Fractionation of Lycopodiaceae Alkaloids and Evaluation of Their Anticholinesterase and Cytotoxic Activities

In view of the abundant evidence that Lycopodiaceae alkaloids, including the well-known huperzine A (HupA), are among the potent acetylcholinesterase (AChE) inhibitors, an attempt was made to search for new compounds responsible for this property. For this purpose, three plant species belonging to the Lycopodiaceae family, commonly found in the Euro-Asia region, were subjected to the isolation of bioactive compounds, their identification and subsequent evaluation of their anticholinesterase and cytotoxic activities. Methanolic extracts of two Lycopodium and one Hupezia species were obtained via optimized pressurized liquid extraction (PLE) and then pre-purified using innovative gradient vacuum liquid chromatography (gVLC). For the first time, three sorbents of different porosity packed in polypropylene cartridges and mobile phase systems of different polarity were used to elute the target compounds. This technique proved to be a rapid tool for the obtainment of alkaloid fractions and allowed one to select the appropriate process conditions to yield potent AChE inhibitors in each of the species studied. More than 100 collected fractions were analyzed via HPLC/ESI-QTOF-MS, which enabled one to detect more than 50 compounds, including several new ones previously unreported. Some of them were present in high purity fractions (60–90% of the established purity). TLC bioautography assays proved that the analyzed species are rich sources of AChE inhibitors, but H. selago showed the highest anti-AChE activity. Additionally, the modified silanized silica gel sorbent used allowed one to isolate L. clavatum alkaloids more efficiently using an aqueous reversed-phase solvent system. Furthermore, the tested extracts from the three plant extracts were found to be safe, as they did not exhibit cytotoxicity to skin fibroblasts.     

Ten new fawcettimine-related alkaloids from three species of Lycopodium

Ten new fawcettimine-related alkaloids, i.e., lycopoclavamines, lycoposquarrosamine-A, and other hydroxylated fawcettimine derivatives, were isolated from three species of Lycopodium (Lycopodium clavatum, Lycopodium serratum, and Lycopodium squarrosum). The structures of the new alkaloids were elucidated by spectroscopic methods and chemical correlation.     

Pressurized liquid extraction and anticholinesterase activity-based thin-layer chromatography with bioautography of Amaryllidaceae alkaloids

Modern extraction technique-pressurized liquid extraction (PLE) was optimised for extraction of lycorine and galanthamine (Amaryllidaceae alkaloids) from Narcissus jonquilla ‘Pipit’. Crude extracts were purified on Oasis MCX cartridges, and the alkaloids eluted with 80-100% recoveries using methanol-10% ammonia solution (3:1, v/v). Quantitative results were obtained by both HPTLC-densitometry on silica gel plates and RP-HPLC with diode array (DAD) on XTerra C₁₈ stationary phase. Both methods were fully validated in terms of specificity, precision (including intra- and inter-day measurements), LOD and LOQ values, correlation of UV spectra and linearity of calibration curves. The methods were also well correlated each other with correlation coefficients (r) 0.98823 and 0.99081, respectively, for the mean values of galanthamine and lycorine.Among the investigated solvents methanol and 1% tartaric acid methanolic solution at default conditions (120 °C, p = 60 bar, time: 10 min, one static cycle) permit the highest yields of the total sum of the alkaloids, whereas for toluene the lowest amounts were measured. Lycorine to galanthamine mean ratios were dependant on the type of solvent used, and in toluene galanthamine and related alkaloids were preferably extracted.In temperature experiments for galanthamine, the levels of this compound increased from the temperature of 20 till 150 °C in the investigated solvent systems, then decreased with slight increase from the temperature of 175 to 200 °C in 1% tartaric acid methanolic solution. When lycorine was analysed, similar trends were observed, however the maximum of the concentration was measured at a temperature about 125 °C. The ratios of the mean values of these two compounds differed in temperature-dependant experiments in both solvent systems.Further more, two TLC with bioautography approaches were used in screening for anticholinesterese properties of the extracts. No qualitative differences were found among the different solvent extracts, and AChE inhibition was correlated with galanthamine and related compounds.In conclusion, optimised PLE was much more effective than previously applied hot-solvent extraction, microwave-assisted extraction (MAE) or ultrasound-assisted extraction (USAE).     

