Selective separation of structure-related alkaloids in Rhizoma coptidis with "click" binaphthyl stationary phase and their structural elucidation with liquid chromatography-mass spectrometry

It is a new task to separate structure-related compounds into a fraction according to their structural characteristics in a complex traditional Chinese medicine (TCM). This method makes separation of the components of the sample simple and structural elucidation easy. In this study, selective separation of alkaloids in Rhizoma coptidis was realized on a "click" binaphthyl column possessing a planar conjugate structure. Three kinds of alkaloids, aporphine, tetrahydroprotoberberine and protoberberine in Rhizoma coptidis showed better retention than other compounds by virtue of π-π interactions with the stationary phase. Moreover, the "click" binaphthyl column could distinguish the aporphine and tetrahydroprotoberberine alkaloids possessing two benzene rings from the protoberberine alkaloids possessing three benzene rings. After separating on the "click" binaphthyl column, the fractions containing the alkaloids were collected and then analyzed with liquid chromatography-mass spectrometry (LC-MS). Totally, 23 alkaloids were identified, and among these alkaloids, three tetrahydroprotoberberine, two aporphine and seven protoberberine alkaloids were first found in Rhizoma coptidis. These newly found alkaloids are minor compounds, and they are always neglected without eliminating the interference of compounds in large amounts by pre-separation on the "click" binaphthyl column. The typical fragmentation pathways of each class of alkaloids were summarized to illustrate their structures. In the MS(2) spectrum, the loss of a molecule of dimethylamine ((CH(3))(2)NH) was observed as the characteristic loss of aporphine alkaloids. All the tetrahydroprotoberberine alkaloids would undergo the Retro-Diels-Alder (RDA) fragmentation reaction in the MS(2) fragmentation. For protoberberine alkaloids, different characteristic fragmentations were observed with different skeleton structures.     

One-pot synthesis of magnetic sulfonated covalent organic polymer for extraction of protoberberine alkaloids in herbs and human plasma

A novel magnetic sulfonated covalent organic polymer was prepared for magnetic solid-phase extraction of protoberberine alkaloids. The magnetic sulfonated covalent organic polymer was rapidly synthesized under mild conditions. The physicochemical properties of the prepared materials were characterized by Fourier-transform infrared spectrometry, transmission electron microscopy, and X-ray photoelectron spectroscopy. Several extraction parameters were systematically investigated, including desorption time, pH of sample solution, acetonitrile content, acetic acid content in the eluent, extraction time, and sample volume. By coupling magnetic solid-phase extraction and high-performance liquid chromatography, an efficient and sensitive method for the extraction and determination of protoberberine alkaloids in complex samples was developed. The proposed method showed great linearity (r > 0.9989), low limits of detection (0.2–0.3 ng/ml), and high precision (relative standard deviations ≤ 5.74%). The proposed method was further applied to the analysis of protoberberine alkaloids in Cortex phellodendri and human plasma samples. The recoveries were 91.50%–110.31% with relative standard deviations less than 6.63% in Cortex phellodendri and 96.12%–111.20% with relative standard deviations lower than 5.56% in plasma samples.     

Structural study of 8-azole derivatives of protoberberine alkaloids: experimental and quantum chemical approach

New derivatives of protoberberine alkaloids were prepared by nucleophilic addition of some azoles (differing in bulkiness) to the iminium functionality of the quaternary protoberberine alkaloids. Compounds were structurally characterized mainly by ¹H and ¹³C NMR spectroscopy, and the structure of 8-carbazol-1-yl-7,8-dihydroberberine was determined using single-crystal X-ray diffraction. Additionally, conformational behaviors of five derivatives varying in bulkiness of the azole moiety have been investigated by low temperature NMR spectroscopy and quantum chemical calculations. Ring current effects of pyrrole and carbazole moieties on selected ¹H NMR resonances have been characterized, visualized, and discussed.     

High performance liquid chromatography-mass spectrometry analysis of protoberberine alkaloids in medicine herbs

RP-HPLC is the main method for the analysis of alkaloids. However, peak tailing is a problem that commonly occurs in the separation of alkaloids. In order to overcome this, three kinds of RP columns were compared for the analysis of protoberberine alkaloids in Coptidis Rhizoma and Phellodendri Cortex in this work. XTerra MS C18 column was the best one which gave the best symmetry factor under the same conditions. With this column, a good separation of the crude extracts of C. Rhizoma and P. Cortex was achieved using 0.1% v/v formic acid buffer and methanol as mobile phase. At the same time, the crude extracts of C. Rhizoma and P. Cortex were analyzed by the LC-ESI-MSn and LC-atmospheric pressure chemical ionization (APCI)-MSn methods. In the analysis of HPLC-ESI/MSn, structures of five protoberberine alkaloids were elucidated, compared to authentic standards, and data from the literature. At the same time, the structure of a novel compound was elucidated. In the HPLC-APCI/ MSn analysis, there was an interesting phenomenon that the relative abundance of the ions M+ and [M + 2]+ was different for different alkaloids. The possible fragmentation pathways of protoberberine alkaloids in APCI/MS analysis were studied for the first time in the present work.     

