Alkaloid from Dicranostigma leptopodum （Maxim） Fedde

Phytochemical investigation of the whole plants of Dicranostigma leptopodum (Maxim) Fedde has lead to the isolation of five alkaloids: dicranostigmine (1), isocorydine (2), corydine (3), protopine (4) and sinoacutine (5). Oxomorphine alkaloid (5) was isolated from the genus Dicranostigma for the first time and the new compound structure (1) was elucidated by various spectroscopic methods including 2D NMR techniques (gCOSY, HMQC, HMBC and NOESY) and HRESI-MS.     

Preparative separation of quaternary ammonium alkaloids from Corydalis yanhusuo W. T. Wang by pH-zone-refining counter-current chromatography

The optimal extraction condition for extracting quaternary ammonium alkaloid dehydrocorydaline from Corydalis yanhusuo W. T. Wang was investigated using orthogonal experimental design. pH‐zone‐refining counter‐current chromatography (CCC) with normal phase elution was successfully applied to preparative separation of alkaloids from the crude extract of Corydalis yanhusuo. The separation was performed with a biphasic solvent system composed of chloroform (CHCl₃)–methanol (MeOH)–water (2:1:1, v/v), in which the lower organic phase containing 10 mM of triethylamine was used as the mobile phase, while the upper aqueous phase containing 10 mM of hydrochloric acid was used as the stationary phase. The separation mechanism of quaternary ammonium alkaloids using pH‐zone‐refining CCC was discussed in comparison with standard high‐speed CCC. In the present study, the separation of 1.200 g of crude sample yielded 129 mg of dehydrocorydaline and 12 mg of palmatine at a high purity of 94 and 92%, respectively. Recovery for dehydrocorydaline and palmatine was 85 and 86%, respectively.     

pH‐Zone‐refining counter‐current chromatography for two new lipo‐alkaloids separated from refined alkaline extraction of Kusnezoff monkshood root

An efficient and refined method for the separation of six aconitine‐type alkaloids from the alkaline prepared “Kusnezoff monkshood root” was established. It is the first study that two new lipo‐alkaloids were successfully isolated from refined sample by pH‐zone‐refining counter‐current chromatography rather than synthetic method. It was of interest that a great deal of lipo‐alkaloids was produced in crude extract from the alkalization of “Kusnezoff monkshood root.” A refined sample method was proposed to enrich two types of alkaloids by liquid–liquid extraction, i.e. lipo‐alkaloids and monoester‐diterpenoid alkaloids. The pH‐zone‐refining counter‐current chromatography was performed with an optimized two‐phase solvent system composed of n‐hexane‐ethyl acetate–methanol–water (3:5:4:5, v/v), where upper organic phase was added to 3 mmol/L triethylamine as a retainer and lower aqueous mobile phase was added to 3 mmol/L hydrochloric acid as an eluter. As a result, six aconitum alkaloids, including two lipo‐alkaloids (8‐lino‐14‐benzoylaconine, 8‐pal‐14‐benzoylaconine), three monoester‐diterpenoid alkaloids (14‐benzoylmesaconine, 14‐benzoylaconine, beyzoyldeoxyaconine), and one aconine alkaloid (neoline) were acquired from the plant at the same time. The anti‐inflammatory activities of the two new lipo‐alkaloids were compared to the six alkaloids in vitro, in cyclo‐oxygen‐ase‐2 inhibition assays. The separation mechanism of six alkaloids by pH‐zone‐refining counter‐current chromatography was illustrated.     

