Effects of the limited analyte solubility on its mobility and zone shape: electrophoretic behavior of sanguinarine and chelerythrine around pH 7

Electrophoretic mobilities and shapes of zones of sanguinarine and chelerythrine in aqueous media around pH 7 are affected by limited solubility of their uncharged forms and by the pH-dependent chemical equilibrium between cationic and uncharged forms of these alkaloids. The sanguinarine solubility in sodium MOPS of pH 7.4 was estimated at 50 micromol x L(-1). Sanguinarine zones in this buffer have the shape of tailed peak with concentration-independent mobility if the injected sanguinarine concentration exceeds this solubility limit only slightly. The chelerythrine solubility is higher because of lower dissociation constants of its cations. Precipitation of sanguinarine and chelerythrine with the phosphate anions decelerates their electrophoretic transport in phosphate buffer. Sanguinarine solubility is 5 micromol x L(-1) at the most in 13 mmol x L(-1) sodium phosphate buffer of pH 7.4. Acidifying of the sample up to pH 3 decreases the tailing of the peaks of sanguinarine and chelerythrine and contributes to the rise of sharp maxima of their migrating zones. Any capillary coating deteriorates the peak shape.     

Capillary zone electrophoretic studies of interactions of some quaternary isoquinoline alkaloids with DNA constituents and DNA

Capillary zone electrophoresis was applied for the investigation of interactions of some quaternary isoquinoline alkaloids, namely sanguinarine, chelerythrine, berberine, and jatrorrhizine, with DNA constituents and with DNA. None of these alkaloids attach covalently to nucleotides or to the whole DNA under physiological conditions. The interaction with DNA constituents is a noncovalent complexation based on weak intermolecular forces. Electrostatic attraction participates in the interaction but other types of intermolecular forces are involved as well. Cations were identified as the most probable interacting forms of the alkaloids. The interaction with compounds derived from purine was always stronger than those derived from pyrimidine. All alkaloids behaved analogously and similarly to ethidium bromide, the classic DNA intercalator. Stability constants K (in l.mol(-1)) for sanguinarine and chelerythrine in phosphate buffer of pH 7.4 (I(S) = 30 mM) ranged from tens to hundreds.     

Capillary electrophoretic determination of sanguinarine and chelerythrine in plant extracts and pharmaceutical preparations

Capillary electrophoresis was employed to determine the principal quaternary benzo[c]phenanthridine alkaloids, sanguinarine and chelerythrine, in two plant extracts and one oral hygiene product. Phosphate-Tris buffer of pH 2.5 was used as a background electrolyte, limits of detection were 3 micromol/l(-1) (sanguinarine) and 2.4 micromol,l(-1) (chelerythrine) using UV detection at 270 nm. The method, which correlated well with HPLC, is suitable for serial determination of sanguinarine and chelerythrine in plant products and pharmaceuticals.     

Site-specific binding of chelerythrine and sanguinarine to single pyrimidine bulges in hairpin DNA

Spectrofluorometric titration, electrospray ionization time-of-flight mass spectrometric and UV melting methods were employed to study the binding of chelerythrine and sanguinarine to bulged DNA. The results showed that both alkaloids bind specifically to single pyrimidine (C, T) bulge sites. The ability of sanguinarine to bind to both regular and bulged hairpins was found to be stronger than that of chelerythrine, but the binding selectivity of chelerythrine toward single-base bulges was much larger than that of sanguinarine. Figure Association constants for chelerythrine and sanguinarine toward regular and single-base bulged hairpins obtained from fluorometric analysis     

Determination of Cytotoxic Activity of Sanguinaria canadensis Extracts against Human Melanoma Cells and Comparison of Their Cytotoxicity with Cytotoxicity of Some Anticancer Drugs

