Phytochemical studies and cytotoxicity evaluations of Colchicum tunicatum Feinbr and Colchicum hierosolymitanum Feinbr (Colchicaceae): two native Jordanian meadow saffrons

As a part of our continuing investigation of Jordanian Colchicum species, the biologically active components of Colchicum hierosolymitanum Feinbr and Colchicum tunicatum Feinbr (Colchicaceae) were pursued. The brine shrimp lethality test (BSLT) was used to direct the fractionation and isolation of active components. Five and four known colchicinoids were isolated and characterized from C. tunicatum and C. hierosolymitanum, respectively. The known colchicinoids, reported for the first time from these two species are: (-)-colchicine (I), 3-demethyl-(-)-colchicine (II), (-)-cornigerine (III), beta-lumicolchicine (IV), and (-)-androbiphenyline (V) from C. tunicatum, and (-)-colchicine (I), 2-demethyl-(-)-colchicine (VI), (-)-cornigerine (III), and beta-lumicolchicine (IV) from C. hierosolymitanum. The chemical structures of the isolated compounds have been elucidated using a series of spectroscopic and spectrometric techniques principally; 1D-NMR (1H and 13C) and low resolution EI-MS and APCIMS. All pure compounds were evaluated for cytotoxicity against three human cancer cell lines; MCF-7 human breast carcinoma, NCI-H460 human large cell lung carcinoma, and SF-268 human astrocytoma. (-)-Colchicine (I) and (-)-cornigerine (III) were found to be the most bioactive of the identified compounds with EC50 values in the range of 0.016-0.097 microM.     

Seasonal variation of colchicine content in Colchicum brachyphyllum and Colchicum tunicatum (Colchicaceae)

As part of our continuing investigation of Jordanian Colchicum species, (-)-colchicine content in C. brachyphyllum Boiss. & Haussk. ex Boiss and Colchicum tunicatum Feinbr (Colchicaceae), growing wild in Jordan, was determined during different growth stages. Using external reference standard, a reverse-phase gradient photo-diode array high performance liquid chromatography (PDA-HPLC) method was adapted. Underground parts in both species and during different growth stages, always showed higher (-)-colchicine content than the above ground parts. In C. brachyphyllum total (-)-colchicine content of underground parts during flowering stage was found to be about 0.15% (wt/wt), while that of aerial parts was only about 0.04% (wt/wt). In C. tunicatum total (-)-colchicine content of underground parts was found to be 0.12% (wt/wt), and 0.13% (wt/wt) during flowering and vegetating stages, respectively, while that of aerial parts was only about 0.04% (wt/wt) and 0.02% (wt/wt), respectively. In C. tunicatum, stems, roots and unripe seeds are the main storages of (-)-colchicine at flowering, vegetating and seeding stages, respectively, while in C. brachyphyllum, corms are the main storage of (-)-colchicine at flowering and seeding stages.     

Comparison of analytical and semi-preparative columns for high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance

The application of analytical and semi-preparative columns in reversed-phase liquid chromatography-solid-phase extraction-nuclear magnetic resonance (HPLC-SPE-NMR) was compared. The work was aiming at separating a higher sample amount in a single run and in this way to reduce the necessary NMR measurement time of separated compounds. Several parameters for compound separation and trapping procedures were optimised: flow rate of HPLC and make-up water pumps, choice of stationary phase cartridges and drying time. The separation and loadability of nine model compounds on analytical and semi-preparative columns was determined, as well as the focussing capacity of SH-type SPE cartridges. It was found that a semi-preparative column--or multiple peak trapping on analytical columns--gave better results than a standard 4.6mm analytical column for non-polar compounds (e.g. flavonoid aglycones, sesquiterpene lactones, non-polar terpenes, logP>2), but for polar compounds (logP<-2) did not offer any advantage over an analytical column, or was even disadvantageous. For intermediately polar compounds (-2相似文献   

A comparative study of upright counter-current chromatography and high-performance liquid chromatograpohy for preparative isolation and purification ofphenolic compounds from Magnoliae officinalis

A comparative study of preparative isolation and purification of the phenolic compounds magnolol and honokiol from the Chinese medicinal plant Magnoliae officinalis by upright counter-current chromatography (CCC) and semi-preparative HPLC is presented. The comparison reveals that with a two-phase solvent system composed of light petroleum (bp 60-90 degrees C)-ethyl acetate-tetrachloromethane-methanol-water (1:1:8:6:1, v/v), 1250 mg of honokiol and 520 mg of magnolol, with a purity of 98.7 and 99.5%, respectively, were obtained from 2.0 g of a crude sample of Magnoliae officinalis in a single CCC separation. In contrast, semi-preparative HPLC allowed isolation and purification of these two phenolic compounds with significantly lower productivity and higher solvent consumption. Structures of the purified compounds were identified by 1H and 13C NMR.     

