Ultrasound-assisted solid phase microextraction-HPLC method based on Fe3O4@SiO2-NH2-molecularly imprinted polymer magnetic nano-sorbent for rapid and efficient extraction of harmaline from Peganum harmala extract.

In the present study, a magnetic molecularly imprinted polymer (MMIP) was synthesized for the extraction of harmaline from Peganum harmala by dispersive solid-phase microextraction (DSPME). The MMIP for selective and intelligent extraction of harmaline with excellent functionality and high selectivity was synthesized using the sol-gel method with functionalized superparamagnetic core-shell nanoparticles, ethylene glycol dimethacrylate (EDMA) as a cross-linker, methacrylic acid (MAA) as a functional monomer, and 2,2-azobisisobutyronitrile (AIBN) as a porogen. To study the properties and morphology of the coated polymer, FT-IR spectroscopy, FESEM, TEM images, and VSM were used. The DSPME-HPLC-UV equipment was used to quantify and analyze the data obtained from harmaline extraction. In this research, the efficiency of the synthesized polymer in harmaline extraction was modeled and optimized using the response surface methodology based on central composite design (RSM-CCD). In addition, for modeling the isotherm of harmaline sorption by the MMIP, Langmuir and Freundlich isotherm equations were used. The obtained results showed that the extraction of harmaline with the MMIP was well described with Freundlich isotherm. The results of the validation of the method showed that the measurement of harmaline in the concentration range of 1.0-4000 ng mL-1 followed a linear relationship (R2 = 9986.0). Moreover, the accuracy or repeatability index (% RSD) was determined to be < 10, and the LOQ and LOD values were 0.526 and 0.158 ng mL-1, respectively. The results of this study showed that the DSPME technique by using the synthesized MMIP as an effective sorbent with high efficiency and capacity could be utilized for pre-concentration and extraction of harmaline from real and complex samples.

Microwave-assisted extraction of three bioactive alkaloids from Peganum harmala L. and their acaricidal activity against Psoroptes cuniculi in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Peganum harmala L. is a perennial herbaceous, glabrous plant that grows in semi-arid conditions, steppe areas and sandy soils. It is used to treat fever, diarrhoea, subcutaneous tumours, arthralgia, rheumatism, cough, amnesia and parasitic diseases in folk medicines. In this paper, we aimed to develop a simpler and faster method for the extraction of three alkaloids from Peganum harmala L. than other conventional methods by optimizing the parameters of a microwave-assisted extraction (MAE) method, and to investigate the acaricidal activities of three compounds against Psoroptes cuniculi. MATERIALS AND METHODS: After optimizing the operating parameters with the single factor experiment and a Box-Behnken design combined with a response-surface methodology, a MAE method was developed for extracting the alkaloids from the seeds, and a high-performance liquid chromatography was used to quantify these compounds. An in vitro experiments were used to study the acaricidal activities. RESULTS: The optimal conditions of MAE method were as follows: liquid-to-solid ratio 31.3:1mL/g, ethanol concentration 75.5%, extraction time 10.1min, temperature 80.7°C, and microwave power 600W. Compared to the heat reflux extraction (HRE, 60min) and the ultrasonic-assisted extraction (UAE, 30min) methods, MAE method require the shortest time (10min) and obtain the highest yield of three compounds (61.9mg/g). Meanwhile, the LT50 values for the vasicine (1.25 and 2.5mg/mL), harmaline (1.25 and 2.5mg/mL), harmine (1.25 and 2.5mg/mL) and MAE extract (100mg/mL) against Psoroptes cuniculi were 12.188h, 9.791h, 11.994h, 10.095h, 11.293h, 9.273h and 17.322h, respectively. CONCLUSIONS: The MAE method developed exhibited the highest extraction yield within the shortest time and thus could be used to extract the active compounds from Peganum harmala L. on an industrial basis. As the active compounds of Peganum harmala L., vasicine, harmalin and harmine presented the marked acaricidal activities against Psoroptes cuniculi, and could be widely applied for the treatments of acariasis in animals.