Characterization of protoberberine alkaloids in Coptidis Rhizoma (Huanglian) by HPLC with ESI‐MS/MS

This study aims to qualitatively analyze protoberberine alkaloids in crude extract of Coptidis Rhizoma using HPLC with ESI‐MS/MS. Possible specific molecular weights of protoberberine alkaloids were firstly deduced according to literatures and were adopted to screen the alkaloids in the HPLC with ESI‐MS of crude extract of Coptidis Rhizoma. As a result, 21 protoberberine alkaloids were found, including compounds of very low concentration and compounds coeluted in one peak. Among these, two compounds were positively identified and verified by comparison with standards. Ten of these compounds were first reported in this study for Coptidis Rhizoma. In addition, chromatographic retention parameters a and c of all compounds were obtained using their retention times under five gradient conditions and were applied to confirm the deduction about the structures of protoberberine alkaloids by tandem mass data.     

Synthesis of protoberberine alkaloids

In this review an analysis is made of literature information on the synthesis of protoberberine alkaloids during 1980–1990.Institute of Chemistry of Plant Substances, Uzbekistan Republic Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 449–468, September–October, 1992.     

Calorimetric study of the effect of protoberberine alkaloids in Coptis chinensis Franch on Staphylococcus aureus growth

The effects of five protoberberine alkaloids (PBAs) from rhizoma of Coptis chinensis Franch on Staphylococcus aureus growth were investigated by calorimetry. The power-time curves of S. aureus with and without PBA were measured in closed glass ampoules in a TAM Air isothermal calorimeter. And, the extent and duration of inhibitory effects on the metabolism were evaluated by growth rate constant (k), half inhibitory ratio (IC₅₀), maximum heat-output (P_max) and peak time (t_p). The obtained calorimetric data showed that the inhibitory action varied for different protoberberine alkaloid. The results also revealed that the sequence of antimicrobial activity of five PBAs was: berberine>coptisine>palmatine>epiberberine>jatrorrhizine. One explanation could be substitutions at several positions in the core structure of berberine possess different antimicrobial activity. In conclusion, it can be proposed that this technique should be as a useful analytical method for determining the bioactivity of PBAs.     

Simultaneous analysis of protoberberine, indolequinoline and quinolone alkaloids in coptis-evodia herb couple and the Chinese herbal preparations by high-performance liquid chromatography-electrospray mass spectrometry

A new and sensitive method has been developed for analysis of protoberberine, indolequinoline and quinolone alkaloids in coptis-evodia herb couple and the Chinese herbal preparations by using HPLC in combination with electrospray mass spectrometry (ESI-MS). Different LC and cone voltages have been optimized prior to this in order to obtain better results and sensitivity. The method was carried out by using a spherigel C₁₈ analytical column with a gradient solvent system of acetate buffer-acetonitrile-methanol. The contents of alkaloids were measured by pseudomolecule ion on the selective ion recording (SIR) mode. Linearity of around three orders in the magnitude of concentration was generally obtained and limits of detection for these compounds were in the range of 9-30 pg.     

Quality evaluation and species differentiation of Rhizoma coptidis by using proton nuclear magnetic resonance spectroscopy

Rhizoma coptidis, a broadly used traditional Chinese medicine, derives from the dried rhizomes of Coptis chinensis Franch, Coptis deltoidea C.Y. Cheng et Hsiao and Coptis teeta Wall. Quantitative determination of protoberberine alkaloids in R. coptidis is critical for controlling its quality. In this study, a rapid, simple and accurate quantitative ¹H NMR (qNMR) method was developed for simultaneous determination of berberine, jatrorrhizine, epiberberine, coptisine, palmatine and columbamine in R. coptidis from the three species. Method validation was performed in terms of selectivity, precision, repeatability, stability, accuracy, robustness and linearity. The average recoveries obtained were in the range of 96.9–102.4% for all the six alkaloids. In addition, the qNMR data were analyzed with analysis of variance (ANOVA), hierarchical clustering analysis (HCA) and principal component analysis (PCA), and the results showed that the contents of the active alkaloids have significant difference among the three species. Compared with the conventional HPLC approach, the proposed qNMR method was demonstrated to be a powerful tool for quantifying the six alkaloids due to its unique advantages of high robustness, rapid analysis time and no need of standard compounds for calibration curves preparation. These findings indicate that this method has potential as a reliable method for quality evaluation of herb medicines, especially for protoberberine alkaloid-containing ones.     

