, to possess highly specific binding capacities in an optimized competitive indirect ELISA. The immunoassays make use of immunogen-coated microtiterplates and a peroxidase-labeled anti-mouse IgG₁ secondary antibody with ABTS as a chromogenic substrate. For X the IC₅₀ value derived from the standard curve was 62.91 ng mL⁻¹, and for both IX and 8-PN 37.15 ng mL⁻¹. The assay was validated for the quantitative analysis of X, IX and 8-PN in urine and serum. A simple sample pretreatment procedure using a diethyl ether extraction was optimized and the recoveries and matrix effects were assessed. The validity of the established assay was tested and mean inter- and intra-assay variations in urine were 2.32% and 1.91%, respectively for X, 6.24% and 2.39%, respectively for IX and 7.18% and 0.74%, respectively for 8-PN. In serum, the mean inter- and intra-assay variations were 8.90% and 1.37%, respectively for X, 6.13% and 1.57%, respectively for IX and 6.13% and 2.43%, respectively for 8-PN. Furthermore, the method demonstrated excellent accuracy and significant correlation with measurements by an established and validated HPLC-MS method.     

Analysis of the Leaves and Cones of Lithuanian Hops (Humulus lupulus L.) Varieties by Chromatographic and Spectrophotometric Methods

This work involves a comprehensive chemical composition analysis of leaf and cone samples of Lithuanian hop varieties. This study aimed to determine the chemometric properties of the leaves and cones of five Lithuanian hop varieties. Determined properties were the following: (a) xanthohumol content, (b) phenolic compounds, (c) flavonoids, (d) radical scavenging activity, and (e) the qualitative composition of volatile compounds. The total content of phenolic compounds in aqueous 75% methanolic extracts varied between 31.4–78.2 mg of rutin equivalents (RE)/g, and the concentration of flavonoids was between 11.0–23.3 mg RE/g. Radical scavenging activity varied between 34.4–87.2 mg RE/g. A QUENCHER analysis procedure showed 91.7–168.5 mg RE/g of the total phenolic compound content, 12.7–21.4 mg RE/g of flavonoids, and 48.4–121.0 mg RE/g of radical scavenging activity. ‘Fredos taurieji’ and ‘Fredos derlingieji’ varieties have shown maximum values of phenolic compounds and radical scavenging activity both in leaf and cone suspensions. These varieties accumulated a higher amount of xanthohumol in leaves. The concentration of xanthohumol in the samples varied between 0.0014–0.2136% of dry mass, with the highest concentration in the cones of ‘Kauno gražieji’. We identified 19 volatile compounds in leaves, and in cones, we identified 32. In both of them, α-humulene and β caryophyllene dominated. ‘Raudoniai’ leaves were exceptional in their aroma due to dominating compound nagina ketone (Kovats index 1306). The QUENCHER procedure has shown a great potential for the unextractable residue of hop raw material. Further investigation and valorization of different hop biomass components, not only cones, are essential.     

手性非水毛细管电泳法测定沙美特罗替卡松粉吸入剂中昔萘酸沙美特罗对映体

昔萘酸沙美特罗是目前治疗哮喘夜间发作和哮喘维持治疗的理想药物之一,它在临床上以外消旋体形式给药。昔萘酸沙美特罗的两个对映体在药理活性和毒理作用等方面差异较大,建立昔萘酸沙美特罗对映体的手性分离分析方法对提高手性药物质量、保证临床用药安全有效具有重要意义。该文以L(+)-酒石酸-硼酸络合酸为手性选择剂,建立了测定沙美特罗替卡松粉吸入剂中昔萘酸沙美特罗对映体含量的非水毛细管电泳法。实验考察了L(+)-酒石酸浓度、硼酸浓度和表观pH(apparent pH, pH^* )对手性分离效果的影响。优化的缓冲溶液为:含120.0 mmol/L L(+)-酒石酸和120.0 mmol/L硼酸的甲醇溶液,pH^* 为0.93;其他实验条件为:未涂层弹性熔融石英毛细管(内径50.0 μm,总长度64.5 cm,有效长度55.5 cm),重力进样17.5 cm×10.0 s,检测波长225 nm,室温,工作电压20.0 kV。在优化的实验条件下,昔萘酸沙美特罗的两个对映体在18.0 min内获得了2.18的分离度;在27.5~800.0 mg/L质量浓度范围内,与峰面积呈现良好的线性关系,相关系数(r)大于0.9990;检出限和定量限分别为7.5和25.0 mg/L;加标回收率为98.1%~101.9%,相对标准偏差为1.2%~1.9%。随机购买市面上出售的沙美特罗替卡松粉吸入剂,对其昔萘酸沙美特罗对映体的含量进行了分析检测。结果显示,昔萘酸沙美特罗对映体1和对映体2的标示量百分含量均为98.7%, RSD分别为2.5%和2.7%。该方法操作简便易行,结果准确可靠,消耗低,可用于市售沙美特罗替卡松粉吸入剂中昔萘酸沙美特罗对映体的含量测定。     

