Simultaneous determination of hydroxyanthraquinones in rhubarb and experimental animal bodies by high-performance liquid chromatography.

A simple and reliable high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of five hydroxyanthraquinones (aloe-emodin, rhein, emodin, chrysophanol, and physcion) in Rhubarb and experimental animal bodies. A Zorbax SB-C18 column (250 mm x 4.6 mm i.d., 5 microm) and a methanol-0.5% acetic acid (85:15, v/v) mobile phase were used for the separation. The detection wavelength of a diode array detector (DAD) was set at 254 nm. Regression equations revealed a linear relationship (R2>0.9996) between the mass of hydroxyanthraquinones injected and the peak areas detected by DAD. The detection limits (S/N=3) ranged from 0.35 ng to 3.13 ng, and the recoveries ranged from 83% to 103% for different hydroxyanthraquinones. This method is simple, sensitive and suitable for the analysis of hydroxyanthraquinones in medicinal materials and pharmacological experiment samples.     

Purification of hydroxyanthraquinones and cinnamic acid from the roots of Rheum officinale Baill

A chromatographic method for isolation and purification of chemical constituents from the well-known traditional Chinese drug Da-huang (roots of Rheum officinale Baill.) was established by using 12% cross-linked agarose gel, Superose 12, as the separation media. A two-step separation procedure is employed. Sixty five percent methanol was used as the eluent for separation of cinnamic acid, rhein, physcion and emodin form Da-huang crude extract. The fraction containing aloe-emodin and chrysophanol was then separated by using 55% methanol containing 0.5% acetic acid as the eluent. As a result, cinnamic acid and five kinds of hydroxyanthraquinones including rhein, aloe-emodin, chrysophanol, physcion and emodin were obtained. The retention behavior of hydroxyanthraquinones on Superose 12 was also studied. The retention of hydroxyanthraquinones on Superose 12 is based on a mixture of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of the hydroxyanthraquinones and the residues of the cross-linking reagents used in the manufacturing process of Superose 12.     

Large-scale separation of hydroxyanthraquinones from Rheum palmatum L. by pH-zone-refining counter-current chromatography

pH-zone-refining counter-current chromatography was successfully applied to purify four hydroxyanthraquinones, rhein, emodin, aloe-emodin and chrysophanol, from three crude extracts of Rheum palmatum L.. After the two-phase solvent system methyl tert-butyl ether-tetrahydrofuran-water at an optimized volume ratio of 2:2:3 (v/v) was equilibrated, trifluoroacetic acid (10 mM) was added to the organic phase as a retainer and ammonia (10 mM), sodium carbonate (15 mM) and sodium hydroxide (15 mM) were added to the aqueous phase as the eluter, respectively, for three individual runs. Three separation runs of 1.25, 1.53 and 1.41 g of the three crude samples yielded four hydroxyanthraquinones: 0.70 g rhein, 0.81 g emodin, 0.41 g aloe-emodin and 0.94 g chrysophanol at a high purity of over 99.0, 98.5, 98.2 and 97.8% (determined by HPLC), respectively. The structures were identified by electrospray ionization MS-MS and (1)H NMR.     

Preparative isolation and purification of hydroxyanthraquinones from Rheum officinale Baill by high-speed counter-current chromatography using pH-modulated stepwise elution.

Analytical and preparative high-speed counter-current chromatography was successfully used for the isolation and purification of hydroxyanthraquinones from Rheum officinale Baill (Dahuang) using pH-modulated stepwise elution. Four major components including chrysophanol, emodin, physcion and aloe-emodin were isolated each at over 98% purity.     

The enhanced electrochemiluminescence of lucigenin by some hydroxyanthraquinones

Several hydroxyanthraquinones (emodin, rhein and physcion) were found to strongly enhance the cathodic electrochemiluminescence (ECL) of the lucigenin by scanning under the mode of differential pulse voltammetry. The enhanced intensity was linear with the concentrations of rhein, emodin and physcion. The linear calibration ranges of 8.0 × 10⁻⁸ to 2.0 × 10⁻⁶, 3.0 × 10⁻⁷ to 8.0 × 10⁻⁶ and 1.0 × 10⁻⁷ to 1.0 × 10⁻⁶ mol/L, the detection limits of 2.1 × 10⁻⁸, 1.6 × 10⁻⁷ and 5.2 × 10⁻⁸ mol/L were obtained for rhein, emodin and physcion, respectively. Potential-resolved ECL was used to study the possible mechanism of the enhancement effect. An electron transfer pathway was found to be involved in the ECL process. It has been confirmed that the reduced hydroxyanthraquinones reacted with dioxygen to give superoxide radical anion which increased the ECL of the lucigenin. Furthermore, these three hydroxyanthraquinones revealed the different enhanced ECL efficiency in the following order: rhein > physcion > emodin.     