Pressurized liquid extraction versus other extraction techniques in micropreparative isolation of pharmacologically active isoflavones from Trifolium L. species

As a new sample preparation technique, pressurized liquid extraction (PLE), in combination with reversed-phase liquid chromatography (RP-LC) and photodiode-array (PDA) detection were used for the isolation and determination of phytoestrogenic isoflavones in hydrolyzed extracts obtained from aerial parts of five Trifolium L. (clover) species. To optimize the effectiveness of PLE procedure, variable extraction parameters: methanol and acetone (or their 75% aqueous solutions), as extraction solvents, temperatures (75, 100 and 125 °C) and the changeable number of static extraction cycles were tested. Additionally, two other micropreparative techniques: ultrasound-assisted extraction (UAE), and conventional solvent extraction (CSE), under optimized conditions, were also used and compared. Optimum extraction efficiency, expressed in the highest yield of biochanin A, formononetin, daidzein and genistein from plant material, with PLE, using methanol-water (75:25, v/v) as an extraction solvent, an oven temperature of 125 °C and three 5-min static extraction cycles, was obtained.     

Bixin extraction from defatted annatto seeds

Bixin is the major carotenoid in the seed of the Annatto plant (Bixa orellana L.). The aim of this study was to obtain extracts containing bixin from seeds that had been partially defatted by supercritical fluid extraction. Pressurized liquid extraction (PLE) and low-pressure solvent extraction (LPSE) methods were used, and the effects of the solvent, temperature, pressure, solvent mass to feed mass (S/F) ratio and ultrasonication were evaluated for the global yield (X₀(%)) and the bixin yield (BY(%)). Extraction conditions producing high yields of bixin were established for both the PLE and LPSE methods. Analysis of variance was used to examine the influence of the individual extraction variables in LPSE and PLE. For LPSE; significant effects were found for solvent, temperature, and the interactions of temperature with solvent and temperature with S/F. Solvent was the only variable that significantly affected X₀(%) and BY(%), for PLE. While ultrasonication did not significantly affect X₀(%) or BY(%), scanning electron microscopy analysis revealed structural changes in the vegetal matrix following this treatment.     

Static and dynamic superheated water extraction of essential oil components from Thymus vulgaris L.

Superheated water extraction (SWE) performed in both static and dynamic condition (S‐SWE and D‐SWE, respectively) was applied for the extraction of essential oil from Thymus vulgaris L. The influence of extraction pressure, temperature, time, and flow rate on the total yield of essential oil and the influence of extraction temperature on the extraction of some chosen components are discussed in the paper. The SWE extracts are related to PLE extracts with n‐hexane and essential oil obtained by steam distillation. The superheated water extraction in dynamic condition seems to be a feasible option for the extraction of essential oil components from T. vulgaris L.     

Chemical composition of mate tea leaves (Ilex paraguariensis): a study of extraction methods

The objective of this work was to investigate the extraction of Ilex paraguariensis leaves by means of three extraction techniques: pressurized liquid extraction (PLE, also called accelerated solvent extraction – ASE), maceration, and sonication. Samples of mate tea leaves were collected from an experiment conducted under agronomic control at Indústria e Comércio de Erva‐Mate Bar?o LTDA, Brazil. Six solvents with increasing polarities (n‐hexane, toluene, dichloromethane, ethyl acetate, acetone, and methanol) were used in this investigation. Chemical analysis of the extracts was performed by GC coupled with a mass spectrometer detector. The identification and quantification were accomplished by coinjections of certified standards. The results showed that no significant differences in the qualities of the extracts were noticed regarding the extraction methods. On the other hand, the PLE technique was found to be more effective for the extractions of caffeine, phytol, palmitic, and stearic acid. The use of PLE led to a significant decrease in the total extraction time, amount of solvent consumption, and manipulation of samples compared to maceration and ultrasound‐assisted extraction methods.     

Rapid quantification of four major bioactive alkaloids in Corydalis decumbens (Thunb.) Pers. by pressurised liquid extraction combined with liquid chromatography-triple quadrupole linear ion trap mass spectrometry

A new method based on pressurised liquid extraction (PLE) followed by liquid chromatography-triple quadrupole linear ion trap mass spectrometry (LC-QTrap-MS) analysis has been developed for the identification and quantification of four major alkaloids in extracts of Corydalis decumbens (Thunb.) Pers. PLE extractions were performed using 90% ethanol; temperature was set at 100 °C and pressure at 1500 psi. HPLC analysis was performed on a Waters XBridge™ C₁₈ column (150 mm × 2.1 mm i.d., 3.5 μm) eluted by a mobile phase of acetonitrile and 0.2% acetic acid. Data acquisition was carried out in multiple reaction monitoring transitions (MRMs) mode, monitoring two MRM transitions to ensure an accurate identification of target compounds in the samples. Additional identification and confirmation of target compounds were performed using the enhanced product ion modus (EPI) of the linear ion trap. The novel LC-QTrap-MS platform offers the best sensitivity and specificity for characterization and quantitative determination of the four alkaloids in C. decumbens (Thunb.) Pers. and fulfils the quality criteria for routine laboratory application.     

