Optimized separation of scopoletin and umbelliferone and cis-trans isomers of ferulic and p-coumaric acids by reverse-phase high-performance liquid chromatography

A systematic optimization strategy for reverse-phase h.p.l.c. is used to produce complete separation of all components of a mixture containing two coumarins, umbelliferone and scopoletin, and the cis-trans isomers of two cinnamic acids, p-coumaric and ferulic. A series of experiments gives retention time data for each of these substances in four citric acid-buffered mobile phases. A mathematical model fitted to this data gives model parameters that provide the basis for a window diagram of relative retentions vs. mobile phase pH. The window diagram gives an estimate of the mobile phase pH required for optimum separation.     

Identification and Quantification of Coumarins by UHPLC-MS in Arabidopsis thaliana Natural Populations

Coumarins are phytochemicals occurring in the plant kingdom, which biosynthesis is induced under various stress factors. They belong to the wide class of specialized metabolites well known for their beneficial properties. Due to their high and wide biological activities, coumarins are important not only for the survival of plants in changing environmental conditions, but are of great importance in the pharmaceutical industry and are an active source for drug development. The identification of coumarins from natural sources has been reported for different plant species including a model plant Arabidopsis thaliana. In our previous work, we demonstrated a presence of naturally occurring intraspecies variation in the concentrations of scopoletin and its glycoside, scopolin, the major coumarins accumulating in Arabidopsis roots. Here, we expanded this work by examining a larger group of 28 Arabidopsis natural populations (called accessions) and by extracting and analysing coumarins from two different types of tissues–roots and leaves. In the current work, by quantifying the coumarin content in plant extracts with ultra-high-performance liquid chromatography coupled with a mass spectrometry analysis (UHPLC-MS), we detected a significant natural variation in the content of simple coumarins like scopoletin, umbelliferone and esculetin together with their glycosides: scopolin, skimmin and esculin, respectively. Increasing our knowledge of coumarin accumulation in Arabidopsis natural populations, might be beneficial for the future discovery of physiological mechanisms of action of various alleles involved in their biosynthesis. A better understanding of biosynthetic pathways of biologically active compounds is the prerequisite step in undertaking a metabolic engineering research.     

Constituents of Flowers of Murraya Paniculata

Three new coumarins, yuehgesin-A (1), -B (2) and -C (3) and 22 compounds—murracarpin (4), mupanidin (5), isomeranzin (6), murralongin (7), scopoletin (10), 7-methoxy-8-(l′-ethoxy-2′-hydroxy-3′-methyl-3′-butenyl)coumarin (11), umbelliferone (12), paniculatin (13), braylin (14), auraptenol (15), meranzin hydrate (16), minumicrolin (17), scopolin (18), caffeine (19), 3,3′,4′,5,5′,6,7-heptamethoxyflavone (20), 4-hy-droxybenzaldebyde (21), p-hydroxybenzoic acid (22), cis- and trans-ferulic acid (23), cis- and transmethyl ferulate (24) and trans-ethylferulate (25)—were isolated and characterized from fresh flowers of Murraya paniculata collected in Taiwan. Their structures were elucidated by spectral methods. The chemotaxonomy of Murraya paniculata is, discussed.     

Cardenolides and coumarins ofCoronilla varia, C. cretica, etc.

Conclusions The cardenolide and coumarin composition ofC. varia L. and the closely related speciesC. hyrcana Pril.,C. cretica L., andC. orientalis Müll have been investigated. It has been shown that they all contain the cardiac glycoside hyrcanoside, with the exception ofC. orientalis, in which another, unidentified, glycoside, has also been found.A comparative study of some properties of hyrcanogenin (⁴-strophanthidin) and pachygenin (⁵-strophanthidin) connected with conformational features has been carried out.In the species studied, no less than five coumarins have been found of which umbelliferone, scopoletin, and daphnoretin have been isolated.Khimiya Prirodnykh Soedinenii, Vol. 5, No. 3, pp. 141–144, 1969     

Quality assessment of traditional Chinese medicine herb couple by high‐performance liquid chromatography and mass spectrometry combined with chemometrics

This study was designed to develop a simple, specific and reliable method to overall analyze the chemical constituents in clematidis radix et rhizome/notopterygii rhizome et radix herb couple using high‐performance liquid chromatography coupled with tandem mass spectrometry and multiple chemometric analysis. First, the separation and qualitative analysis of herb couple was achieved on an Agilent Zorbax Eclipse Plus C₁₈ column (250 mm × 4.6 mm, 5 μm), and 69 compounds were unambiguously or tentatively identified. Moreover, in quantitative analysis, eight ingredients including six coumarins and two triterpenoid sapogenins were quantified by high‐performance liquid chromatography coupled with tandem mass spectrometry. In terms of good linearity (r² ≥ 0.9995) with a relatively wide concentration range, recovery (85.40–102.50%) and repeatability (0.99–4.45%), the validation results suggested the proposed method was reliable, and successfully used to analyze ten batches of herb couple samples. Then, hierarchical cluster analysis and principal component analysis were used to classify samples and search significant ingredients. The results showed that ten batches of herb couple samples were classified into three groups, and six compounds were found for its better quality control.     