Development of an HPLC‐DAD method for the determination of five alkaloids in Stephania yunnanensis Lo and in rat plasma after oral dose of Stephania yunnanensis Lo extracts

For the rational utilization and the quantitative quality control of the Stephania yunnanensis Lo, an HPLC‐DAD method was developed for the quantitative and simultaneous determination of five alkaloids in rat plasma (stepharine, sinomenine, palmatine, isocorydine and tetrahydropalmatine), which were the main active chemical constituents of this plant and belong to four kinds of isoquinoline‐type alkaloids (protoberberine, morphine, aporphine and protaporphine alkaloids). The contents of five alkaloids ranged from 0.09 to 2.32% (w/w). The method validation was tested for the linearity (r² > 0.9975), precision (intra‐day RSD < 4.8% and inter‐day RSD < 4.9%), extraction recovery (85.49 ± 2.29% to 99.21 ± 1.48%) and stability (98.5 ± 5.3% to 101.2 ± 3.4%). We developed an HPLC‐DAD method to simultaneously measure these alkaloids in rat plasma after oral administration of the extract of this plant to rats. The results supported the hypothesis that isoquinoline alkaloids were the compounds responsible for the main pharmacological activities for anti‐inflammatory and analgesic.     

Establishment of a search library about benzylisoquinoline alkaloids based on selective separation on the binaphthyl column and standard analysis on C18 column

A search library about benzylisoquinoline alkaloids was established based on preparation of alkaloid fractions from Rhizoma coptidis, Cortex phellodendri, and Rhizoma corydalis. In this work, two alkaloid fractions from each herbal medicine were first prepared based on selective separation on the “click” binaphthyl column. And then these alkaloid fractions were analyzed on C18 column by liquid chromatography coupled with tandem mass spectrometry. Many structure‐related compounds were included in these alkaloids fractions, which led to easy separation and good MS response in further work. Therefore, a search library of 52 benzylisoquinoline alkaloids was established, which included eight aporphine, 19 tetrahydroprotoberberine, two protopine, two benzyltetrahydroisoquinoline, and 21 protoberberine alkaloids. The information of the search library contained compound names, structures, retention times, accurate masses, fragmentation pathways of benzylisoquionline alkaloids, and their sources from three herbal medicines. Using such a library, the alkaloids, especially those trace and unknown components in some herbal medicine could be accurately and quickly identified. In addition, the distribution of benzylisoquinoline alkaloids in the herbal medicines could be also summarized by searching the source samples in the library.     

Novel molecularly imprinted magnetic nanoparticles for the selective extraction of protoberberine alkaloids in herbs and rat plasma

In this work, a novel magnetic nanomaterial functionalized with a molecularly imprinted polymer was prepared for the extraction of protoberberine alkaloids. Molecularly imprinted polymers were made on the surface of Fe₃O₄ nanoparticles by using berberine as template, acetonitrile/water as porogen, acrylamide as functional monomer and ethylene glycol dimethacrylate as cross‐linker. The optimized molar ratio of template/functional monomer was 1:7. The polymeric magnetic nanoparticles were characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. The stability and adsorption capacity of the molecularly imprinted polymers were investigated. The molecularly imprinted polymers were used as a selective sorbent for the magnetic molecularly imprinted solid‐phase extraction and determination of jatrorrhizine, palmatine, and berberine. Extraction parameters were studied including loading pH, sample volume, stirring speed, and extraction time. Finally, a magnetic molecularly imprinted solid‐phase extraction coupled to high‐performance liquid chromatography method was developed. Under the optimized conditions, the method showed good linear range of 0.1–150 ng/mL for berberine and 0.1–100 ng/mL for jatrorrhizine and palmatine. The limit of detection was 0.01 ng/mL for berberine and 0.02 ng/mL for jatrorrhizine and palmatine. The proposed method has been applied to determine protoberberine alkaloids in Cortex phellodendri and rat plasma samples. The recoveries ranged from 87.33–102.43%, with relative standard deviation less than 4.54% in Cortex phellodendri and from 102.22–111.15% with relative standard deviation less than 4.59% in plasma.     

Preparation of a stationary phase with quaternary ammonium embedded group for selective separation of alkaloids based on ion‐exclusion interaction

A stationary phase (named QA C10) with quaternary ammonium embedded between a propyl and a decyl chain was synthesized by immobilization of N,N‐dimethyldecylamine on chloropropyl–silica surface. A set of representative neutral, basic, and acidic compounds was employed to evaluate its chromatographic properties. The results illustrated that QA C10 was a mixed‐mode stationary phase possessing both hydrophobic and ionic characteristics. The QA C10 stationary phase was further used for selective separation of alkaloids from Cortex phellodendri. Under acidic condition, alkaloids could be eluted in first 8 min, while other neutral and acidic fractions were retained better on QA C10 column. Then, obtained alkaloid fraction was analyzed by LC‐MS/MS and 22 alkaloids were identified. Our study confirmed the advantages and application potential of the QA C10 stationary phase for alkaloids separation.     

