For the 50th anniversary of the laboratory of alkaloid chemistry,Institute of the Chemistry of Plant Substances of the Uzbekistan Republic Academy of Sciences

The present review gives the main advances made in the laboratory of alkaloid chemistry of the Institute of Plant Substances of the Usbekistan Republic Academy of Sciences [IKhRV AN RUz] (Tashkent) over 50 years in the field of the chemistry of diterpene, isoquinoline, quinoline, steroid, indole, spiropiperidine, sulfur-containing, tropane, pyrrolidine, quinazoline, quinolizidine, amaryllis, and pyrrolizidine bases and possible routes for the biosynthesis of some of these groups of alkaloids.Institute of Chemistry of Plant Substances, Uzbekistan Republic Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 3–33, January–February, 1993.     

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.     

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     

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.     

植物内生真菌生物碱活性成分的研究进展

大量的生物碱从植物内生真菌中分离了出来, 并表现出良好的生物活性. 对植物内生真菌所产生的生物碱, 按照酰胺类、有机胺类、吡咯类、喹啉和异喹啉类、吲哚类、吡啶类、喹唑啉类进行了综述, 并且介绍了这些生物碱的生物活性.     

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.     

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.     

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.     

Isoquinoline Alkaloid Contents in Macleaya cordata Extracts and Their Acetylcholinesterase and Butyrylcholinesterase Inhibition

An important strategy for treating neurodegenerative disorders is to maintain the levels of acetylcholine in the synaptic cleft by blocking the cholinesterases. Searching for new effective compounds with inhibited acetylcholinesterase and butyrylcholinesterase activity is one of the most significant challenges of the modern scientific research. The aim of this study was the optimization of the condition for cholinesterase activity determination by high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) in terms of concentrations of enzymatic reaction mixture components, temperature of incubation, and incubation time. In vitro investigation of acetylcholinesterase and butyrylcholinesterase activity inhibition by some isoquinoline alkaloids and extracts obtained from the aerial part and roots of Macleaya cordata collected in May, July, and September. Acetylcholinesterase and butyrylcholinesterase activity inhibition of the extracts obtained from the plant had not been tested previously. The application of the HPLC method allowed eliminating absorption of interfering components, for example, alkaloids such as sanguinarine and berberine. The HPLC method was successfully applied for the evaluation of the acetylcholinesterase inhibitory activity in samples such as plant extracts, especially those containing colored components adsorbing at the same wavelength as the adsorption wavelength of 5-thio-2-nitro-benzoic acid, which is the product of the reaction between thiocholine (product of the hydrolysis of acetyl/butyrylthiocholine reaction) with Ellman’s reagent. Moreover, liquid chromatography coupled with a triple quadrupole mass spectrometer (LC–QqQ–ESI–MS/MS) analysis allowed evaluating the identification of relevant bioactive compounds in the obtained plant extracts. The investigated alkaloids, especially sanguinarine and chelerythrine, and all the Macleaya cordata extracts, especially the extract obtained from the aerial part collected in May, exhibited very high cholinesterase activity inhibition. HPLC-DAD was also applied for the kinetics study of the most active alkaloids sanguinarine and chelerythrine. Our investigations demonstrated that these plant extracts can be recommended for further in vivo experiments to confirm their cholinesterase inhibition activity.     

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.     

Alkaloids produced by endophytic fungi: a review

In recent years, a number of alkaloids have been discovered from endophytic fungi in plants, which exhibited excellent biological properties such as antimicrobial, insecticidal, cytotoxic, and anticancer activities. This review mainly deals with the research progress on endophytic fungi for producing bioactive alkaloids such as quinoline and isoquinoline, amines and amides, indole derivatives, pyridines, and quinazolines. The biological activities and action mechanisms of these alkaloids from endophytic fungi are also introduced. Furthermore, the relationships between alkaloid-producing endophytes and their host plants, as well as their potential applications in the future are discussed.     

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.     