半制备型液相色谱法分离纯化茶叶鲜叶中7种儿茶素类化合物

建立了从茶叶鲜叶中分离纯化7种儿茶素类化合物(没食子儿茶素(GC)、表没食子儿茶素(EGC)、儿茶素(C)、表没食子儿茶素没食子酸酯(EGCG)、表儿茶素(EC)、表没食子儿茶素3-O-(3-O-甲基)没食子酸酯(EGCG3"Me)和表儿茶素没食子酸酯(ECG))的半制备色谱法。铁观音鲜叶经甲醇超声浸提、浓缩、氯仿萃取后,向水相中加入碱式醋酸铅沉淀,得到茶多酚粗品。分别以甲醇-水、乙腈-水作为流动相,采用半制备色谱法纯化7种儿茶素类化合物,纯度均达到90%。此外,利用同样的方法分离纯化另外两种茶叶鲜叶中的7种儿茶素类化合物,得到相似的结果。该方法以溶剂提取、离子沉淀结合半制备色谱,适于简单、高效地同时分离制备多种儿茶素类化合物。     

Identification of shikonin and its ester derivatives from the roots of Echium italicum L.

Nine shikonin pigments: shikonin (S), acetylshikonin (AS), propionylshikonin (PS), isobutyrylshikonin (IBS), tiglylshikonin (TS), 3,3-dimethylacrylshikonin (DAS), angelylshikonin (ANS), 2-methyl-n-butyrylshikonin (MBS) and isovalerylshikonin (IVS) were identified in the root epidermis of Echium italicum L. for the first time. A new thin-layer chromatographic (TLC) method for the separation of enantiomers alkannin and shikonin proved only shikonin after saponification of the root extract, and was afterwards esterified with the corresponding acyl chloride to acquire seven standard compounds (all except ANS). The developed isocratic high-peformance liquid chromatographic (HPLC) methods with VIS and mass spectrometry (MS) detection, allowed for the first time simultaneous separation of all nine compounds with similar structures including positional and geometric isomers in a short time. Structures of the main five compounds (AS, IBS, ANS, MBS, IVS) isolated from the extract by a new semi-preparative HPLC on C18 have additionally been confirmed by ¹H and ¹³C nuclear magnetic resonance spectra, which were reported for AS and MBS for the first time.     

Degraded and oxetane-bearing limonoids from the roots of Melia azedarach

Brine shrimp lethality test (BST)-guided fractionation of a methanol extract of the roots of Melia azedarach resulted in the isolation of two new limonoids, 9alpha-hydroxy-12alpha-acetoxyfraxinellone (1) and 7,14-epoxy-azedarachin B (2), together with the known compounds, 12alpha-hydroxyfraxinellone (4), 9alpha-hydroxyfraxinellone (5), azedarachin B (6), and neoazedarachin B (7). The structures of 1 and 2 were elucidated by analysis of spectroscopic data and comparison of their NMR data with those of the known compounds. Compounds 1, 2 and 4-7 exhibited significant activity in the BST, in particular, azedarachin B (6) showed remarkable BST activity with an LC(50) value of 0.0098 microM.     

Brine shrimp lethality test active constituents and new highly oxygenated seco-prezizaane-type sesquiterpenes from Illicium merrillianum

In the study of bioactive substances in Illicium plants, the methanol extract of I. merrillianum showed brine shrimp lethality test (BST) activity at 200 microg/ml. Bioassay-guided fractionation of the BST active fractions resulted in the isolation of 4-O-methyleudesm-11-en-4alpha-ol, eudesmol-11-en-4alpha-ol and (-)-hinokinin as potent BST active compounds. On the other hand, four new highly oxygenated seco-prezizaane-type sesquiterpenes, merrilliortholactone (1), 2alpha-hydroxycycloparvifloralone (2), 2alpha-hydroxycycloparviflorolide (3), and 2alpha-hydroxyanisatin (4) were isolated from the BST-inactive polar fractions. The structures of new compounds were elucidated by extensive analyses of spectral data. Furthermore, the absolute configuration of 3 was established by the modified Mosher's method. Compounds 1--4 showed neither BST activity at 100 microg/ml nor neurite outgrowth-promoting activity.     