Efficient Solvent‐Free Synthesis of Benzothiazine‐Fused Pyrrolo[3,4‐c]coumarins: Cycloaddition Reactions between Coumarin‐Based Dihydrobenzothiazoles and Isocyanides

A new and unusual synthesis of benzothiazine‐fused pyrrolo[3,4‐c]coumarins, involving the ring‐opening of coumarin‐based dihydrobenzothiazoles and subsequent [4+1] cycloaddition reaction with isocyanides, was described. Thus, simple heating of various 3‐(2,3‐dihydro‐2‐methylbenzo[d]thiazol‐2‐yl)coumarins with isocyanides produced the title compounds in good yields under solvent‐free conditions.     

Nutritional composition,antioxidant activity and isolation of scopoletin from Senecio nutans: support of ancestral and new uses

Continuing with our study characterising Senecio nutans Sch. Bip., we have isolated and identified a simple coumarin, scopoletin, that could be relevant for the biological properties of the species related with the ancestral medical uses. This is the first report of scopoletin from S. nutans. In addition, the extract was analysed for its antioxidant activity using the ABTS and FRAP method as well as providing the first nutritional analyses of this plant from northern Chile highlands.     

Simultaneous isolation of seven compounds from Glehnia littoralis roots by off‐line overpressured layer chromatography guided by a TLC antioxidant autographic assay

A combinative method using overpressured layer chromatography (OPLC) and TLC bioautography against O₂^?? was developed to separate antioxidants from Glehnia littoralis roots. Through target‐directed isolation by the TLC bioautographic method, seven compounds including five antioxidants were rapidly isolated by OPLC and identified as 1‐linoloyl‐3‐palmitoylglycerol, facarindiol, panaxynol, isoimperatorin, β‐sitosterol, scopoletin, and umbelliferone from G. littoralis roots. This OPLC method is a very suitable separation technique for light‐sensitive polyacetylenes (panaxynol and facarindiol) with higher isolated yields compared to conventional open column chromatography. This is the first report on the separation of polyacetylenes by OPLC.     

Quantitative determination of seven chemical constituents and chemo‐type differentiation of chamomiles using high‐performance thin‐layer chromatography

A simple and rapid high‐performance thin‐layer chromatographic method was developed for the separation and determination of six flavonoids (rutin, luteolin‐7‐O‐β‐glucoside, chamaemeloside, apigenin‐7‐O‐β‐glucoside, luteolin, apigenin) and one coumarin, umbelliferone from chamomile plant samples and dietary supplements. The separation was achieved on amino silica stationary phase using dichloromethane/acetonitrile/ethyl formate/glacial acetic acid/formic acid (11:2.5:3:1.25:1.25 v/v/v/v/v) as the mobile phase. The quantitation of each compound was carried out using densitometric reflection/absorption mode at their respective absorbance maxima after postchromatographic derivatization using natural products reagent (1% w/v methanolic solution of diphenylboric acid‐β‐ethylamino ester). The method was validated for specificity, limits of detection and quantification, precision (intra‐ and interday) and accuracy. The limits of detection and quantification were found to be in the range from 6–18 and 16–55 ng/band for six flavonoids and one coumarin, respectively. The intra‐ and interday precision was found to be <5% RSD and recovery of all the compounds was >90%. The data acquired from high‐performance thin‐layer chromatography was processed by principal component analysis using XLSTAT statistical software. Application of principal component analysis and agglomerative hierarchial clustering was successfully able to differentiate two chamomiles (German and Roman) and Chrysanthemum.     

Studies of coumarins from the Chinese drug Qianhu, XXVII: structure of a new simple coumarin glycoside from Bai-Hua Qianhu, Peucedanum praeruptorum

A new simple coumarin glycoside, named praeroside VI (1), along with six known coumarins, were isolated from an aqueous extract of Bai-Hua Qianhu, the root of Peucedanum praeruptorum DUNN. (Umbelliferae). Spectroscopic analysis and chemical studies were carried out to investigate the structure of the new coumarin, which was concluded to be 1. Additionally, two simple glycosidic coumarins and four simple nonglycosidic coumarins were identified as apiosylskimmin (2), hymexelsin (3), umbelliferone (4), scopoletin (5), isofraxidin (6) and 8-carboxy-7-hydroxy coumarin (7), respectively. This is the first reported identification of compound 7 as a constituent of plant materials.     