The orientation of protoberberine alkaloids and their binding activities to human serum albumin by surface-enhanced Raman scattering

Raman and surface-enhanced Raman scattering (SERS) technique are reliably used to compare relative intensity shifts and to investigate the adsorption geometry of protoberberine alkaloids on Ag nanoparticles. We report joint application of fluorescence and SERS spectroscopy to study the interaction between protoberberine alkaloids and human serum albumin (HSA). We propose SERS technique to improve the quenching interaction caused by protoberberine alkaloids which are used to be applied in recognition process of fluorescent drugs with large biomolecules. The fluorescence results show that the fluorescence intensity of HSA is significantly decreased in presence of protoberberine alkaloids. The SERS technique demonstrates obvious advantages over direct measurements in discriminating and identifying pharmaceutical molecules. By means of this method, we are able to detect important information concerning the orientation of protoberberine alkaloids when interacting with HSA. We also show that the nitrogen atom is free, but a benzene ring and two adjacent methoxy groups are involved in the spontaneously electrostatic inducement and subsequently binding with HSA.     

Convenient synthesis of 2,3,9,10-tetraoxygenated protoberberine alkaloids and their 13-methyl alkaloids

New and convenient synthesis of 2,3,9,10-tetraoxygenated protoberberine alkaloids and their 13-methyl alkaloids through the same intermediates was developed. Acylation of the brominated benzylphenethylamine (13) with alpha-chloro-alpha-(methylthio)acetyl chloride, followed by cyclization with stannic chloride, furnished the key intermediates 4-methylthio-3-phenethylisoquinolin-3-ones (14), which were methylated to provide their methyl derivatives (17). Both isoquinolin-3-ones (14, 17) were easily transformed into protoberberine alkaloids (16) and their 13-methyl alkaloids (21) in good yield.     

Simultaneous determination of the content of isoquinoline alkaloids in Dicranostigma leptopodum (Maxim) Fedde and the effective fractionation of the alkaloids by high‐performance liquid chromatography with diode array detection

A simple and efficient method was developed for the simultaneous determination of eight isoquinoline alkaloids in methanol extracts of Dicranostigma leptopodum (Maxim) Fedde and the effective fractionation of the alkaloids of D. leptopodum by high‐performance liquid chromatography with diode array detection. The chromatographic conditions were optimized on a SinoChrom ODS‐BP column to obtain a good separation of the four types of alkaloid analytes, including two aporphines (isocorydine, corydine), two protopines (protopine and allocryptopine), a morphine (sinoacutine), and three quaternary protoberberine alkaloids (berberrubine, 5‐hydroxycoptisine, and berberine). The separation of these alkaloids was significantly affected by the composition of the mobile phase, and particularly by its pH value. Acetonitrile (A) and 0.2% phosphoric acid solution adjusted to pH 6.32 with triethylamine (B) were selected as the mobile phase with a gradient elution. With this method, a new quaternary protoberberine alkaloid was isolated and the two structural isomers (isocorydine and corydine) were baseline separated. The appropriate harvest period for D. leptopodum was also recommended based on our analysis. The method for the effective fraction of the alkaloids of D. leptopodum was optimized under this method with regard to the varying significant pharmacological activities of the alkaloids.     

Copper(II)-cetyltrimethylammonium chloride-chromal blue G ternary complex and its application to the spectrophotometric determination of copper(II)

A sensitive spectrophotometric method for the determination of copper(II) based on a ternary complex with chromal blue G, a triphenylmethane reagent in the presence of cetyltrimethylammonium chloride, is described. The sensitivity of color reaction between copper and chromal blue G has been greatly increased by the sensitizing action of cetyltrimethylammonium chloride, a cationic surfactant. The color development of the ternary complex can be utilized in the highly sensitive spectrophotometric determination of copper. The molar absorptivity of the binary complex between copper and chromal blue G ε_630nm = 9.56 × 10³liters · mol⁻¹ · cm⁻¹ is enchanced on ternary complex formation to ε_{542 nm} = 4.78 × 10⁴liters · mol⁻¹ · cm⁻¹. The ternary complex gave a maximal absorbance at 542 nm in the pH range 9.8–11. Beer's law is obeyed up to at least 1.2 ppm of copper. The maximal absorbance of the ternary complex was found to develop within 5 min and then it remains constant for several hours. The formation constant of the ternary complex is calculated to be 8.6 × 10¹⁰ under these conditions.     