Purification method for the isolation of monophosphate nucleotides from Champagne wine and their identification by mass spectrometry

Monophosphate nucleotides are difficult to identify in Champagne wine because they are present in small concentrations in a complex mixture. A method for the isolation, separation and identification of reference compounds, which achieved on average 79% recovery (except for cytidine derivatives), was developed and applied to wine. Some monophosphate nucleotides were then isolated from a Champagne wine aged on lees for 8 years, by ultrafiltration followed by a semi-preparative HPLC step using a strong anion-exchange column. The fraction obtained was subjected to HPLC in a reversed-phase column to remove the salt previously introduced, before identification of compounds by HPLC coupled to a mass spectrometer. For the first time in wine, 5'-IMP, 5'-AMP, 5'-CMP, 5'-GMP, 5'-UMP and the 3'- and/or 2'-isomers of the four latter compounds were identified by comparing their HPLC and electrospray ionization mass spectrometry data with those of reference nucleotides.     

Analytical and semi-preparative HPLC enantioseparation of novel pyridazin-3(2H)-one derivatives with α-aminophosphonate moiety using immobilized polysaccharide chiral stationary phases

The direct HPLC enantioseparation of a novel series of chiral pyridazin-3(2H)-one derivatives with α-aminophosphonate moiety was performed on two immobilized polysaccharide chiral stationary phases (Chiralpak IA, Chiralpak IC) using n-hexane (n-Hex)/dichloromethane (DCM) mobile phase with 5% alcohol additive. Good baseline separation of the enantiomers was achieved using amylose tris-(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA) on analytical scale. The analytical method was further scaled up to semi-preparative loading to obtain small amounts of both the enantiomers of pyridazin-3(2H)-one derivative. The semi-preparative resolution of all compounds was successfully achieved with n-hexane/dichloromethane/ethanol (EtOH) as mobile phase using a semi-preparative Chiralpak IA column. The first fractions were isolated with purities of >99.9% (enantiomeric excess (e.e.), and the second fractions were obtained with purities of >98.2% (enantiomeric excess). The assignment of the absolute configuration was established for the F1 fraction of compound a-2 by single-crystal X-ray diffraction method.     

Rapid preparative isolation of a new phenylpropanoid glycoside and four minor compounds from Sparganium stoloniferum using high-speed counter-current chromatography as a fractionation tool

A rapid high-speed counter-current chromatography (HSCCC) method was used to isolate five minor compounds from rhizome of Sparganium stoloniferum namely San Leng in Chinese, including two phenylpropanoid glycosides, sparganiaside A (1) and 1-O-feruloyl-3-p-coumaroylglycerol (2), and three aromatic acids, vanillic acid (3), p-hydroxylcinnamic acid (4), and p-hydroxybenzoic acid (5), of which, compound 1 was a new one. Five compounds were preparatively enriched at top efficiency by one-step HSCCC operation in the isolation procedure. A suitable solvent system composed of chloroform-methanol-water (4:3.5:1.8, v/v/v) was used. And the operation time was less than 4 h. The purities of compounds (1-5) in the enriched fractions were determined to be 75.8%, 66.3%, 90.6%, 79.9%, and 98.2%, respectively. The mean recoveries of the five compounds were 84.8%, 87.3%, 81.8%, 90.3%, and 92.7%, respectively. Compounds 1-4 were further purified by semi-preparative high-performance liquid chromatography (HPLC). This is the first report on the use of HSCCC as a fractionation tool for preparative isolation of minor compounds from S. stoloniferum. The method was proved to be rapid, convenient, high yield, and low cost. HSCCC was shown to be a quick and effective tool in isolation of natural products even though the compounds were not abundant.     