非水毛细管电泳测定黄连饮片中5种生物碱

建立了一种非水毛细管电泳(NACE)同时测定黄连饮片生品与炮制品中小檗碱、巴马汀、药根碱、木兰碱和黄连碱含量的方法。分别考察了非水溶剂、缓冲液体系及其浓度和pH、运行电压、运行温度和检测波长等条件对实验结果的影响。在优化的实验条件下,选择非水毛细管电泳分离模式,以40 mmol/L乙酸钠-40 mmol/L乙酸铵的无水甲醇缓冲溶液(pH 5.8)为电泳介质,未涂渍标准熔融石英毛细管(64.5 cm×75 μm,有效长度56 cm)为分离通道,检测波长为254 nm,分离电压为25 kV,压力进样(5 kPa×6 s),柱温为20 ℃。结果显示,5种生物碱在20 min内可实现基线分离,加标回收率为98.37%～101.03%。该方法简单、准确,重现性较好,可用于黄连饮片内在质量的评价和控制。     

非水毛细管电泳法测定藤黄中藤黄酸的含量

藤黄酸(gambogic acid, GA)等环氧杂蒽酮类化合物的水溶性差,可通过非水毛细管电泳(non-aqueous capillary electrophoresis, NACE)分析。本文系统地考察了添加20%~60%(v/v)的甲醇或乙腈的运行电解质溶液对藤黄提取液中藤黄酸分离的影响。比较了不同的运行电解质溶液、运行电解质溶液浓度、pH、添加剂 β-环糊精的浓度、分离温度及分离电压的影响,建立了测定藤黄药材中藤黄酸含量的非水毛细管电泳方法。在40%乙腈、10 mmol/L β-环糊精、20 mmol/L四硼酸钠(pH 9.86)为运行电解质溶液、分离电压为10 kV、分离温度为30 ℃、检测波长为280 nm的条件下进行测定。结果表明,藤黄酸在2~2000 mg/L范围内线性关系良好,相关系数为0.9996,检出限(S/N=3)为2 mg/L。将本方法应用于越南、泰国、缅甸、印度4个产地的藤黄药材中藤黄酸的含量测定,测得含量为1.67~472.40 mg/g(相对标准偏差(RSD)为1.12%~2.60%),其中越南产藤黄中藤黄酸含量低,其他产地藤黄中藤黄酸的含量高。实际藤黄样品中藤黄酸的加标回收率为95.2%~105.6%。非水毛细管电泳方法简单、快速、重现性好,可用于藤黄药材中藤黄酸的含量测定。     

Baseline separation of α and β‐acids homologues and isomers in hop (Humulus lupulus L.) by CD‐MEKC‐UV

An alternative method for simultaneous baseline separation of α and β‐acids homologues and isomers in hop by CD‐MEKC with UV detection was proposed. The optimized background electrolyte was composed of 30 mmol/L sodium tetraborate solution, 45 mmol/L sodium dodecyl sulfate, 20 mmol/L β‐cyclodextrin and 10% v/v acetonitrile. The instrumental conditions were evaluated by using a 3³ Box‐Benhken experimental design. In order to demonstrate the applicability of the method, 21 hop samples from different varieties were analyzed. The repeatability intra‐ and interday tests were performed and relative standard deviations lower than 7% for area and migration times were observed. The present method comprehended 8 min analysis time and revealed to be faster and more efficient when compared to previous reports from literature.     

