Bacillus subtilis stimulates plant growth and production of bioactive components in saffron.

The effects of B. subtilis on the morphology and physiology of saffron were investigated using two types of soils. Three different bacterial suspensions were applied at 14-day intervals to treat saffron. Morphological attributes were recorded, and the amounts of α-crocin and safranal in the stigma extracts were quantified. The longest stigma, petal, and leaf were observed in the treated groups with 105 and 108 cfu/ml. The highest weight of stigma per corm belonged to the treated groups with 102 cfu/ml in unsterile soil and 105 and 108 cfu/ml in sterile soil. Treatment with 102 and 108 cfu/ml caused a significant increase in safranal production in sterile and unsterile peat/perlite. While treatment with 105 and 108 cfu/ml in sterile peat/perlite and exposure to 102 cfu/ml in unsterile peat/perlite soil resulted in an increase in α-crocin. The data showed that B. subtlis triggers the morphological and physiological processes in saffron.

In vitro Cytotoxic Screening of Different Parts from Ornithogalum bungei on Selected Cancer Cells.

BACKGROUND: Natural products comprise a large section of pharmaceutical agents in the field of cancer therapy. In the present study, the organic extracts and fractions of various parts of Ornithogalum bungei were investigated for in vitro cytotoxic properties on three human cancer cell lines, hepatocellular carcinoma (HepG2), prostate cancer (PC3), and leukemia (K562) cells. METHODS: The present experimental study was conducted at Tehran University of Medical Sciences (Tehran, Iran) during 2017-2019. Separately extracted plant materials, including bulbs, stems, and flowers of O. bungei were assessed by the tetrazolium dye-based colorimetric assay (MTT). The selected extracts were submitted to fractionation using vacuum liquid chromatography and after MTT assay, the half maximal inhibitory concentration (IC50 (value for each fraction was determined. The data were analyzed using One-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered statistically significant. RESULTS: The cytotoxicity of the bulb's methanol extract and the dichloromethane extract of aerial parts increased in a concentration-dependent manner. Additionally, cell viability decreased in a dose-dependent manner. In the HepG2 cell line, the best IC50 values of fractions from DCM extracts of aerial parts were determined to be 19.8±10.2 µg/mL after 24 hours of exposure and 19.39±6.4 µg/mL following 48 hours of exposure. In the PC3 cell line, after 48 hours of exposure, the IC50 values of fractions were unaccountable, while the percentage of inhibition for A6 to A11 in 24 hours of exposure was more than 40 µg/mL. CONCLUSION: O. bungei growing in Iran showed significant potentials as a cytotoxic agent with selective effects on different cancer cell lines.

Isolation and Characterization of Acetylcholinesterase Inhibitors from Piper longum and Binding Mode Predictions.

Restoration of cholinergic function is considered a rational approach to enhance cognitive performance. Acetylcholinesterase inhibitors are still the best therapeutic option for Alzheimer's disease. The fruits of Piper longum have been used in traditional medicines for the treatment of memory loss. It was demonstrated that the dichloromethane extract of these fruits is able to inhibit acetylcholinesterase. Thus, the aim of this study was to identify the contained acetylcholinesterase inhibitors. The active zones were presented via TLC-bioautography, and five compounds were isolated in the process of a bioassay-guided phytochemical investigation. Their structures were characterized as piperine, methyl piperate, guineenisine, pipercide, and pellitorine using spectroscopy and spectrometry methods (UV, IR, MS, 1H-, and 13C-NMR). In vitro acetylcholinesterase inhibitory activities of the isolates and their IC50 values were determined via a colorimetric assay. Three of them exhibited enzyme inhibitory activities, with piperine being the most potent compound (IC50 of 0.3 mM). In order to investigate the binding mode of the tested compounds, docking studies were performed using the X-ray crystal structure of acetylcholinesterase from Tetronarce californica with the Protein Data Bank code 1EVE. The content of the active compounds in the extract was determined by a developed HPLC method. Piperine was present in the maximum quantity in the fruits (0.57%), whereas methyl piperate contained the minimum content (0.10%).

Isolation, in vitro evaluation and molecular docking of acetylcholinesterase inhibitors from South African Amaryllidaceae.