Triterpenoids from swallow roots--a convenient HPLC method for separation

A convenient semi-preparative high-performance liquid chromatography (HPLC) method for separating a mixture of triterpenoids (alpha-amyrin, beta-amyrin, and lupeol) and their corresponding acetates from the swallow roots (Decalepis hamiltonii Wight and Arn), which are known to have potential bioactive properties, is described. The swallow roots are found to be one of the richest natural sources for these compounds. The hexane extract of the dried spent root on column chromatography yields mixtures (i.e., triterpenoids and their acetates) containing at least three compounds in each. These could not be further separated using the routine chromatographic techniques, such as classical column chromatography and preparative thin-layer chromatography using various solvent systems. Therefore, the optimal conditions are determined on reversed-phase HPLC for their separation and are characterized using spectral data, particularly by nuclear magnetic resonance with physical and chemical properties.     

黄花棘豆种子中化学成分的研究

用半制备HPLC方法从黄花棘豆(OxytropisochrocephalaBunge)种子水溶性部分中分到一个新的黄酮苷和4个已知化合物。经HPLC保留时间、红外光谱、紫外光谱、核磁共振氢谱、质谱等方法推定新黄酮苷的结构2为5-甲氧基-7-羟基-3-氧-β-D-半乳糖-4'-氧-β-D-葡萄糖黄酮醇苷。     

Purification and quantification of destruxins A and B from Metarhizium anisopliae

Purification of insecticidal cyclodepsipeptides, destruxins A (DA) and B (DB), from Metarhizium anisopliae fermentation broth was performed. The isolation scheme of these destruxins using ion-exchange chromatography, silica gel chromatography, and semi-preparative HPLC chromatography is presented. The quality of the semi-preparative products was unique. Over 90% purity (based on HPLC chromatograms) was achieved for both DA and DB. Purified destruxins were further identified employing the ¹H NMR and fast atom bombardment MS methodology. The HPLC quantification methods for DA and DB in the fermentation broth have been established. The use of the purified destruxins as analytical standards demonstrated good correlation.     

Microfluidic reactions using [11C]carbon monoxide solutions for the synthesis of a positron emission tomography radiotracer

Microfluidic technology has been used to perform [(11)C]carbonylation reactions using solutions containing [(11)C]CO in the form of the complex, copper(i)tris(3,5-dimethylpyrazolyl)borate-[(11)C]carbonyl (Cu(Tp*)[(11)C]CO). The synthesis of the model compound [(11)C]N-benzylbenzamide and the known tracer molecule [(11)C]trans-N-[5-(2-flurophenyl)-2-pyrimidinyl]-3-oxospiro[5-azaisobenzofurane-1(3H),1'-cyclohexane]-4'-carboxamide ([(11)C]MK-0233), a ligand for the neuropeptide Y Y5 receptor, have been performed using this technique. Following semi-preparative HPLC purification and reformulation, 1262 ± 113 MBq of [(11)C]MK-0233 was produced at the end of the synthesis with a specific activity of 100 ± 30 GBq μmol(-1) and a >99% radiochemical purity. This corresponds to a decay corrected radiochemical yield of 7.2 ± 0.7%. Using a 3 mL vial as the reaction vessel, and following semi-preparative HPLC purification and reformulation, 1255 ± 392 MBq of [(11)C]MK-0233 was produced at the end of the synthesis with a specific activity of 100 ± 15 GBq μmol(-1) and a >99% radiochemical purity. This corresponds to a decay corrected radiochemical yield of 7.1 ± 2.2%.     

Rapid structural determination of new trace cassaine-type diterpenoid amides in fractions from Erythrophleum fordii by liquid chromatography-diode-array detection/electrospray ionization tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance

Electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn), and combination with HPLC (HPLC/ESI-MSn), have been extensively applied to on-line analysis of natural products. Hyphenation of liquid chromatography to nuclear magnetic resonance spectroscopy (HPLC/NMR) has been developed in the last decade, which is utilized for the analysis of metabolites and drug impurities. In the study reported here, the fragmentation behaviors of eight cassaine-type diterpenoid amides from Erythrophleum fordii were investigated by ESI-MSn. The fragmentation rules and NMR spectral characteristics are summarized, and the relationship among the rules, characteristics and the structures is described. According to the fragmentation rules and NMR spectral characteristics, seven trace constituents and two formerly obtained compounds of cassaine-type diterpenoid amides in the fractions from E. fordii were structurally characterized on the basis of HPLC/HRMS, HPLC-DAD/ESI-MSn, HPLC/1H NMR and HPLC/1H-1H COSY rapidly. Among them, constituents 1-5 are new compounds, and 6 and 7 are reported from E. fordii for the first time. The aim is to develop an effective analytical method for structural identification of new trace natural products in plants.     