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.     

Sodium dodecyl sulfate sensitized switchable solvent liquid‐phase microextraction for the preconcentration of protoberberine alkaloids in Rhizoma coptidis

A sodium dodecyl sulfate sensitized switchable solvent liquid‐phase microextraction method was developed and applied to the preconcentration of active alkaloids in Rhizoma coptidis followed by high performance liquid chromatography determination. Before extraction, nonionic triethylamine was converted to its cationic form in the presence of carbon dioxide. Then, the ionic solvent carrying target analytes was once more reverted to its nonionic form by adding sodium hydroxide, as well as phase separation and analytes enrichment were realized simultaneously. Several parameters affecting the approach, such as concentration of sodium dodecyl sulfate, extraction solvent volume, sodium hydroxide concentration, sample phase pH, injection solvent type, and extraction time, were investigated and optimized. The possible microextraction mechanism of double micelle supramolecular inclusion was explored. Under the optimum conditions, the enrichment factors of four protoberberine alkaloids were from 101.8 to 152.0. The linear ranges (with r² ≥ 0.990) were 0.032–4.23, 0.031–4.33, 0.0026–10.04, and 0.0013–4.13 μg/mL for epiberberine, coptisine, palmatine, and berberine, respectively. The detection limits were in the range of 0.16–0.32 ng/mL. Satisfactory accuracies (recoveries 98.8–104.6%) and precisions (RSDs 1.9–10.9%) were also obtained. The results showed that the approach is rapid, effective, eco‐friendly, and easy‐to‐handle for the enrichment and detection of active alkaloids in Rhizoma coptidis.     

Three‐phase solvent systems for the comprehensive separation of a wide variety of compounds from Dicranostigma leptopodum by high‐speed counter‐current chromatography

A three‐phase solvent system was efficiently applied for high‐speed counter‐current chromatography to separate secondary metabolites with a wide range of hydrophobicity in Dicranostigma leptopodum. The three‐phase solvent system of n‐hexane/methyl tert‐butyl ether/acetonitrile/0.5% triethylamine (2:2:3:2, v/v/v/v) was selected for high‐speed counter‐current chromatography separation. The separation was initiated by filling the column with a mixture of intermediate phase and lower phase as a stationary phase followed by elution with upper phase to separate the hydrophobic compounds. Then the mobile phase was switched to the intermediate phase to elute the moderately hydrophobic compounds, and finally the polar compounds still retained in the column were fractionated by eluting the column with the lower phase. In this research, 12 peaks were eluted out in one‐step operation within 110 min, among them, eight compounds with acceptable purity were obtained and identified. The purities of β‐sitosterol, protopine, allocryptopine, isocorydione, isocorydine, coptisine, berberrubine, and berberine were 94.7, 96.5, 97.9, 86.6, 98.9, 97.6, 95.7, and 92.8%, respectively.     

Large‐scale separation of antipsychotic alkaloids from Rauwolfia tetraphylla L. by pH‐zone‐refining fast centrifugal partition chromatography