Melanoma is an enormous global health burden, and should be effectively addressed with better therapeutic strategies. Therefore, new therapeutic agents are needed for the management of this disease. The aim of this study was the investigation of cytotoxic activity of some isoquinoline alkaloid standards and extracts obtained from Sanguinaria canadensis—collected before, during, and after flowering—against three different human melanoma cells (A375, G361, SK-MEL-3). The cytotoxicity of these extracts was not previously tested on these melanoma cell lines. Determination of alkaloid contents was performed by HPLC-DAD using Polar RP column and mobile phase containing acetonitrile, water, and 1-butyl-3-methylimidazolium tetrafluoroborate. The cytotoxicity of alkaloid standards was investigated by determination of cell viability and calculation of IC₅₀ values. Significant differences were observed in the alkaloids content and cytotoxic activity of the extracts, depending on the season of collection of the plant material. In the Sanguinaria canadensis extracts high contents of sanguinarine (from 4.8543 to 9.5899 mg/g of dry plant material) and chelerythrine (from 42.7224 to 6.8722 mg/g of dry plant material) were found. For both of these alkaloids, very high cytotoxic activity against the tested cell lines were observed. The IC₅₀ values were in the range of 0.11–0.54 µg/mL for sanguinarine and 0.14 to 0.46 µg/mL for chelerythrine. IC₅₀ values obtained for Sanguinaria canadensis extracts against all tested cell lines were also very low (from 0.88 to 10.96 µg/mL). Cytotoxic activity of alkaloid standards and Sanguinaria canadensis extracts were compared with the cytotoxicity of anticancer drugs—etoposide, cisplatin, and hydroxyurea. In all cases except the one obtained for cisplatin against A375, which was similar to that obtained for Sanguinaria canadensis after flowering against the same cell line, IC₅₀ values obtained for anticancer drugs were higher than the IC₅₀ values obtained for sanguinarine, chelerythrine, and Sanguinaria canadensis extracts. Our results showed that Sanguinaria canadensis extracts and isoquinoline alkaloids, especially sanguinarine and chelerythrine, could be recommended for further in vivo experiments in order to confirm the possibility of their application in the treatment of human melanomas.     

Capillary electrophoresis with LED-induced native fluorescence detection for determination of isoquinoline alkaloids and their cytotoxicity in extracts of Chelidonium majus L

In this study, we introduced a simple and sensitive method of capillary electrophoresis with ultraviolet light-emitting diode-induced native fluorescence (UV-LEDIF) detection for the determination of isoquinoline alkaloids in extracts of Chelidonium majus L. Samples were extracted with acidic methanol and the extracts were directly analysed by CE. Simultaneous determination of protopine, chelidonine, coptisine, sanguinarine, allocryptopine, chelerythrine and stylopine was performed in 20mM phosphate buffer (pH 3.1). The baseline separation of these alkaloids was finished within 20 min. As these alkaloids have native fluorescence, they were directly detected using the commercially available UV light emitting diode without troublesome fluorescent derivatisation. Satisfactory LOD values were obtained for the studied compounds considering their appearance in natural extracts. Lower limits of detection were 0.05 μg/mL for protopine, 0.06 μg/mL for stylopine and allocryptopine, 0.07 μg/mL for chelidonine, 0.22 μg/mL for sanguinarine, 1.7 μg/mL for chelerythrine and 5.5 μg/mL for coptisine. The developed method was successfully applied to determine the contents of seven alkaloids in the aerial parts of Chelidonium majus L, which varied from 0.025 to 0.763% (w/w). Also, to demonstrate the potential of the proposed CE method, an estimation of the cytotoxic properties of selected Celandine alkaloids in a natural extract was carried out.     

A simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence detection for the analysis of chelerythrine and sanguinarine in Chinese herbal medicines

Laser-induced fluorescence (LIF) is a highly sensitive detection method for capillary electrophoresis (CE). However, it usually requires analyte to be derivatized, unless the wavelength of native fluorescence of analyte matches the laser's. That limits its application in drug analysis. In this work, we introduced a rapid, simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence (NACE-LIF) detection for the analysis of chelerythrine and sanguinarine for the first time. As these two alkaloids have some native fluorescence, they were directly detected using a commercially available Ar⁺ laser without troublesome fluorescent derivatization. The fluorescence was enhanced by nonaqueous media. Compared with previously reported UV detection method, lower limit of detection (LOD) is achieved thanks to the high sensitivity of LIF detection (2.0 ng/mL for chelerythrine and 6.3 ng/mL for sanguinarine). Moreover, with NACE, the baseline separation of these alkaloids is finished within 3.5 min. This method is successfully applied to determine the contents of chelerythrine and sanguinarine in Macleaya cordata (Willd.) R. Br. and Chelidonium majus L.     