Two New Lycopodium Alkaloids from Lycopodium obscurum

Two new Lycopodium alkaloids, (+)‐cermizine D N‐oxide ( 1 ) and (8β)‐8‐(acetyloxy)obscurumine A ( 2 ), along with five known compounds, were isolated from the crude alkaloid portion of Lycopodium obscurum. Their structures were elucidated on the basis of spectroscopic data and chemical correlation. All of these alkaloids were tested in an assay for acetylcholine esterase (AChE) inhibitory activity.     

Phenolic Profile,Antioxidant Capacity and Antimicrobial Activity of Nettle Leaves Extracts Obtained by Advanced Extraction Techniques

Nettle is a widely known plant whose high biological activity and beneficial medicinal effects are attributed to various bioactive compounds, among which polyphenols play an important role. In order to isolate polyphenols and preserve their properties, advanced extraction techniques have been applied to overcome the drawbacks of conventional ones. Therefore, microwave-assisted extraction (MAE) has been optimized for the isolation of nettle leaves polyphenols and it was compared to pressurized liquid extraction (PLE) and conventional heat-reflux extraction (CE). The obtained extracts were analyzed for their individual phenolic profile by UPLC MS² and for their antioxidant capacity by ORAC assay. MAE proved to be the more specific technique for the isolation of individual phenolic compounds, while PLE produced extracts with higher amount of total phenols and higher antioxidant capacity. Both techniques were more effective compared to CE. PLE nettle extract showed antimicrobial activity against bacteria, especially against Gram-negative Pseudomonas fragi ATCC 4973 and Campylobacter jejuni NCTC 11168 strains. This suggests that PLE is suitable for obtaining a nettle extract with antioxidant and antimicrobial potential, which as such has great potential for use as a value-added ingredient in the food and pharmaceutical industry.     

Pressurized liquid extraction of mate tea leaves

The objective of this work is to investigate the influence of process parameters on the pressurized liquid extraction (PLE) of Ilex paraguariensis leaves. A factorial 2⁶⁻² experimental design was employed using responses as the extraction yield and the chromatographic profile of the extracts. The extraction time, polarity of solvent, amount of sample, numbers of PLE cycles, flushing volume and extraction temperature were selected as independent variables (factors). Results obtained indicated that the solvent polarity was the most significant variable in the study, while the amount of sample and extraction temperature also showed significant effect. The other variables did not present significant influence in the yield of extraction. GC/MS analysis of the extract enabled the identification of saturated hydrocarbons, fatty acids, fatty acid methyl esters, phytosterols and theobromine in the extracts. Quantitative analysis of four compounds presented in the extracts (caffeine, phytol, vitamin E and squalene) was performed by the GC/MS in the SIM mode.     

Structure Elucidation and Cholinesterase Inhibition Activity of Two New Minor Amaryllidaceae Alkaloids

Two new minor Amaryllidaceae alkaloids were isolated from Hippeastrum × hybridum cv. Ferrari and Narcissus pseudonarcissus cv. Carlton. The chemical structures were identified by various spectroscopic (one- and two-dimensional (1D and 2D) NMR, circular dichroism (CD), high-resolution mass spectrometry (HRMS) and by comparison with literature data of similar compounds. Both isolated alkaloids were screened for their human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBuChE) inhibition activity. One of the new compounds, a heterodimer alkaloid of narcikachnine-type, named narciabduliine (2), showed balanced inhibition potency for both studied enzymes, with IC₅₀ values of 3.29 ± 0.73 µM for hAChE and 3.44 ± 0.02 µM for hBuChE. The accommodation of 2 into the active sites of respective enzymes was predicted using molecular modeling simulation.     