The Interplay between Solvation and Stacking of Aromatic Rings Governs Bright and Dark Sites of Benzo[g]coumarins

Coumarins are classic, strongly polarized fluorophores with multiple applications, and significant efforts have been put into modifying their emission characteristics and elucidating their photophysics. Expecting that π-expansion of these donor–acceptor chromophores could modify their ground- and excited-state characteristics, the authors performed combined, detailed photophysical and computational studies of linearly π-expanded coumarins, that is, 8-dialkylamino-3-carboxyalkyl-benzo[g]coumarins. The investigation led to the conclusion that emission is only possible thanks to the stabilizing effect of the solvent and that breaking of the lactone ring leads to the conical intersection with the ground state and induces the radiationless decay of the electronic excitation. Aiming at the fine-tuning the excited state properties through the construction of covalently linked dye assemblies, the authors designed and synthesized a new bis(benzo[g]coumarin), built from two similar moieties that exhibit different degrees of polarization due to the electron donor at position 8: one possesses a dialkylamino, and the other a weaker amide donor. Comprehensive studies have shown that the observed weak fluorescence of the system is the result of the interplay between the solvation-induced separation of the benzo[g]coumarin moieties, which stabilizes the emitting locally excited singlet state and the π-stacking interactions, favoring their sandwiched orientation and leading to the non-emissive charge-transfer state.     

Isolation and Simultaneous Determination of Coumarin Compounds in Radix Angelica dahurica

For the first time simple, rapid, and systematic methods have been established for preparative isolation and purification of coumarin compounds in an important traditional Chinese Medicine, Radix Angelica dahurica, and for simultaneous determination of several of the compounds in the medicine. Bergapten, imperatorin, and cnidilin, three of the biologically active coumarin compounds, were isolated from the chloroform-soluble fraction of the ethanol extract of Radix Angelica dahurica. After further purification by open column ODS chromatography the purified components were simultaneously determined, with two other coumarins (osthole and isoimperatorin), by reversed phase high-performance liquid chromatography (RP-HPLC) on a C₁₈ column, with methanol–water, 66:34 (v/v), as mobile phase at a flow rate of 0.8 mL min⁻¹. The compounds were detected by UV absorption at 310 nm. Calibration plots for all the coumarins had correlation coefficients close to unity. Limits of detection (S/N = 3) were <92 ng mL⁻¹ and limits of quantification (S/N = 10) were <259 ng mL⁻¹. Mean recovery of the coumarins was in the range 96.7–101.9% and the intra-day and inter-day precision, as relative standard deviation, was <2.3 and <2.9%, respectively. This simple, sensitive, and reproducible method can be used for quality control of Radix Angelica dahurica.     

CE with Field-Enhanced Stacking for Rapid and Sensitive Determination of Umbelliferone,Rutin and Aesculetin in Prunella vulgaris

A new capillary electrophoresis procedure with field-enhanced stacking concentration for the analysis of umbelliferone, rutin and aesculetin in Prunella vulgaris is established for the first time. After optimization of the separation and concentration conditions, the three analytes can be separated within 6 min and quantified with high sensitivity. The method was useful for qualitative and quantitative analysis of umbelliferone, rutin and aesculetin in Prunella vulgaris with recovery of 97.5 to 105.3%.

     

Anticomplement and antitussive activities of major compound extracted from Chimonanthus nitens Oliv. leaf

Chimonanthus nitens Oliv. leaf (CNOL), as a traditional Chinese medicine, has been widely used for the treatment of influenza and colds over a long history. However, the mechanism of colds related to the effects of CNOL have been little studied. In this study, the anticomplement and antitussive activities of different polarity extracts of CNOL were evaluated. Ethyl acetate extract (EAE) among different extracts not only significantly decreased cough times by 21–58% (P < 0.01), but also had anticomplement effects demonstrated by the CH₅₀ values of 0.100 mg/ml. A total of 28 constituents (10 coumarins, 13 flavonoids and five phenolics) were identified in EAE based on the ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry technique. Eight compounds in EAE were evaluated by an ammonia-induced cough model to reveal the antitussive mechanisms and classical anticomplement pathway. The results indicated that the antitussive effects of scopoletin, kaempferol-3-O-rutinoside and kaempferol may depend on central mechanisms and that flavonoids such as compounds of kaempferol-3-O-rutinoside and kaempferol have better anticomplementary activity than coumarins like compounds of scopolin, scopoletin and isofraxidin. Taken together, kaempferol-3-O-rutinoside and kaempferol could be important chemical markers in the present study that might be used to evaluate the quality and biological activity of CNOL.     