pH‐zone‐refining centrifugal partition chromatography was successively applied in the large‐scale separation of close R_f antipsychotic indole alkaloids directly from CHCl₃ fraction of Rauwolfia tetraphylla leaves. Two experiments with increasing mass from 500 mg to 3 g of crude alkaloid extracts ( 1 C) of R. tetraphylla were carried out in normal‐displacement mode using a two‐phase solvent system composed of methyl tert‐butyl ether/ACN/water (4:1:5, v/v/v) where HCl (12 mM) was added to the lower aqueous stationary phase as a retainer and triethylamine (5 mM) to the organic mobile phase as an eluter. The two centrifugal partition chromatography separations afforded a total of 162.6 mg of 10‐methoxytetrahydroalstonine ( 1 ) and 296.5 mg of isoreserpiline ( 2 ) in 97% and 95.5% purity, respectively, along with a 400.9 mg mixture of α‐yohimbine and reserpiline ( 3 and 4 ). Further, this mixture was resolved over medium pressure LC using TLC grade silica gel H (average particle size 10 μm), which afforded 160.4 mg of α‐yohimbine ( 3) and 150.2 mg of reserpiline ( 4) in >95% purities. The purity of the isolated antipsychotic alkaloids was analyzed by high‐performance LC and their structures were characterized on the basis of their 1D, 2D NMR and electrospray ionization‐mass spectroscopic data.     

Alkaloids ofDicentra

Methanolic extraction ofDicentra spectabilis L., collected in the flowering period in the botanical garden of Pyatigorsk Pharmaceutical Institute, has yielded 0.17% of combined alkaloids from the epigeal part and 0.25% from the roots. By the separation of these materials, dihydrosanguinarine, sanguinarine, scoulerine, cheilanthifoline, corydine, and protopine have been obtained.Dicentra peregrina Rudolph, collected in the flowering period on the island of Sakhalin, has yielded 1.8% of combined alkaloids from the epigeal part and 1.51% from the roots. From these combined materials have been isolated isocorydine, corydine, dicentrine, protopine, dihydrosanguinarine, sanguinarine, cheilanthifoline, bicuculline, lederine, scoulerine, isoboldine, predicentrine, reticuline, and allocryptopine.Institute of the Chemistry of Plant Substances. Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 79–81, January–February, 1984.     

Online high‐pH reversed‐phase liquid chromatography × low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension for the analysis of alkaloids in Macleaya cordata (willd.) R. Br

An online high‐pH reversed‐phase liquid chromatography× low‐pH reversed‐phase liquid chromatography tandem electrospray ionization mass spectrometry combined with pulse elution gradient in the first dimension was constructed to separate and identify alkaloids from Macleaya cordata (willd.) R. Br. The modulation was performed by using a dual second dimensional columns interface combined with a make‐up dilution pump, which is responsible for dilution and neutralization of the first dimensional effluent, and the dual second dimensional columns integrated the trapping and the separation function to reduce the second dimension system dead volume. Taking advantage of the dissociable characteristics of alkaloids, mobile phases with different pH values were applied in the first dimension (pH 9.0) and the second dimension (pH 2.6) to improve the orthogonality of two‐dimension separation. Besides, the pulse elution gradient in first dimension and second dimensional gradient were carefully optimized and much better separation was achieved compared to the separation with the traditional two‐dimensional liquid chromatography approach. Finally, mass measurement was performed for alkaloids in M. cordata (willd.) R. Br. by coupling proposed two‐dimensional liquid chromatography system with triple quadrupole mass spectrometry, and 39 alkaloids were successfully identified by comparing the obtained result with the former reported results.     

Separation and mechanism elucidation for six structure-like matrine-type alkaloids by micellar liquid chromatography

A mixed micellar liquid chromatography (MLC) method, the mobile phase consisting of anionic surfactant SDS and nonionic surfactant Brij35, was firstly developed for the separation and determination of six structure-like matrine-type alkaloids, including matrine, oxymatrine, sophocarpine, oxysophocarpine, sophoridine, and oxysophoridine. The factors influencing the resolution of the six alkaloids were systematically investigated and optimized, including the micellar composition and concentration, column temperature, the type and amount of organic solvent, and the pH values in the mobile phases. Under the optimized separation conditions, the six matrine-type alkaloids could be easily isocratically eluted with a baseline separation within 22 min. Under the designated conditions (SDS concentration from 10 to 50 mM, Brij35 from 5 to 30 mM, pH 3 and 5% 1-propanol), the hydrophobic selectivity was negatively correlated with the concentration of Brij35 but not with SDS. The functional group selectivity of the carbonyl group, double bond, and diastereomers, all decreased with the increase in percentage of SDS in the mixed micellar phase, because the strong electrostatic force masks other molecular forces which can discriminate the retention of the analytes. Therefore, such a combination in surfactants of MLC is a powerful strategy to increase the selectivity by adjusting the balance among the various molecular interaction forces influencing analytes' retention. Finally, the developed method was successfully used to separate and determine the contents of main alkaloids in Sophora medicinal plants, S. flavescens Ait. In summary, the mixed MLC is a valuable approach to separate and determine the structure-like multi-component natural samples.     