Inhibition of acetylcholinesterase (AChE) is considered a promising strategy for the treatment of Alzheimer's disease (AD) and dementia. Members of the Amaryllidaceae family are well known for their pharmacologically active alkaloids, including galanthamine, which is used to treat AD. The aim of this study was to evaluate the potential of South African Amaryllidaceae species to inhibit AChE, to isolate the active compounds, and probe their ability to bind the enzyme using molecular docking. The AChE inhibitory activity of extracts of 41 samples, representing 14 genera and 28 species, as well as isolated compounds, were evaluated in vitro using a qualitative thin layer chromatography (TLC) bio-autography assay and Ellman's method in a quantitative 96-well microplate assay. Targeted isolation of compounds was achieved with the aid of preparative-high perfomance liquid chromatography-mass spectrometry. The structures of the isolates were elucidated using nuclear magnetic resonance spectrocopy, and were docked into the active site of AChE to rationalise their biological activities. The most active species were found to be Amaryllis belladonna L (IC50 14.3 ± 2.6 µg/mL), Nerine huttoniae Schönland (IC50 45.3 ± 0.4 µg/mL) and Nerine undulata (L.) Herb. (IC50 52.8 ± 0.5 µg/mL), while TLC bio-autography indicated the presence of several active compounds in the methanol extracts. Four compounds, isolated from A. belladonna, were identified as belladine, undulatine, buphanidrine and acetylcaranine. Acetylcaranine and undulatine were previously isolated from A. belladonna, while belladine and buphanidrine were reported from other South African Amaryllidaceae species. Using Ellman's method, acetylcaranine was found to be the most active of the isolates towards AChE, with an IC50 of 11.7 ± 0.7 µM, comparable to that of galanthamine (IC50 = 6.19 ± 2.60 µM). Molecular docking successfully predicted the binding modes of ligands within receptor binding sites. Acetylcaranine was predicted by the docking workflow to have the highest activity, which corresponds to the in vitro results. Both qualitative and quantitative assays indicate that several South African Amaryllidaceae species are notable AChE inhibitors.

Preparative isolation of oleocanthal, tyrosol, and hydroxytyrosol from olive oil by HPCCC.

For the provision of oleocanthal (OLC), a phenolic compound with very promising pharmacological properties, isolation from olive oil is a very important option. Due to the compound's sensitivity to decomposition upon exposure to oxygen and light, a very gentle isolation method has been developed under use of high performance countercurrent chromatography (HPCCC). By partition of olive oil between hexane and methanol, an extract enriched in phenolics was prepared and subjected to a two-step HPCCC separation under use of heptane-EtOAc-MeOH-H2O mixtures in normal-phase and reverse phase mode, respectively. With this method, the isolation of tyrosol, hydroxytyrosol, and the mixture of (3S,4E)- and (3S,4Z)-OLC was achieved in approx. 70 min for each step. By one- and two-dimensional NMR-experiments and LC-MS, the equilibrium of (3S,4E)- and (3S,4Z)-OLC in such olive oil extracts has unambiguously been proven for the first time.

Compounds from gum ammoniacum with acetylcholinesterase inhibitory activity.

The use of herbal medicinal preparations in dementia therapy has been studied based on experience from traditional medicine. A dichloromethane extract of gum ammoniacum, the gum-resin from Dorema ammoniacum D. Don had shown acetylcholinesterase (AChE) inhibitory activity in a previous study. The aim of this study was the isolation and characterization of the active compounds from this resin. The extract was investigated by a respective colorimetric microplate assay and the active zones were identified via TLC bioautography and isolated using several chromatographic techniques. The structures of the active components were characterized by one- and two-dimensional (1)H and (13)C NMR spectroscopy and mass spectrometry as (2'S,5'S)-2'-ethenyl-5'-(3-hy-droxy-6-methyl-4-oxohept-5-en-2-yl)-7-methoxy-2'-methyl-4H-spiro[chromene-3,1'-cyclopentane]-2,4-dione (1), which is an analogue of doremone A and a new natural compound, and as (2'S,5'R)-2'-ethenyl-5'-[(2R,4R)-4-hydroxy-6-methyl-3-oxohept-5-en-2-yl]-7-methoxy-2'-methyl-4H-spiro[chromene-3,1'-cyclo-pentane]-2,4-dione (2 = doremone A), (4E,8E)-1-(2,4-dihydroxyphenyl)-5,9,13-trimethyltetradeca-4,8,12-trien-1-one (3 = dshamirone), and 4,7-dihydroxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-2H-chromen-2-one (4 = am-moresinol). Dshamirone turned out to be the most active compound with an IC50 value for AChE inhibitory activity of 23.5 µM, whereas the other substances showed weak activity. The concentrations of the analytes in the resin were determined by HPLC as 3.1%, 4.6%, 1.9%, and 9.9%, respectively.