固相萃取-高效液相色谱法测定啤酒中的黄腐酚

采用Waters Sep-Pak C18固相萃取小柱对啤酒样品进行分离纯化,建立了啤酒中黄腐酚的固相萃取-高效液相色谱检测方法。选用色谱柱Zorbax Eclipse XDB-C18柱(250 mm×4.6 mm,5 μm),以甲醇和0.1%甲酸水溶液为流动相进行梯度洗脱,柱温25 ℃,流速0.4 mL/min,检测波长370 nm。在此条件下,黄腐酚分离良好且无杂质峰干扰,在0.5～500 μg/L的范围内线性关系良好(r21),在高、中、低浓度下的加标回收率为91.21%～95.58%,相对标准偏差小于2%。方法的检出限为0.24 μg/L,定量限为0.80 μg/L。该方法简便快速、结果准确、重现性好,是检测啤酒中黄腐酚含量的有效方法。     

Determination of Organophosphorus Pesticides in Leeks (Allium porrum L.) by GC-FPD

A method was validated for the analysis of organophosphorus pesticides in leeks. The leek sample was cut into about 2 cm lengths and heated in a microwave oven for 50 s to inactivate enzymes. The sample was extracted with 50 mL acetonitrile using an Ultra-turrax T18 blender and cleaned up by solid phase extraction. The target analytes were determined by GC with a flame photometric detector. Average recoveries were 73–118% and limits of detection ranged from 1.1 to 10.0 μg kg^?1. The uncertainty of the analysis for each pesticide was evaluated as below 16%. The method was applied to determine organophosphorus pesticides in real samples. The GC–MS was used as confirmatory tool for positive samples.     

Simultaneous Determination of the Active Ingredients in Abelmoschus manihot (L.) Medicus by CZE

A capillary zone electrophoretic method has been developed for the quantitative analysis of four active compounds, quercetin-3-O-robinobioside (LXY3), hyperin (LXY4), isoquercetin (LXY5) and myricetin (LXY7) in A. manihot (L.) Medicus with UV detection at 254 nm. The capillary temperature was kept constant at 25 °C. The effects of buffer composition, pH, and concentration and running voltage on migration were studied systematically. Optimum separation was achieved with 20 mmol L^?1 Borax–NaOH buffer (pH 9.50) at 25 °C and 25 kV. Regression equations revealed good linear relationship between the peak area of each compound and its concentration. All the correlation coefficients were higher than 0.9990. The relative standard deviations of migration time and the peak area were <0.53% and 5.34% (inter-day), and <0.56% and 3.81% (intra-day). The contents of the four compounds in A. manihot (L.) Medicus were successfully determined with satisfactory repeatability and recovery.     

Multielement Determination in Turmeric (Curcuma longa L.) Using Different Digestion Methods

The antioxidant, anti-inflammatory and antiseptic properties of turmeric (Curcuma longa L.) derive from its rich nutritional composition making it interesting for medicinal uses, besides being used as spice in cooking. To complete the picture on the composition of turmeric, not only the organic compounds need to be known, but also the elemental composition covering essential and potentially toxic elements. The samples were digested in a microwave assisted digestion system using different reagent mixtures. The best digestion mixture was semi-concentrated nitric acid combined with hydrogen peroxide. After optimization of the sample preparation method, the contents of Ag, Al, As, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, Ga, K, Li, Mg, Mn, Mo, Na, Ni, Pb, Rb, Se, Sr, Te, Tl, V and Zn in curcuma were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES), as well as by inductively coupled plasma mass spectrometry (ICP-MS). Even if the general composition found is in line with the scarce data in literature, clear differences can be seen between the analyzed samples, considering provenience, production procedures, and harvesting year as potential influencing factors. Whereas all samples contained less As and Pb than regulated by WHO, one limit exceeding was found for Cd.