A Rapid Nano-Liquid Chromatographic Method for the Analysis of Cannabinoids in Cannabis sativa L. Extracts

Cannabis sativa L. is an herbaceous plant belonging to the family of Cannabaceae. It is classified into three different chemotypes based on the different cannabinoids profile. In particular, fiber-type cannabis (hemp) is rich in cannabidiol (CBD) content. In the present work, a rapid nano liquid chromatographic method (nano-LC) was proposed for the determination of the main cannabinoids in Cannabis sativa L. (hemp) inflorescences belonging to different varieties. The nano-LC experiments were carried out in a 100 µm internal diameter capillary column packed with a C18 stationary phase for 15 cm with a mobile phase composed of ACN/H₂O/formic acid, 80/19/1% (v/v/v). The reverse-phase nano-LC method allowed the complete separation of four standard cannabinoids in less than 12 min under isocratic elution mode. The nano-LC method coupled to ultraviolet (UV) detection was validated and applied to the quantification of the target analytes in cannabis extracts. The nano-LC system was also coupled to an electrospray ionization–mass spectrometry (ESI-MS) detector to confirm the identity of the cannabinoids present in hemp samples. For the extraction of the cannabinoids, three different approaches, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), and an extraction procedure adapted from the French Pharmacopeia’s protocol on medicinal plants, were carried out, and the results achieved were compared.     

Evaluation of surfactant assisted pressurized liquid extraction for the determination of glycyrrhizin and ephedrine in medicinal plants

Surfactant assisted pressurized liquid extraction (PLE) with a laboratory made system was applied for the extraction of glycyrrhizin in Radix glycyrrhizae/liquorice and ephedrine in Ephedra sinica. The proposed system set-up for this current work was simpler as no heating and back pressure regulator was required. Extraction with surfactant assisted PLE was carried out dynamically at a flow of 1.5 mL min⁻¹, at room temperature, under an applied pressure of 10-20 bar with an extraction time of 45-50 min. The extraction efficiencies of the proposed method using surfactants such as sodium dodecyl sulfate (SDS) and Triton X-100 were compared with sonication using organic solvent for different batches of medicinal plants materials. For the determination of glycyrrhizin in R. glycyrrhizae, the extraction efficiencies of surfactant assisted PLE with SDS and Triton X-100 was observed to be comparable with sonication. The method precision was found to vary from 1.6 to 2.6% (R.S.D., n = 6) on different days. For ephedrine in E. sinica, surfactant assisted PLE with SDS was found to give higher extraction efficiencies compared to Triton X-100. The overall method precision for surfactant assisted PLE with SDS for ephedrine in E. sinica was found to vary from 1.5 to 4.1% (R.S.D., n = 6) on different days. The marker compounds present in the various medicinal plant extracts were determined by gradient elution HPLC. Our data showed the possibility of PLE at room temperature and the advantages of eliminating the use of organic solvents in the extraction process.     

Selective pressurized liquid extraction of polybrominated diphenyl ethers in fish

A fast and simple method for the analysis of polybrominated diphenyl ethers (PBDEs) in fish samples was developed using a one-step extraction and clean-up by means of pressurized liquid extraction (PLE) combined with gas chromatography-ion trap tandem mass spectrometry (GC-ITMS-MS). The selective PLE method provided to obtain ready-to-analyse extracts without any additional clean-up step, using a sorbent as fat retainer inside the PLE cell. Several PLE operating conditions, such as solvent type, extraction temperature and time, number of cycles and type of fat retainer, were studied. Using Florisil as fat retainer, maximum recoveries of PBDEs (83-108%) with minimum presence of matrix-interfering compounds were obtained using a mixture of n-hexane:dichloromethane 90:10 (v/v) as solvent, an extraction temperature of 100 °C and a static extraction time of 5 min in combination with three static cycles. Quality parameters of the method were established using standards and fish samples. Limits of detection and quantification ranged from 10 to 34 pg g⁻¹ wet weight and between 34 and 68 pg g⁻¹ wet weight, respectively. In addition, good linearity (between 1 and 500 ng ml⁻¹) and high precision (RSD % < 15%) were achieved. The method was validated using the standard reference material SRM-1945 (whale blubber) and was then applied to the analysis of PBDEs in fish samples.     

Lannotinidines A-G, new alkaloids from two species of Lycopodium

Seven new Lycopodium alkaloids, lannotinidines A-G (1-7), have been isolated from the club moss Lycopodium annotinum and L. annotinum var. acrifolium. Stereochemistry of 1-7 was elucidated by combination of NOESY correlations and chemical transformation. Lannotinidines B-E (2-5) elevated NGF mRNA expression.     