Separation of atropisomeric 1,4,5,6-tetrahydropyrimidinium salts by chiral HPLC and determination of their enantiomerization barriers

Chiral HPLC separation of a series of novel atropisomeric quaternary (1) and ternary (2) 1,2-disubstituted 1,4,5,6-tetrahydropyrimidinium salts bearing disymmetric aryl groups in positions 1 and/or 2 is described. A screening of different polysaccharide stationary phases (OD-R, OJ-R and AD-RH) and chromatographic conditions allowed for partial or baseline resolution of 16 over 26 compounds. When a semi-preparative separation was achieved, the corresponding enantiomerization barriers were determined employing the off-column method. The experimental data were compared inter se in order to establish the factors influencing the magnitude of the barriers and with those corresponding to the parent amidines.     

New insecticidal amides from petroleum ether extract of dried Piper nigrum L. whole fruits

The petroleum ether extract of dried ground whole fruits of Piper nigrum L. afforded 20 compounds (1-20) including two new insecticidal amides named as pipnoohine (1), and pipyahyine (2), seven reported for the first time from this plant (12, 13, 15-17, 19, 20), and eleven known compounds (3-11, 14, 18). The structure of 1 has been elucidated as (2E,4E,12Z)-N-(4-methylpentyl)octadeca-2,4,12-trienamide and that of 2 as (2E,4E,11E)-12-(benzo[1,3]dioxol-5-yl)-N-(3-methylbutyl)dodeca-2,4,11-trien-amide through extensive ID-, 2D-NMR spectral studies and chemical reactions. The known compounds have been identified through comparison of their spectral data with those reported in literature. 1 and 2 exhibited toxicity at 35.0 and 30.0 ppm respectively against fourth instar larvae of Aedes aegypti L. by WHO method.     

Use of solid-phase microextraction for measuring oil-water partition coefficients and correlation with high-performance liquid chromatographic methods for lipophilicity

For flavour compounds, lipophilicity is often estimated by the partition coefficient between oil and water (log Koil-water), which is highly relevant to food. A modification of the shake-flask method is reported here where compounds are quantified in the two phases using solid-phase microextraction (SPME). SPME's highly sensitivity to non-polar compounds facilitates quantification in the water phase. Twelve flavour compounds representing a broad range of lipophilicities and functional groups were analysed by two methods. Their log Koil-water was determined using SPME quantitation and their log k(w) using a reversed-phase HPLC methodology. The isocratic capacity factor at 60% methanol and predicted log P value also showed high correlation factors with other methods. The octadecyl silylated surface of the HPLC column provides a matrix that interacts with lipophilic compounds where the retention time is the indication of lipophilicity. Both methods gave reproducible results (median 3% and 4% RSD) and similar but not identical values for lipophilicity. The relationship between the two methods is log k(w) =0.85 log Koil-water +0.48 with a correlation coefficient of 0.94. The new SPME detection method, with the ability to quantify limonene and 2-pentylfuran at 1 ppm in the water phase, is preferred for flavour compound analysis due to the applicability of oil-water partitioning in food.     

Isolation of isomangiferin from honeybush (Cyclopia subternata) using high-speed counter-current chromatography and high-performance liquid chromatography

Isomangiferin was isolated from Cyclopia subternata using a multi-step process including extraction, liquid–liquid partitioning, high-speed counter-current chromatography (HSCCC) and semi-preparative reversed-phase high-performance liquid chromatography (HPLC). Enrichment of phenolic compounds in a methanol extract of C. subternata leaves was conducted using liquid–liquid partitioning with ethyl acetate–methanol–water (1:1:2, v/v). The enriched fraction was further fractionated using HSCCC with a ternary solvent system consisting of tert-butyl methyl ether–n-butanol–acetonitrile–water (3:1:1:5, v/v). Isomangiferin was isolated by semi-preparative reversed-phase HPLC from a fraction containing mostly mangiferin and isomangiferin. The chemical structure of isomangiferin was confirmed by LC–high-resolution electrospray ionization MS, as well as one- and two-dimensional NMR spectroscopy.