Thin-layer chromatography of mycotoxins

TLC has become an extremely powerful, rapid and in most instances inexpensive separation technique in mycotoxicology. This review presents achievements of its applications in this field. General technical aspects of the TLC of mycotoxins that are discussed include extraction and clean-up procedures, adsorbents and solvent systems, detection methods, two-dimensional TLC, high-performance TLC (HPTLC), quantitation and preparative TLC (PLC). Special applications of TLC deal with multi-mycotoxin analyses and with structurally related or individual mycotoxins (aflatoxins, sterigmatocystins, versicolorins, ochratoxins, rubratoxins, patulin, penicillic acid, mycophenolic acid, butenolide, citreoviridin, trichothecenes, cytochalasans, tremorgenic toxins, epipolythiopiperazine-3,6-diones, hydroxyanthraquinones, zearalenone, citrinin, secalonic acids, cyclopiazonic acid, PR toxin, roquefortine, xanthomegnin, viomellein and naphtho-gamma-pyrones).     

Fluorescence quenching and molecular docking study on the binding of four hydroxyanthraquinones to FTO

In this work, the binding of four hydroxyanthraquinones (HAQs) to fat mass and obesity-associated (FTO) protein has been studied using fluorescence, UV-vis absorption and circular dichroism spectroscopy as well as molecular docking analysis. Analysis of fluorescence data showed that the binding of HAQs to FTO occurred via a static quenching mechanism. Thermodynamic analysis and molecular docking results suggested that hydrophobic force played a major role in stabilising the HAQ-FTO complex. Circular dichroism spectra indicated that the secondary structure of FTO was changed by the addition of HAQs. Results also showed that emodin had the strongest quenching and binding ability among four HAQs.     

Bromination of emodin

Using emodin as an example, it has been shown that in the bromination of hydroxyanthraquinones the qualitative composition and quantitative ratio of the reaction products depend on the nature of the brominating agent and the solvent, the ratio of the rea Lants, and the temperature regime. In order to obtain bromo-3-methyl-1,6,8-trihydroxyanthraquinone it is recommended to use dioxane dibromide in acid solution as the brominating agent, and to obtain 5-bromo-3-methyl-1,6,8-trihydroxyanthraquinone the same reagent in dioxane solution. The optimum conditions for obtaining 3-bromomethyl-1,6,8-trihydroxyanthraquinone by the methods of initiated bromination and photobromination have been selected. S. M. Kirov Kazakh State University, Alma-Ata. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 618–621, September–October, 1990.     

Bromination of emodin

Using emodin as an example, it has been shown that in the bromination of hydroxyanthraquinones the qualitative composition and quantitative ratio of the reaction products depend on the nature of the brominating agent and the solvent, the ratio of the rea Lants, and the temperature regime. In order to obtain bromo-3-methyl-1,6,8-trihydroxyanthraquinone it is recommended to use dioxane dibromide in acid solution as the brominating agent, and to obtain 5-bromo-3-methyl-1,6,8-trihydroxyanthraquinone the same reagent in dioxane solution. The optimum conditions for obtaining 3-bromomethyl-1,6,8-trihydroxyanthraquinone by the methods of initiated bromination and photobromination have been selected.S. M. Kirov Kazakh State University, Alma-Ata. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 618–621, September–October, 1990.     

Use of high-performance thin layer chromatography by the American Herbal Pharmacopoeia

TLC characterizations are among the key identity tests in most pharmacopoeial monographs. Pharmacopoeial standards are typically used by industry as a basis for meeting QC requirements and current good manufacturing practices (cGMPs). TLC is a relatively low-cost, highly versatile tool for developing specifications for raw materials, as well as for the various preparations for which pharmacopoeial standards are created. In addition to its use in the development of identity tests, TLC is a valuable tool for screening plant samples that pharmacopoeias must review in the development of monographs and botanical reference materials (BRMs). Specifically, HPTLC is the ideal TLC technique for these purposes because of its increased accuracy, reproducibility, and ability to document the results, compared with standard TLC. Because of this, HPTLC technologies are also the most appropriate TLC technique for conformity with GMPs. This article highlights the manner in which HPTLC is used by the American Herbal Pharmacopoeia (AHP) in the development of AHP monograph identity standards, the identification of adulterating species, and the development of AHP-verified BRMs.     