Capillary electrophoretic studies of acid-base properties of sanguinarine and chelerythrine alkaloids

Capillary zone electrophoresis with UV detection was used for determination of dissociation constants of alkaloids sanguinarine and chelerythrine. Despite the limited solubility of the uncharged forms of the alkaloids resulting in insufficient analytical signal at higher pH the reliable dissociation constants were obtained when acidified samples containing low amount of the alkaloid were injected into the capillary. The precipitation of the alkaloid in the capillary induced by injecting sample of low pH into the background electrolyte of higher pH does not affect the mobility of the alkaloid if its concentration injected exceeds the solubility only to a small extent. Dissociation constants (pK(R+)) of sanguinarine and chelerythrine calculated to 8.3 +/- 0.1 and 9.2 +/- 0.1, respectively, are relevant to Good buffers of ionic strength of 30 mM.     

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.     

Synthesis of indolo[2,1-a]isoquinolines by CF3COOH-induced cyclization

Indolo[2,1-a]isoquinoline alkaloids and related compounds have been known to have interesting biological activities, such as antileukemic and antitumor activities. We found that 1-(3,4-dimethoxyphenethyl)indole gave 2,3-dimethoxyindolo[2,1-a]isoquinoline and 1-(3,4-dimethoxyphenylacetyl)indole gave 2,3-dimethoxy-6-oxoindolo[2,1-a]isoquinoline, respectively, by an intramolecular cyclization carried out in boiling trifluoroacetic acid.     

Characterization of the early stages of programmed cell death in maize root cells by using comet assay and the combination of cell electrophoresis with annexin binding

An emerging topic in plant biology is whether plant cells display similar elements of programmed cell death (PCD) as animal cells do. We have studied cell death in maize roots exposed to cold stress by using fluorescence microscopy, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), DNA gel electrophoresis, single cell gel electrophoresis (SCGE), cell electrophoresis, and annexin binding techniques. The results showed that cell death in maize root cells triggered by cold stress was accompanied by a subset of features characteristic of animal PCD such as nuclear condensation and fragmentation, and oligonucleosomal DNA fragmentation. In addition to DNA laddering and TUNEL positivity, a "comet" pattern indicative of DNA breakage appeared as short as after one day of treatment. The maize root cell PCD process was also accompanied by an increase in negative surface charge of the dying cells due to exposure of phosphatiolylserine (PS) from inner to outer membrane. After annexin binding, however, the enhanced electrophoretic mobility (EPM) of the dying cells decreased nearly to normal values. This result suggests that the combination between cell electrophoresis and annexin binding provides a quantitative method for monitoring PS exposure during plant PCD.     

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.     

Simultaneous determination of seven main alkaloids of Chelidonium majus L. by ultra‐performance LC with photodiode‐array detection

A simple and rapid method for the simultaneous determination of seven isoquinoline alkaloids, protopine, chelidonine, coptisine, stylopine, sanguinarine, berberine, and chelerythrine, in Chelidonium majus L. (Ch. majus) samples by ultra‐performance LC method with photodiode array detection is described. The baseline separation of these compounds was performed with (A) acetonitrile–(B) ammonium acetate (10 mM, adjusted to pH 3.0 with acetic acid) as the mobile phase using a C18 RP column (2.1×100 mm, 1.7 μm). Optimized conditions resulted in excellent peak shapes. The seven alkaloids were completely separated within 20 min. Good linear behaviors (r≥0.9992) over the investigated concentration ranges were observed for all the analytes. Validation proved the repeatability of the method was good and recovery was satisfactory. The validated method was successfully applied for 20 batches of Ch. majus. These results demonstrated that the ultra‐performance LC photodiode array method proposed was very useful in the analysis and quality control of Ch. majus.     

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.     

Highly stereoselective construction of 4,6-cis-substituted quinolizidine ring core: an application to enantioselective total synthesis of the marine alkaloid clavepictines A, B and pictamine

The enantioselective total synthesis of the marine alkaloids clavepictines A, B and pictamine has been achieved through the highly stereocontrolled quinolizidine ring closure of the conformationally constrained piperidine ring system (2), which bears the chiral centers and appropriate functionality needed for the synthesis of target alkaloids. The absolute stereochemistry of clavepictines and pictamine was verified to be 3R, 4S, 6S, 9aS by the present synthesis.