二氢苯骈[c]菲啶类生物碱的制备及其抗乙肝病毒的活性

二氢苯骈[c]菲啶类生物碱是自然界中一类重要的生物活性成分,但含量很低.利用硼氢化还原方法,对博落回粗提物中的血根碱和白屈菜红碱混合物进行还原、分离,得到了二氢血根碱和二氢白屈菜红碱;并初步评价了其抗乙肝病毒活性.结果表明,利用硼氢化还原方法制备二氢苯骈[c]菲啶类生物碱的产率较高,还原产物具有一定的抗乙肝病毒活性.     

Seasonal variation in alkaloid composition and antiproliferative activity of <Emphasis Type="Italic">Stylophorum lasiocarpum</Emphasis> (Oliv.) Fedde

Stylophorum lasiocarpum (Oliv.) Fedde (Papaveraceae) belongs to traditional Chinese medicine herbs but there was minimal information on the content of alkaloids in this plant. Extracts from the aerial part and roots were examined by liquid chromatography with UV and mass spectrometric detection, with nineteen alkaloids identified. Changes in alkaloid content over the entire vegetation period of a one- and two-year old plant were studied. The protoberberine alkaloids, coptisine and stylopine, were found to be the main substances in extracts of the aerial part irrespective of the plant’s age and time of harvest. Variable amounts of protopine, sanguinarine, chelerythrine, chelirubine, macarpine, chelilutine and berberine were also recorded in the aerial part. The roots contained significantly larger quantities of all alkaloids than the aerial part with the levels of most alkaloids varying from May to October, peaking in the middle of the vegetation period. Coptisine was the dominant alkaloid in all samples. The antiproliferative activities of the root extract and of seven individual alkaloids were tested on A375 human malignant melanoma cells. The significant dose-dependent toxicity of the root extract was attributed largely to the quaternary benzo[c]phenanthridine alkaloids, macarpine and sanguinarine.     

Electrochemistry of Benzophenanthridine Alkaloids. Formation and Characterization of Redox Active Films from Products of Sanguinarine and Chelerythrine Oxidation

The behavior of sanguinarine and chelerythrine alkaloids upon electrochemical oxidation on solid electrodes in buffered aqueous medium is reported in this study. Electro‐oxidation of sanguinarine and chelerythrine results in the formation of redox active electropolymerized films. The films were characterized by cyclic voltammetry, EQCM and surface enhanced Raman scattering spectroscopy excited in near‐infrared range. The electropolymerization is suggested to proceed via formation of alkaloids' ortho‐benzoquinone derivatives.     

Alkaloids induce programmed cell death in bloodstream forms of trypanosomes (Trypanosoma b. brucei)

The potential induction of a programmed cell death (PCD) in Trypanosoma b. brucei by 55 alkaloids of the quinoline, quinolizidine, isoquinoline, indole, terpene, tropane, steroid, and piperidine type was studied by measuring DNA fragmentation and changes in mitochondrial membrane potential. For comparison, the induction of apoptosis by the same alkaloids in human leukemia cells (Jurkat APO-S) was tested. Several alkaloids of the isoquinoline, quinoline, indole and steroidal type (berberine, chelerythrine, emetine, sanguinarine, quinine, ajmalicine, ergotamine, harmine, vinblastine, vincristine, colchicine, chaconine, demissidine and veratridine) induced programmed cell death, whereas quinolizidine, tropane, terpene and piperidine alkaloids were mostly inactive. Effective PCD induction (EC(50) below 10 microM) was caused in T. brucei by chelerythrine, emetine, sanguinarine, and chaconine. The active alkaloids can be characterized by their general property to inhibit protein biosynthesis, to intercalate DNA, to disturb membrane fluidity or to inhibit microtubule formation.     