Separation and purification of six components from the roots of Rheum officinale Baill. by supercritical fluid chromatography

The dried roots of Rheum officinale Baill., named Dahuang in Chinese, has many pharmacological effects and has been widely used for the treatment of many diseases. To develop a harmless and eco-friendly method for the separation of components in Dahuang is of great interest for quality control and pharmacological study of Dahuang. A method for separation and purification of components in Dahuang using supercritical fluid chromatography (SFC) is established in this work. Samples were prepared by extraction of Dahuang powders with 20% H₂SO₄ and benzene under reflux. The extracts were obtained by evaporating benzene extracts and separating by SFC on YMC-Diol column using supercritical CO₂ with CH₃OH 4% (v/v) as the modifier. The flow rate of the mobile phase was 15?mL/min, the column pressure was 13?MPa, and the column temperature was 318?K. Cinnamic acid and five kinds of hydroxyanthraquinones including rhein, emodin, aloe emodin, chrysophanol, and physcion were obtained by SFC. The purities of the obtained compounds were all above 97% as determined by high performance liquid chromatography and their chemical structures were identified by nuclear magnetic resonance and mass spectrum. The thermodynamics of chromatographic process was also studied and it revealed that the SFC separation process of these compounds on YMC-Diol column was controlled by enthalpy.     

Qualitative and quantitative analyses of aloe-emodin,rhein, and emodin in qi yin granules by high-performance thin-layer chromatography

The aim of this study was to perform qualitative and quantitative analyses of aloe-emodin, rhein, and emodin in three prepared samples of compound qi yin granules by high-performance thin-layer chromatography (HPTLC) and to establish an analytical method. TLC was used to qualitatively analyze the three major components of the compound: aloe-emodin, rhein, and emodin. HPTLC was performed to determine the contents of the three components. HPTLC analysis showed that using Anhui Liangchen high-efficiency silica gel G plate was the optimal stationary phase and the upper layer solution of a petroleum ether–ethyl acetate–formic acid (15.5:5:1, V/V) mixed solution was the optimal developing agent. The composition of the samples for testing was basically the same, but the content was different. In summary, this study used HPTLC to qualitatively and quantitatively analyze aloe-emodin, rhein, and emodin in compound qi yin granules. It can lay the foundation for improving the quality control and standards of compound qi yin granules.

     

Hydrolytical instability of hydroxyanthraquinone glycosides in pressurized liquid extraction

Hydroxyanthraquinones represent a group of pharmacologically active compounds characteristic for plants of the Rumex and Rheum genera. These compounds in the human intestine act as laxative compounds. As they cause the greatest side effects and are often abused by the public, their accurate analysis in plants and plant-derived laxatives is much needed. To isolate compounds from plants, pressurized liquid extraction (PLE) is frequently applied. The technique has been regarded, so far, as very effective, even in isolation of sensitive compounds for which exposure time in high temperature has a negative impact. This work demonstrates some interesting and surprising results accompanying PLE of hydroxyanthraquinones from the Rumex crispus L. root using methanol/water mixtures as extractant. The presented results demonstrate that glycoside forms of hydroxyanthraquinones (emodin-8-O-β-D-glucopyranoside, chrysophanol-8-O-β-D-glucopyranoside, and physcion-8-O-β-D-glucopyranoside) are hydrolytically unstable even in the short-lasting PLE. The increase of water concentration in the extractant leads to the increase of the transformation degree of the glycoside forms to the corresponding aglycones (emodin, chrysophanol, and physcion), increasing the concentration of the latter. The rise in the PLE temperature accelerates the hydrolytical degradation of the glycoside forms. The extension of the extraction time also intensifies this process. The presented results show that extraction of glycosides using extractants containing water can lead to false conclusions about the chemical composition of plants.

Surface-initiated molecularly imprinted polymeric column: In situ synthesis and application for semi-preparative separation by high performance liquid chromatography

In this work, we prepared a monolithic and surface initiated molecularly imprinted polymeric (MIP) column for HPLC and explored its application for template separation from plant extract. The silica beads (40-60 μm) were coupled with initiator on the surface and then packed in to a stainless steel HPLC column. The pre-polymerization mixture (the template, functional monomer and crosslinker were emodin, acrylamide and divinylbenzene, respectively) was injected into the column and polymerized by thermal initiation. The prepared MIP column exhibited excellent retention capability and large imprinted factor for template (the retention time and imprinted factor for emodin on MIP column were 16.26 min and 7.21, respectively). Moreover, the emodin-molecularly imprinted polymeric column could be applied to separate emodin from alcohol extract of Rheum palmatum L. at semi-preparative scale and 1.2 mg of emodin was obtained in 4 h.     