HPLC-MS and MECC analysis of coumarins

Summary MECC separation of 11 coumarins has been achieved by use of a running electrolyte at pH 10.4 prepared from 50 mM boric acid, 10 mM sodium tetraborate and 100 mM sodium hydroxide. The buffer solution contained 50 mM SDS and, as organic modifier 1%n-propanol. The applied voltage was 25 kV and the temperature of the capillary was kept constant at 20°C. HPLC baseline separation of the coumarin mixture was obtained by use of a reversed-phase column and an acetonitrile-water solvent gradient. UV detection was performed at 205 nm. Peak assignment and purity control were achieved by HPLC-mass spectrometry with either an electrospray interface or an atmospheric-pressure chemical-ionization interface. Compounds were detected in either negative- or positive-ion modes. These MECC and HPLC-MS methods are suitable for ‘fingerprint’ analysis of a number of coumarin-containing plants, e.g. Fr.Ammi visnagae, Rd.Scopoliae and Rd.Imperatoriae.     

高速逆流色谱法分离制备华山参中东莨菪内酯

采用高速逆流色谱法从华山参乙醇提取液的氯仿萃取物中分离得到了一种高纯度的香豆素类化合物,东莨菪内酯．所用体系为正己烷：乙酸乙酯：甲醇：水（体积比3：5：3：5）,上相做固定相,下相做流动相．分离所得东莨菪内酯的纯度经高效液相色谱检测达到97％．该方法操作简单、方便、快捷,可用于华山参中香豆素类化合物——东莨菪内酯的分离制备．     

Isocratic HPLC Separation of Scopoletin and Cis/Trans Isomers of Ferulic Acid as Well as Isoscopoletin and Cis/Trans Isomers of Isoferulic Acid

Abstract

Cis/trans isomers of ferulic and isoferulic acids and their corresponding coumarins, scopoletin and isoscopoletin, were separated by isocratic High Performance Liquid Chromatography using RP-8 (5 μm) as a stationary phase and aqueous methanol or aqueous acetonitrile as a mobile phase. The UV spectrum of cis-isoferulic acid was obtained by a photodiode array detector.     

Synthesis of novel coumarin and benzocoumarin derivatives and their biological and photophysical studies

Several derivatives of coumarin‐3N‐carboxamides ( 3‐21 ) have been prepared via the reaction of the coumarin‐3‐carbonyl chloride ( 1 ) with a number of nucleophiles. Novel double‐headed coumarin‐3N‐carboxamides ( 26‐33 ) were also produced using the same method. The Pechmann‐Duisberg reaction was applied to prepare new benzo[f]‐ benzo[h]coumarins and 4‐(chloromethyl)‐pyrano[3,2‐c]coumarin‐2‐one ( 36‐42 ). The reaction of 1‐chloromethylbenzo[f]coumarins ( 36 ) with cyanide anion under different reaction conditions was also investigated in order to assess its suitability for nucleophilic substitution reactions as well as ring transformation products ( 43‐49 ). Synthesis of 1‐((benzo[d]thiazol‐2‐yl)methyl)‐9‐hydroxybenzo[ f ]coumarin ( 50 ) represented the first example of methylene bridge‐head heterocyclecontaining benzo[f]coumarin. Some of the newly prepared coumarins exhibited anti‐bacterial activity against Gram Positive and Gram negative bacteria. Compound 36d was found to be active against all the screened bacteria. Photophysical studies were performed on selected fluorescent benzo[f]‐ and benzo[h]coumarin and the quantum yields were also calculated. All new compounds were characterized by IR, MS, ¹H and ¹³C NMR, as well as elemental analysis.     

Microwave‐promoted Synthesis of Novel Fused Osthole Analogues

Osthole is a natural coumarin derivative and has a broad scope of biological activities. Two series of novel fused osthole analogues were designed, and synthesized through a highly efficient microwave‐promoted synthetic protocol via the reaction of 4‐hydroxycoumarins and β‐ketoesters. The reaction conditions including solvent, catalyst, microwave power and irradiation time were also optimized. The pyrano[3,2‐c]chromene‐2,5‐diones and furo[3,2‐c]coumarins were obtained through two distinct intramolecular cyclization processes, and the proposed mechanism was also discussed.     

Coumarin glycosides ofHaplophyllum perforatum

From the epigeal part ofHaplophyllum perforatum we have isolated the coumarins scopoletin (I), scopoletin 7-0--D-glucopyranoside (II) and the new coumarin glycoside haploperoside A (III), mp 212–213°C, [] _D ²² –37° (c 0.24, CH₃OH). The acid hydrolysis of (III) formed (I) and the monosaccharides D-glucose and L-rhamnose. Partial hydrolysis of (III) with 10% acetic acid led to (II) and L-rhamnose. On the basis of the results of a study of UV, IR, and PMR spectra, and also periodate oxidation and polarimetric analysis the structure of 6-methoxy-7-[0--L-rhamnopyranosyl-(21)--D-glucopyranosyloxy] coumarin has been established for (III). Details of the IR, UV, PMR, and mass spectra are given.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenni, No. 2, pp. 168–172, March–April, 1980.