Identification and metabolite profiling of alkaloids in aerial parts of Papaver rhoeas by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry

Papaver plants can produce diverse bioactive alkaloids. Papaver rhoeas Linnaeus (common poppy or corn poppy) is an annual flowering medicinal plant used for treating cough, sleep disorder, and as a sedative, pain reliever, and food. It contains various powerful alkaloids like rhoeadine, benzylisoquinoline, and proaporphine. To investigate and identify alkaloids in the aerial parts of P. rhoeas, samples were collected at different growth stages and analyzed using liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry. A liquid chromatography with mass spectrometry method was developed for the identification and metabolite profiling of alkaloids for P. rhoeas by comparing with Papaver somniferum. Eighteen alkaloids involved in benzylisoquinoline alkaloid biosynthesis were used to optimize the liquid chromatography gradient and mass spectrometry conditions. Fifty‐five alkaloids, including protoberberine, benzylisoquinoline, aporphine, benzophenanthridine, and rhoeadine‐type alkaloids, were identified authentically or tentatively by liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry in samples taken during various growth stages. Rhoeadine alkaloids were observed only in P. rhoeas samples, and codeine and morphine were tentatively identified in P. somniferum. The liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry method can be a powerful tool for the identification of diverse metabolites in the genus Papaver. These results may help understand the biosynthesis of alkaloids in P. rhoeas and evaluate the quality of this plant for possible medicinal applications.     

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.     

Quantitative determination of isoquinoline alkaloids and chlorogenic acid in Berberis species using ultra high performance liquid chromatography with hybrid triple quadrupole linear ion trap mass spectrometry

Berberis species are well known and used extensively as medicinal plants in traditional medicine. They have many medicinal values attributable to the presence of alkaloids having different pharmacological activities. In this study, a method was developed and validated as per international conference on harmonization guidelines using ultra high performance liquid chromatography with hybrid triple quadrupole‐linear ion trap mass spectrometry operated in the multiple reaction monitoring mode for nine bioactive compounds, including protoberberine alkaloids, aporphine alkaloids and chlorogenic acid. This method was applied in different plant parts of eight Berberis species to determine variations in content of nine bioactive compounds. The separation was achieved on an ACQUITY UPLC CSH? C₁₈ column using a gradient mobile phase at flow rate 0.3 mL/min. Calibration curves for all the nine analytes provided optimum linear detector response (with R² ≥0.9989) over the concentration range of 0.5–1000 ng/mL. The precision and accuracy were within RSDs ≤2.4 and ≤2.3%, respectively. The results indicated significant variation in the total contents of the nine compounds in Berberis species.     

Two-dimensional RPLC-RPLC system with different pH in two dimensions for separation of alkaloids from Corydalis yanhusuo W. T. Wang

An effective method utilizing the same RP chromatographic column with different pH in first and second LC dimensions has been developed for separation of the basic compounds from traditional Chinese medicines (TCMs). In this work, the alkaloids in Corydalis yanhusuo which is an important TCM were selected as a model to develop the method. The additives and pH values of the mobile phase were optimized in this work. To investigate the feasibility of this method, off-line mode separation was performed in the experiments. According to the UV-absorption intensity, there were eight fractions collected in acidic conditions. All the fractions were analyzed in basic conditions. The results showed that the chromatographic selectivities were significantly different in the separations performed with acidic and alkaline elution systems. Complementary separation was achieved in this work. It is demonstrated that this method would be an effective tool for alkaloids research. Based on the different pH of the mobile phase in this method, it could also be suitable to analyze compounds which were sensible to the pH of the solution.     

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.     

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.