多壁碳纳米管表面大黄素印迹材料制备及吸附性能研究

以表面硅烷修饰的碳纳米管为基材,甲基丙烯酸为功能单体,二甲基丙烯酸乙二醇酯为交联剂,采用表面印迹技术成功制备了大黄素印迹复合材料。采用扫描电镜和透射电镜研究该印迹复合材料的形貌,结果表明,在碳纳米管表面接枝了1层稳定的约40nm厚的印迹层。在pH=8.5时,该印迹材料对大黄素具有较大吸附容量和良好选择吸附性能,最大理论吸附量(Qmax)为20.8mg/g。以该印迹材料作为固相萃取吸附剂,在85%乙醇水溶液淋洗和10%乙酸的乙醇溶液洗脱下,可实现对猕猴桃根样品粗提取液中大黄素固相萃取分离富集,回收率达到85%以上。     

The carbon-13 nuclear magnetic resonance spectra of anthraquinone,eight polyhydroxyanthraquinones and eight polymethoxyanthraquinones

The ¹³C NMR spectra of anthraquinone, eight hydroxyanthraquinones and eight methoxyanthraquinones are reported. Peak assignment for the carbon atoms of these compounds is achieved by using decoupling techniques and by intercomparison of the variously-substituted derivatives. Carbonyl chemical shifts in the hydroxyanthraquinones can be rationalized in terms of cross conjugation and intramolecular hydrogen bonding and in terms of cross conjugation in the methoxyanthraquinones. Hydroxy and methoxy substituent chemical shifts are also reported.     

Application of spectroscopic and computational methods in the study of the electronic excitations of partially silylated hydroxyquinones

The silylation of hydroxynaphthoquinones and hydroxyanthraquinones with N-methyl-N-(tert-butyldimethylsilyl)-2,2,2-trifluoroacetamide, in one step reaction, under mild conditions, has led, in nearly quantitative yields, to the isolation of hydrolytically stable partially silylated products (only of the hydroxyl groups), which differ dramatically in color from the starting materials. The spectroscopic investigation of the silylated products in solution as well as the correlation of the observed data with computationally derived results have been carried out within a study aiming at the understanding of the visible maxima shift to either the ultraviolet or near-infrared region of the electromagnetic spectrum, which would imply availability of the silylated products in a variety of related applications.     

Isolation and determination of alizarin in cell cultures of Rubia tinctorum and emodin in Dermocybe sanguinea using solid-phase extraction and high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of the non-glycosidic anthraquinones alizarin (1,2-dihydroxy-9,10-anthracenedione), emodin (1,3,8-trihydroxy-6-methyl-9,10-anthracenedione) and anthraquinone (9,10-anthracenedione). The anthraquinones were separated by isocratic elution on a 125 × 4.6 mm I.D. column containing ODS Hypersil 5 reversed-phase material using methanol-5% acetic acid (pH 3.0) (70:30) as the mobile phase. Free alizarin was determined in plant cell suspension cultures of Rubia tinctorum and free emodin in mushrooms (Dermocybe sanguinea). The effective extraction of anthraquinones from plant cells was achieved with 80% (v/v) ethanol after incubation for 10 h at 80°C. Prepurification and concentration of anthraquinones in the plant cell and mushroom extracts were effected by a solid-phase technique using C₈ cartridges.     

Development and validation of a simple thin layer chromatographic method for the analysis of artemisinin in Artemisia annua L. plant extracts

Owing to the development of parasite resistance to standard antimalarial treatments like chloroquine and sulfadoxine-pyrimethamine, the demand for Artemisia annua, a key ingredient for new and highly effective antimalarial drugs, is huge. Therefore selective and precise methods to determine the content of artemisinin in dry plant material and in raw impure extracts are needed. In this work a method is described for the clear separation and extraction of artemisinin from other plant components in the Artemisia annua L. plant by thin-layer chromatography (TLC). To obtain optimal extraction and recovery efficiency, several parameters were evaluated, including choice of extraction solvent, TLC plate type and sensitivity between UV and visible light. Method validation was performed on both the dry plant material and non-purified plant extracts. Toluene presented the highest extraction efficiency compared with petroleum ether, hexane and methanol. Reversed-phase plates showed more concentrated spots than normal-phase plates, while the sensitivity of the analysis in UV was comparable to that in visible light but less precise. The impure plant extracts were analyzed by both TLC and HPLC-UV at 215 nm and both methods met the requirements for linearity, selectivity, precision and accuracy. Hence, the proposed TLC method can easily be used for both qualitative and quantitative control of the raw plant extract in areas where advanced methods are scarce.