Studies on kinetics of complexation of lanthanide α-hydroxycarboxylates with 1,10-phenanthroline

Kinetics of the coordination reaction of lanthanide (La^III, Eu^III) α-hydroxycarboxylates [LnL₃(H₂O)₂] with 1,10-phenanthroline (phen) in methanol-water (v/v, 3:2) were studied at 25°C by calorimetric titration. A one-step reaction process in accordance with the rate law has been suggested. The reaction is found to be first order for both lanthanide α-hydroxycarboxylates and phen. We have evaluated rate constants of the reactions. It is found that a linear free energy relationship exists between the stability constants of the lanthanide-α-hydroxycarboxylate-phen ternary complex and the rate constants. It is also found that a linear free energy relationship exists between the rate constants of La-hydroxycarboxylate with phen and the acid strength of α-hydroxy-acid as primary ligand, but the linear free energy relationship does not exist in the Eu-α-hydroxycarboxylate-phen ternary complex. The influence of other factors upon the reaction rate constants was also discussed.     

Capillary isotachophoresis of some isoquinoline alkaloids

The isotachophoretic behavior of quaternary benzo/c/phenanthridine, protoberberine and aporphine alkaloids in different electrolyte systems is described. The concentration of the leading ion and the pH value of the leading electrolyte affect the relative effective mobilities of the alkaloids. The system of pH 4.7, containing the leading ion K⁺ (0.005 M) counter ion acetate, and the terminating ion β-alanine (0.02 M), has been selected for the quantitative determination of the studied alkaloids in model mixtures and plant extracts.     

A review on analytical methods for natural berberine alkaloids

Berberine alkaloids, a group of protoberberine alkaloids under the classification of isoquinoline alkaloids, include berberine, coptisine, palmatine, columbamine, dehydrocorydaline, jatrorrhizine, and epiberberine from natural sources. Studies have shown that berberine alkaloids have various pharmacological functions, such as antibacterial, antiviral, blood pressure‐lowering, hypoglycaemic, antiarrhythmia, and anticancer effects. Therefore, it is worthwhile to develop analytical methods to investigate the pharmacokinetics and activity mechanisms of berberine alkaloids and to study berberine alkaloids more comprehensively. Current analytical methods for berberine alkaloids include liquid chromatography, thin‐layer chromatography, ultraviolet spectroscopy, capillary electrophoresis, and gas chromatography. The most widely used detection method is mass spectrometry. In order to provide a systematic and comprehensive summary and to serve as a reference for the future pharmacokinetics studies and analysis of berberine alkaloids, analytical methods for natural berberine alkaloids that have been used in the past ten years are reviewed here.     

Chemical Constituents from the Stems of Mahonia Japonica

A new dihydroberberine alkaloid, 7,8‐dihydro‐8‐methoxyberberine ( 1 ), along with six known compounds including two dihydroberberine alkaloids, 7,8‐dihydro‐8‐hydroxyberberine ( 2 ) and oxyberberine ( 3 ) and four protoberberine alkaloids, berberine ( 4 ), palmatine ( 5 ), jatrorrhizine ( 6 ) and columbamine ( 7 ), were isolated from the stems of Mahonia japonica. These compounds were characterized and identified by physical and spectral evidence.     

Quantitative Determination of Three Protoberberine Alkaloids in Jin-Guo-Lan by HPLC-DAD

An accurate and simple HPLC method for the simultaneous determination of three protoberberine alkaloids (columbamine, jatrorrhizine, and palmatine) contained in Chinese medicine Jin-Guo-Lan (Tinospora sagittata Oliv. and Tinospora capillipes Gagnep) is presented in this study. The herb samples from six main origins and three herb markets were investigated. The separation was performed on a YMC-C₁₈ ODS column at 30°C with a gradient elution program. Acetonitrile and phosphate buffer (0.02 mol L⁻¹ sodium dihydrogen phosphate and 0.01 mol L⁻¹ triethylamine, pH 3) were used as mobile phases and the flow rate was set at 1 mL min⁻¹. The recovery of the method was in the range of 99.43–100.96%, and all the alkaloids showed good linearity (r ² > 0.9997) in the relatively wide concentration ranges. The developed method was applied to the determination of these alkaloids in the collected herb samples, and the results showed that the contents of these components in Jin-Guo-Lan varied greatly from habitat to habitat. It was demonstrated that the proposed method was helpful for the quality evaluation of Chinese medicine Jin-Guo-Lan.     

Isoquinoline alkaloids: a 15N NMR and x‐ray study. Part 2

Continuing our systematic ¹⁵N NMR study of isoquinoline alkaloids, we report a contribution extending our previous paper. The ¹⁵N NMR chemical shifts and ¹⁵N,¹H long‐range coupling pathways of tertiary and quaternary isoquinoline alkaloids of several constitutional types are presented. The selected compounds belong to the protoberberine, proaporphine, pavinane, rhoeadine and phtalideisoquinoline classes of alkaloids and were investigated by gradient‐selected inverse‐detected multiple bond correlation experiments (GHMBC and GSQMBC). In addition, x‐ray data and the principal geometric parameters of stylopine, mecambridine, norchelerythrine, isothebaine and mecambrine are reported and discussed. Copyright © 2002 John Wiley & Sons, Ltd.