Harmaline and hispidin from Peganum harmala and Inonotus hispidus with binding affinity to Candida rugosa lipase: In silico and in vitro studies.

The inhibitory effect of phenolic compounds and alkaloids of Inonotus hispidus and Peganum harmala on Candida rugosa lipase was investigated, also, their antioxidant activities using DPPH, ABTS and phosphomolybdenum were studied in this paper. The phenolic extracts have shown a stronger antiradical activity than the alkaloids extracts. The enzymatic inhibition produced by these extracts is described here for the first time. The results have shown that the phenolic and the alkaloid extracts are good inhibitors of C. rugosa lipase. Thus, the inhibitor molecules (harmaline and hispidin) have been isolated from P. harmala and I. hispidus. Their structures were elucidated by (1)H NMR analysis. Molecular docking has been achieved using AutoDock Vina program to discuss the nature of interactions and the mechanism of inhibition. Therefore, these isolated molecules could be used in the treatment of candidiasis.

Direct infusion ESI-IT-MSn alkaloid profile and isolation of tetrahydroharman and other alkaloids from Bocageopsis pleiosperma maas (Annonaceae).

INTRODUCTION: The Annonaceae family is known as a promising abundant source of secondary metabolites, especially annonaceous acetogenins, terpenoids and isoquinoline-derived alkaloids. Although widely investigated from the phytochemical viewpoint, this family still presents some largely unexplored genera, e.g. the Bocageopsis. OBJECTIVE: To investigate the alkaloid content of Bocageopsis pleiosperma Maas using direct infusion electrospray ionisation ion trap tandem mass spectrometry (ESI-IT-MS(n)) analysis. METHODOLOGY: Dichloromethane extracts of aerial parts were subjected to acid-base partitioning to yield the alkaloidal fractions. These fractions were analysed by direct infusion into a (+)ESI-IT-MS(n) system. The alkaloidal fraction from the leaves was also obtained on a large scale and subjected to chromatographic separation. RESULTS: The tentative MS(n) -based identification of alkaloids in leaves, twigs and trunk bark showed that aporphine alkaloids were restricted to the leaves and twigs, tetrahydroprotoberberine alkaloids were only found in the twigs and trunk bark while benzylisoquinoline alkaloids were found in the leaves, twigs and trunk bark. Chromatographic separation of the leaf alkaloidal fraction yielded the aporphine alkaloids nornuciferine, asimilobine and isoboldine, the ß-carboline alkaloid tetrahydroharman and some mixtures containing benzylisoquinoline and aporphine alkaloids, all described for the first time in the Bocageopsis genus. Furthermore, tetrahydroharman has not previously been reported in the Magnoliales order. CONCLUSION: Direct infusion ESI-IT-MS(n) analysis of alkaloids allowed fast recognition of alkaloidal classes previously reported in the Annonaceae family, aiding the chromatographic step and allowing a selective isolation of compounds previously not identified in the Bocageopsis genus.

Herbicidal effects of harmaline from Peganum harmala on photosynthesis of Chlorella pyrenoidosa: probed by chlorophyll fluorescence and thermoluminescence.

The herbicidal effects of harmaline extracted from Peganum harmala seed on cell growth and photosynthesis of green algae Chlorella pyrenoidosa were investigated using chlorophyll a fluorescence and thermoluminescence techniques. Exposure to harmaline inhibited cell growth, pigments contents and oxygen evolution of C. pyrenoidosa. Oxygen evolution was more sensitive to harmaline toxicity than cell growth or the whole photosystem II (PSII) activity, maybe it was the first target site of harmaline. The JIP-test parameters showed that harmaline inhibited the donor side of PSII. Harmaline decreased photochemical efficiency and electron transport flow of PSII but increased the energy dissipation. The charge recombination was also affected by harmaline. Amplitude of the fast phase decreased and the slow phase increased at the highest level of harmaline. Electron transfer from QA(-) to QB was inhibited and backward electron transport flow from QA(-) to oxygen evolution complex was enhanced at 10 µg mL(-1) harmaline. Exposure to 10 µg mL(-1) harmaline caused appearance of C band in thermoluminescence. Exposure to 5 µg mL(-1) harmaline inhibited the formation of proton gradient. The highest concentration of harmaline treatment inhibited S3QB(-) charge recombination but promoted formation of QA(-)YD(+) charge pairs. P. harmala harmaline may be a promising herbicide because of its inhibition of cell growth, pigments synthesis, oxygen evolution and PSII activities.