LC-NMR, NMR, and LC-MS identification and LC-DAD quantification of flavonoids and ellagic acid derivatives in Drosera peltata.

The herb of Drosera peltata, commonly named the shield sundew, is used as an antitussive in phytotherapy, although the plants' composition has not been determined in detail so far. Hence, in this study, we present a validated, sensitive, reliable, and cheap narrow-bore LC-DAD method for the simultaneous quantification of flavonoids and ellagic acid derivatives in this herbal drug. In addition, the structures of 13 compounds have been elucidated by LC-MS, LC-NMR, and offline NMR experiments after isolation: herbacetin-3-O-glucoside (1), gossypitrin (2), ellagic acid (3), quercetin-7-O-glucoside (4), isoquercitrin (5), kaempferol-3-O-(6″-O-galloyl)-glucoside (6), herbacetin-7-O-glucoside (7), astragalin (8), gossypetin (9), herbacetin (10), quercetin (11), 3,3'-di-O-methyl ellagic acid (12), and kaempferol (13). Compounds 1, 2, 4, 5, 6, 7, and 10 have been identified in D. peltata for the first time, and compounds 1, 4, 6, 7, and 10 have not been detected in any Drosera species before.

Combination of bioautography with HPTLC-MS/NMR: a fast identification of acetylcholinesterase inhibitors from galbanum(†).

INTRODUCTION: In the search for new natural compounds with acetylcholinesterase (AChE) inhibitory activity this study focused on galbanum, the oleo gum-resin from Ferula gummosa Boiss., which had shown AChE inhibitory activity in a screening. OBJECTIVE: The isolation of bioactive compounds from plant extracts usually is laborious and time consuming. In an approach to accelerate the characterisation of compounds with AChE inhibitory activity, the potential of a combination of HPTLC bioautography with HPTLC-MS/NMR for the fast identification of active compounds in galbanum was studied. METHOD: Pre-fractionation of the dichloromethane extract was performed by vacuum liquid chromatography. The resulting fractions were separated by HPTLC and active zones determined by bioautography. A TLC-MS interface was used to elute the single zones from the plates directly into a mass spectrometer. The interface was also used to extract the two major active zones from HPTLC plates for off-line one- and two-dimensional NMR and quadrupole time of flight (QTOF) MS. RESULTS: The isolated compounds were identified as 7-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl]oxy}-2H-chromen-2-one (auraptene) and 7-{[(1R,4aR,6S,8aS)-6-hydroxy-5,5,8a-trimethyl-2-methylenedecahydronaphthalen-1-yl]methoxy}-2H-chromen-2-one (farnesiferol A). This is the first report of these substances in F. gummosa. Their median inhibitory concentration (IC50 ) values for AChE inhibition were determined as 47 and 17 µg/mL in comparison with physostigmine as a positive control (IC50 : 0.8 µg/mL) and their concentrations in galbanum were quantified by HPLC as 3.5% and 7.9%, respectively. CONCLUSION: The study showed that HPTLC-MS/NMR can be considered as a fast and high-confidence method for dereplication of natural compounds. From the correlation of the concentration of the elucidated compounds and their IC50 values for AChE inhibition it can be concluded that auraptene and farnesiferol A are contributing to this activity of galbanum.

Screening of medicinal plants from Iranian traditional medicine for acetylcholinesterase inhibition.

To find new herbal compounds with an acetylcholinesterase (AChE) inhibitory effect, this study focused on herbal drugs and resins which have been used in Iranian traditional medicine for the treatment of cognitive disorders. Forty drugs were selected from authoritative written documents of Iranian traditional medicine. Each drug was extracted by accelerated solvent extraction using dichloromethane followed by methanol. The 80 extracts were screened for AChE inhibitory activity by a TLC bioautography method. The inhibiting effect of the 32 most active extracts was measured by a microplate colorimetric assay. Due to the best activity, the seeds of Peganum harmala L. were investigated in detail. From the TLC bioautography assay the alkaloids harmaline and harmine were identified as active compounds. This result was confirmed by means of HPLC-DAD. The IC(50) values were 41.2 µg/mL for the methanol extract, 95.5 µg/mL for the dichloromethane extract, 8.4 µg/mL for harmaline and 10.9 µg/mL for harmine. The concentrations of active compounds in the extracts were determined by a fast and precise HPLC method. As the amounts of harmaline and harmine in the extracts were correlated with the IC(50) values of the extracts, it can be concluded that these two alkaloids are responsible for the AChE inhibitory activity of P. harmala.