Quaternary benzophenanthridine alkaloids 9,10-demethylene derivatives of sanguinarine

Summary In the isolation and purification of the benzophanthridine alkaloids sanguinarine and chelerythrine two minor bases — 9,10-demethylenesanguinarine and 9,10-demethylene-9,10-dehydrosanguinarine — were isolated, and it was shown that the former is an artefact produced from sanguinarine in the process of isolation and also when the sulfates of the alkaloids are subjected to prolonged storage.Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 764–767, November-December, 1978. Original article submitted August 10, 1978.     

Analysis of Amaryllidaceae alkaloids from Chlidanthus fragrans by GC-MS and their cholinesterase activity

The underivatized alkaloid mixture extracted from the bulbs of Chlidanthus fragrans Herb. was investigated by capillary GC/MS for the first time. Fifteen known Amaryllidaceae alkaloids of five structure types were identified. The main alkaloids were tazzetine (9, tazettine-type), chlidanthine (2, galanthamine-type), belladine (8, belladine-type) and lycorine (12, lycorine-type). The alkaloid extract from the bulbs showed promising human blood acetylcholinesterase (IC50 = 20.1 +/- 2.9 microg/mL) and human plasma butyrylcholinesterase (IC50 = 136.8 +/- 6.9 microg/mL) inhibitory activity.     

Capillary electrophoretic study of the synergistic biological effects of alkaloids from Chelidonium majus L. in normal and cancer cells

In this study, the synergistic biological action of five celandine alkaloids in normal and cancer cells was investigated by capillary electrophoresis with light-emitting diode-induced native fluorescence detection. The specific capacity of each alkaloid to penetrate into the cells was estimated by monitoring alkaloid concentration decreases in the cell medium during incubation with murine fibroblast NIH/3T3, mouse melanoma B16F10, and human breast cancer MCF7 cell lines. Mixtures of isoquinoline alkaloids containing protopine, chelidonine, sanguinarine, allocryptopine, and stylopine were applied to cell cultures for 20 and 40 min, and the content of alkaloids in the cell media was measured by capillary electrophoresis (CE). CE separation of isoquinoline alkaloids was performed in 30 mM phosphate buffer (pH 2.5). As these alkaloids have native fluorescence, they were directly detected using the commercially available UV light-emitting diode without troublesome fluorescent derivatization. The results showed a differential ability of celandine alkaloids to penetrate into the normal and cancer cell interior, which was inversely proportional to their cytotoxic activity. While the most effective transport of celandine alkaloids from the cell medium to the cell interior was observed for normal murine fibroblast NIH/3T3 cells (about 55% of total content), cytotoxicity tests demonstrated selective and profound apoptotic effects of a five-alkaloid combination in the mouse melanoma B16F10 cell line.     

EFFECTS OF IONIC STRENGTH and pH ON THE BINDING OF SANGUINARINE TO DEOXYRIBONUCLEIC ACID

Abstract— The interaction of sanguinarine with DN A has been studied in buffers of various ionic strengths and pH values where the physicochemical properties of DNA remain unchanged. Spectrophotometric analysis using absorption and fluorescence techniques indicates that the complex formed between sanguinarine and DNA is a function of ionic strength and pH. Increasing the salt concentration minimizes the importance of intercalator charge and extrapolation to 1 M [Na+] salt reveals the intercalative abilities, as reflected in binding constants, to be of the same order of magnitude as that of ethidium bromide. The fluorescence of sanguinarine bound to DNA is quenched and can be decreased by [Na +], [Mg²+] and [Ca²+] ions. The influence of pH on the fluorescence spectrum of sanguinarine with increasing concentration of DNA was studied. It is concluded that the binding of sanguinarine to DNA contains a large favourable non-electrostatic interaction and the alkaloid binds more DNA in buffer of low ionic-strength and acidic pH.     

Physicochemical characterization of the Strychnos alkaloids by capillary zone electrophoresis.