A dihydro-pyrido-indole potently inhibits HSV-1 infection by interfering the viral immediate early transcriptional events.

In our continued quest for identifying novel molecules from ethnomedicinal source we have isolated an alkaloid 7-methoxy-1-methyl-4,9-dihydro-3H-pyrido[3,4-b]indole, also known as Harmaline (HM), from an ethnomedicinal herb Ophiorrhiza nicobarica. The compound exhibited a potent anti-HSV-1 activity against both wild type and clinical isolates of HSV-1. Further we demonstrated that HM did not interfere in viral entry but the recruitment of lysine-specific demethylase-1 (LSD1) and the binding of immediate-early (IE) complex on ICP0 promoter. This leads to the suppression of viral IE gene synthesis and thereby the reduced expression of ICP4 and ICP27. Moreover, HM at its virucidal concentration is nontoxic and reduced virus yields in cutaneously infected Balb/C mice. Thus, the interference in the binding of IE complex, a decisive factor for HSV lytic cycle or latency by HM reveals an interesting target for developing non-nucleotide antiherpetic agent with different mode of action than Acyclovir.

Main alkaloids of Peganum harmala L. and their different effects on dicot and monocot crops.

Alkaloids with allelopathic activity are not as well-known as other allelochemicals. Our study revealed that total alkaloids from seeds of the medicinal plant Peganum harmala L. possessed significant growth inhibitory effect on four treated plants, with dicot plants (lettuce and amaranth) being more sensitive than the tested monocot plants (wheat and ryegrass). Further investigation led to the isolation of harmaline and harmine as the main active ingredients in the total alkaloids of P. harmala seeds. Harmaline exerted potent inhibitory effects on seedling growth of treated plants, especially dicots, inhibiting root elongation of lettuce and amaranth by 31% and 47% at a very low concentration (5 µg/mL), whereas harmine exhibited much weaker non-selective inhibitory effect on the plants. Considering the high yield and poor utilization of P. harmala in China, we anticipate that this plant could be exploited as an alternative weed management tool in the future.

Separation and purification of harmine and harmaline from Peganum harmala using pH-zone-refining counter-current chromatography.

pH-zone-refining counter-current chromatography was successfully applied to the separation of alkaloids from a crude extract of Peganum harmala L. using a multilayer coil planet centrifuge. The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether/THF/water (2:2:3 by volume) where triethylamine (10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 1.2 g of the crude extract, 554 mg harmine and 325 mg harmaline were obtained each with a purity of over 96% as determined by HPLC. The structures of the isolated compounds were identified by electron ionization MS (EI-MS), (1)H NMR, and (13)C NMR.

Alkaloids from the seeds of Peganum harmala showing antiplasmodial and vasorelaxant activities.

Bioassay-guided purification from the seeds of Peganum harmala led to the isolation of harmine (1), harmaline (2), vasicinone (3), and deoxyvasicinone (4). Harmine (1) and harmaline (2) showed a moderate in vitro antiplasmodial activity against Plasmodium falciparum. Quinazoline alkaloid, vasicinone (3), showed a vasorelaxant activity against phenylephrine-induced contraction of isolated rat aorta.

A study on the antitumoral and differentiation effects of peganum harmala derivatives in combination with ATRA on leukaemic cells.