Pressurized liquid extraction in determination of polychlorinated biphenyls and organochlorine pesticides in fish samples

Pressurized liquid extraction (PLE) is a relatively new technique applicable for the extraction of persistent organic pollutants from various matrices. The main advantages of this method are short time and low consumption of extraction solvent. The effects of various operational parameters (i.e. temperature of extraction, number of static cycles and extraction solvent mixtures) on the PLE efficiency were investigated in this study. Fish muscle tissue containing 3.2% (w/w) lipids and native polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs) and other related compounds was used for testing. Purification of crude extracts was carried out by gel permeation chromatography employing Bio-Beads S-X3. Identification and quantitation of target indicator PCBs and OCPs was performed by high-resolution gas chromatography (HRGC) with two parallel electron-capture detectors (ECDs). Results obtained by the optimized PLE procedure were compared with conventional Soxhlet extraction (the same extraction solvent mixtures hexane–dichloromethane (1:1 v/v) and hexane–acetone (4:1 v/v) were used). The recoveries obtained by PLE operated at 90–120 °C were either comparable to “classic” Soxhlet extraction (for higher-chlorinated PCB congeners and DDT group) or even better (for lower chlorinated analytes). The highest recoveries were obtained for three static 5 min extraction cycles.     

Optimization of Pressurized Liquid Extraction and In Vitro Neuroprotective Evaluation of Ammodaucus leucotrichus. Untargeted Metabolomics Analysis by UHPLC-MS/MS

Ammodaucus leucotrichus is a spontaneous plant endemic of the North African region. An efficient selective pressurized liquid extraction (PLE) method was optimized to concentrate neuroprotective extracts from A. leucotrichus fruits. Green solvents were tested, namely ethanol and water, within a range of temperatures between 40 to 180 °C. Total carbohydrates and total phenolics were measured in extracts, as well as in vitro antioxidant capacity (DPPH radical scavenging), anticholinesterase (AChE) and anti-inflammatory (LOX) activities. Metabolite profiling was carried out by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-q-TOF-MS/MS), identifying 94 compounds. Multivariate analysis was performed to correlate composition with bioactivity. A remarkable effect of the temperature using water was observed: the higher temperature, the higher extraction yield, the higher total phenolic content, as well as the higher total carbohydrates content. The water extract obtained at 180 °C, 10.34 MPa and 10 min showed meaningful anti-inflammatory (IC50_LOX = 39.4 µg/mL) and neuroprotective activities (IC50_AChE = 55.6 µg/mL). The Principal Components Analysis (PCA) and the cluster analysis correlated these activities with the presence of carbohydrates and phenolic compounds.     

Pressurized-Liquid Extraction as an Efficient Method for Valorization of Thymus serpyllum Herbal Dust towards Sustainable Production of Antioxidants

The aim of this study was to valorize Thymus serpyllum herbal dust, a particular fraction distinguished as an industrial waste from filter-tea production. Pressurized liquid extraction (PLE) was used with the aim of overcoming certain obstacles of conventional extraction techniques in terms of shortening extraction time, reducing solvent consumption and energy costs, using “green” solvents and obtaining high yield and quality products. In order to optimize PLE of T. serpyllum herbal dust, the preliminary screening of the independent variables in order to define the most influential parameters and their domain was done first. After the screening, the optimization study using the face-centered central composite experimental design (CCD) with response surface methodology (RSM) was implemented. Additionally, taking into account the high awareness of the positive influence of antioxidants on the human health and associating it with high content of polyphenolic compounds in various members of Lamiaceae family, PLE has proven to be a great approach for antioxidants recovery from T. serpyllum herbal dust.     

Comparison of Extraction Techniques and Surfactants for the Isolation of Total Polyphenols and Phlorotannins from the Brown Algae Lobophora variegata

Abstract

Surfactant-mediated extraction (SME), pressurized liquid extraction (PLE), and enzyme-assisted extraction (EAE) have been compared to improve the isolation of phlorotannins from the brown algae Lobophora variegata. Enzymatic treatment with Alcalase 2.4?L FG, Carezyme 4500?L, protease from Streptomyces griseus, pectinase from Aspergillus niger, Celluclast 1.5?L, protease from Bacillus licheniformis; surfactant extraction with triacetin and guaiacol and PLE with ethanol:water as extracting solvent, have been studied in terms of total phenolic content by the Folin–Ciocalteu method and total phlorotannin content using the DMBA assay. The results showed that SME yields the highest amount of phenols and phlorotannins by using food grade guaiacol as the surfactant. An extraction protocol was developed to maximize the amount of extract obtained from L. variegata. The effects of various parameters such as the type of surfactant, efficacy of surfactant, and optimum pH, on the extraction efficiency of polyphenols were examined. The simultaneous use of the enzyme and surfactant was also investigated. However, a synergistic effect between the enzymes and the surfactant for the extraction of polyphenols has not been observed. Considering total phenols and total phlorotannins in the extract, the extraction yield were obtained for total phenols as SME?>?EAE?>?PLE and for total phlorotannins as SME?>?PLE?>?EAE.