A capillary zone electrophoresis (CZE) method has been developed for investigating the physicochemical characteristics of five Strychnos alkaloids in Strychnos nux-vomica L. Firstly, the dissociation constants of the five Strychnos alkaloids were determined, based on the relation between the effective mobility of the solutes and the buffer pH. The mathematical relationship was strictly deduced from the fundamental electrophoretic theory and the dissociation equilibrium. Secondly, an equation describing the relation between the migration time of alkaloids of similar structure and their molecular weights was developed and used to predict the migration order and to calculate the electrosomotic velocity. The results predicted by the theory agreed with those from experiments.     

Alkaloid Composition of Chelidonium majus L. Studied by Different Chromatographic Techniques

The greater celandine (Chelidonium majus L.) is a well known source of isoquinoline alkaloids with therapeutic value. The aim of our work was to develop a simple and fast method to determine the alkaloid composition in Chelidonium plant organs. TLC-densitometry seemed to be the most convenient analytical technique for routine, fast investigations and its precision was controlled by using HPLC. A densitometric method using Silica gel 60 F₂₅₄ with chloroform-methanol 60:30 (v/v) and methylene chloride-methanol 97:3 (v/v) as mobile phases has been developed to quantify the main alkaloids (chelidonine, chelerythrine, sanguinarine, coptisine and berberine). The application of TLC permitted utilizing the fluorescence of alkaloids without purification and made the detection extremely sensitive. The samples were further investigated by our reliable HPLC method. Analysis was performed on a C₁₈ column with acetonitrile-methanol-30 mM ammonium formate (pH = 2.8) 14.7:18.0:67.3 (v/v/v) as mobile phase and alkaloids were determined at 280 nm by using external standards. Our TLC-densitometric method elaborated is a simple technique for accurate and reproducible determination of Chelidonium alkaloids. The results of the two chromatographic methods showed good agreement.     

Determination of Dissociation Constants of Strychnos Alkaloids from Strychnos nux-vomica L. by Capillary Electrophoresis

An accurate capillary electrophoresis method was developed for the determination of dissociation constants of five Strychnos alkaloids from Strychnos nux-vomica L. The method relies on measuring the effective mobility of the solute as a function of the buffer pH. The mathematical relationship was strictly derived from the fundamental electrophoresis theory and the dissociation equilibrium of a weak base without any simplifications. Careful optimization of the running buffer permitted base-line resolution of the five structurally similar alkaloids.     

Combined LIBD and XAFS investigation of the formation and structure of Zr(IV) colloids

The solubility of Zr(OH)4(am)--in other words hydrated Zr(IV) oxyhydroxide--is determined by means of coulometric titration (CT), and colloids are detected by laser-induced breakdown when the solubility limit is exceeded. Our results at pH 3-8 demonstrate that the solubility of Zr(OH)4(am) is several orders of magnitude higher than reported classical solubility data for acidic solutions, determined from undersaturation with a less soluble microcrystalline Zr(IV) oxide precipitate. Analysis of extended X-ray absorption fine structure (EXAFS) data shows that the microcrystalline colloids in a 0.1 mol l(-1) Zr aqueous solution at pH 0.2 contain tetrameric units, similar to those present in the structure of ZrOCl2.8H2O. Characterization of the CT solutions by means of EXAFS shows that oligomeric species form as the solubility limit is approached. The current lack of data on equilibrium constants for polynuclear hydroxide complexes prohibits the use of a realistic speciation model to describe the solubility of pH-dependent Zr(OH)4(am). However, the solubility curve is obtained using the mononuclear hydrolysis constants estimated in the present paper, along with the solubility constant (log K'sp=-49.9+/-0.5 in 0.5 mol l(-1) NaCl; log K degrees(sp)=-53.1+/-0.5 at I=0).     

Palladium-catalyzed tandem reaction to construct benzo[c]phenanthridine: application to the total synthesis of benzo[c]phenanthridine alkaloids

A concise and efficient synthesis of benzo[c]phenanthridines was accomplished by the palladium-catalyzed ring-opening coupling of azabicyclic alkene with o-iodobenzoates, followed by tandem cyclization. The strategy was successfully applied in the total synthesis of benzo[c]phenanthridine alkaloids such as sanguinarine, chelerythrine, nitidine and avicine.