Plant derived agents may exert a new approach to the treatment of leukaemia. The present study was an evaluation of proliferation, cytotoxicity and differentiation of harmine and harmaline on HL60 cells, alone or in combination with ATRA and G-CSF. Counting of cells, viability, MTT assay, morphology, NBT reduction and flow cytometry analysis were performed using CD11b and CD 14 monoclonal antibodies. The data showed that harmine and harmaline reduced proliferation in dose and time dependent manner. Optimal antiproliferative concentration of these agents was chosen. However, both agents in higher doses were cytotoxic. Combination of ATRA, G-CSF and each agent alone, particularly harmaline in optimal dose, resulted in partially additive decrease in cell proliferation. Cells treated with both harmaline and ATRA demonstrated some morphological changes and NBT positivity, but the extent of changes observed following treatment with harmaline was less than ATRA. Flow cytometric analysis showed that ATRA induced a neutrophilic differentiation, while harmaline led to a predominantly monocytic differentiation. Combination of harmine and harmaline with ATRA and G-CSF did not change the extent of differentiation, and the cells differentiated into the neutrophilic lineage. This shows that the direction of differentiation is dominantly determined by ATRA. These preliminary data implies a new approach in treatment of leukemia.

Vasorelaxant effects of harmine and harmaline extracted from Peganum harmala L. seeds in isolated rat aorta.

The present work describes the mechanisms involved in the vasorelaxant effect of harmine and harmaline. These alkaloids induce in a dose-dependent manner the relaxation in the aorta precontracted with noradrenaline or KCl. However, the removal of endothelium or pre-treatment of intact aortic ring with L-NAME (inhibitor of NOSe synthetase) or with indomethacin (non-specific inhibitor of cyclo-oxygenase), reduces significantly the vasorelaxant response of harmaline but not harmine. According to their IC50 values, prazosin (inhibitor of alpha-adrenorecepteors) reduces the vasorelaxant effect only of harmaline, whereas, pre-treatment with IBMX (non-specific inhibitor of phosphodiesterase) affects both the harmaline and harmine-responses. Inhibitions of L-type voltage-dependent Ca2+ channels (VOCs) in endothelium-intact aortic rings with diltiazem depress the relaxation evoked by harmaline as well as by harmine. Pre-treatment with harmaline or harmine (3, 10 or 30 microM) shifted the phenylephrine-induced dose response curves to the right and the maximum response was attenuated indicating that the antagonist effect of both alkaloids on alpha1-adrenorecepteors was non-competitive. These two alkaloids also exert an antioxidant activity by scavenging the free radical generated by DPPH. Therefore, the present results suggest that the vasorelaxant effect of harmaline but not harmine is related to its action on the prostacyclin pathway and on the endothelial cells to release NO. However, both alkaloids can act as blockers VOCs, as inhibitors of phosphodiesterase resulting in an increase of the second messenger (cAMP and cGMP) levels and finally reduce the levels of free radicals in tissues.

Affinitive separation and on-line identification of antitumor components from Peganum nigellastrum by coupling a chromatographic column of target analogue imprinted polymer with mass spectrometry.

A coupled LC-MS (liquid-phase chromatography and mass spectrometry) system consisting of a combination of a column of molecularly imprinted polymer (MIP) and a MS detector was used for affinitive separation and on-line identification of the antitumor components, harmine and harmaline, from the methanol extract of Peganum nigellastrum seeds. Three molecularly imprinted polymers were synthesized with porogens bearing different hydrogen bonding capacities with harman, the structural analogue of harmaline, and harmine as the template. The affinity and selectivity of the anti-harman MIPs for the targets, harmine and harmaline, were investigated chromatographically, and the influences of the porogens and sample loads on the retention of the target compounds were also discussed. In addition, the target binding capacities of the MIPs were evaluated by frontal chromatography. When the MIPs were further used in a LC-MS system to separate the extract of herb, it was observed that imprinting with different porogens would cause the MIPs to exhibit different tendencies to adsorb the matrix components from the herb. Though the MIP prepared with a porogen of less hydrogen bonding capacity possessed higher selectivity and stronger affinity for the targets, matrix components in the herb extract interfered with the chromatographic performance more seriously when it was used as the LC solid phase in the LC-MS system for selective extraction of harmaline and harmine from the crude herb extract. Positively, the MIPs were stable and reproducible in the separation test, and the imprinting columns could efficiently separate the antitumor components from the herb extract after the sample was simply pretreated. The work in this paper would be helpful for the further extraction and identification of certain pharmacophoric compounds in herbs by a LC-MS system using MIPs as the HPLC solid phase.