Modeling the liquid-liquid chromatography separation of cannabinoids from hemp extracts.

The separation of cannabinoids from hemp materials is nowadays one of the most promising industrial applications of liquid-liquid chromatography (LLC). Despite various experimental research efforts to purify cannabinoids, there are currently few works on process modeling. Thus, this study aimed to explore a straightforward approach to model the LLC separation of cannabinoids from two hemp extracts with different compositions. The feed materials were simplified to mixtures of preselected key components (i.e., cannabidiol, tetrahydrocannabinol, cannabigerol, and cannabinol). The elution profiles of cannabinoids were simulated using the equilibrium-cell model with an empirical nonlinear correlation. The model parameters were derived from the elution profiles of single-solute pulse injections. For the validation of the proposed approach, LLC separations with the two hemp extracts were performed in descending mode with the solvent system composed of hexane/methanol/water 10/8/2 (v/v/v). The injected sample concentrations were gradually increased from 5 to 100 mg/mL. The results showed that the approach could describe reasonably well the elution behavior of the cannabinoids, with deviations of only 1-2 min between simulated and experimental elution times. However, to improve the prediction accuracy, the model parameters can be refitted to the elution profiles of 3-4 systematically selected pulse injections with specific hemp extracts.

Spent Material Extractives from Hemp Hydrodistillation as an Underexplored Source of Antimicrobial Cannabinoids.

Hemp (Cannabis sativa L.) has been used for millennia as a rich source of food and fibers, whereas hemp flowers have only recently gained an increased market interest due to the presence of cannabinoids and volatile terpenes. Currently, the hemp flower processing industry predominantly focuses on either cannabinoid or terpene extraction. In an attempt to maximize the valorization of hemp flowers, the current study aimed to evaluate the phytochemical composition and antimicrobial properties of several extracts obtained from post-distillation by-products (e.g., spent material, residual distillation water) in comparison to the essential oil and total extract obtained from unprocessed hemp flowers. A terpene analysis of the essential oil revealed 14 monoterpenes and 35 sesquiterpenes. The cannabinoid profiling of extracts showed seven acidic precursors and 14 neutral derivatives, with cannabidiol (CBD) reaching the highest concentration (up to 16 wt.%) in the spent material extract. The antimicrobial assessment of hemp EO, cannabinoid-containing extracts, and single compounds (i.e., CBD, cannabigerol, cannabinol, and cannabichromene) against a panel of 20 microbial strains demonstrated significant inhibitory activities against Gram-positive bacteria, Helicobacter pylori, and Trichophyton species. In conclusion, this work suggests promising opportunities to use cannabinoid-rich materials from hemp flower processing in functional foods, cosmetics, and pharmaceuticals with antimicrobial properties.

Model-based design of gradient elution in liquid-liquid chromatography: Application to the separation of cannabinoids.

Liquid-liquid chromatography (LLC) is a separation technique that utilizes a biphasic solvent system as the mobile and stationary phases. The components are separated solely due to their different distributions between the two liquid phases. Gradient change in the mobile phase composition during the chromatographic process is a powerful method for improving the resolution of separation or shortening the process time. Gradient elution readily applies to LLC with biphasic solvent systems in which the stationary phase composition remains nearly constant when the mobile phase composition changes. This work proposes a model-based approach to optimize gradients in LLC and circumvent tedious trial-and-error experiments. The solutes' distribution constant depends on the mobile phase composition. Thus, the distribution constants were described as a function of the content of one of the solvents (= modifier) in the mobile phase. The dispersive and mass-transfer effects in the tubing and the column are modeled with a stage model. Only a few experiments are required to determine the model parameters. After the validation of the model and its parameters, the model can be used for LLC gradient optimization. The proposed approach was demonstrated for a gradient LLC separation of a mixture of four cannabinoids. Two different gradient shapes, one-step and linear gradient, were considered. For a pre-selected minimal purity requirement, the gradient was optimized for maximum process efficiency, defined as the product of productivity and yield. An experiment conducted with the optimized gradient conditions was in good agreement with the simulation, showing the potential of the proposed method.

Continuous multi-column capture of monoclonal antibodies with convective diffusive membrane adsorbers.

Downstream processing is the bottleneck in the continuous manufacturing of monoclonal antibodies (mAbs). To overcome throughput limitations, two different continuous processes with a novel convective diffusive protein A membrane adsorber (MA) were investigated: the rapid cycling parallel multi-column chromatography (RC-PMCC) process and the rapid cycling simulated moving bed (RC-BioSMB) process. First, breakthrough curve experiments were performed to investigate the influence of the flow rate on the mAb dynamic binding capacity and to calculate the duration of the loading steps. In addition, customized control software was developed for an automated MA exchange in case of pressure increase due to membrane fouling to enable robust, uninterrupted, and continuous processing. Both processes were performed for 4 days with 0.61 g L-1 mAb-containing filtrate and process performance, product purity, productivity, and buffer consumption were compared. The mAb was recovered with a yield of approximately 90% and productivities of 1010 g L-1 d-1 (RC-PMCC) and 574 g L-1 d-1 (RC-BioSMB). At the same time, high removal of process-related impurities was achieved with both processes, whereas the buffer consumption was lower for the RC-BioSMB process. Finally, the attainable productivity for perfusion bioreactors of different sizes with suitable MA sizes was calculated to demonstrate the potential to operate both processes on a manufacturing scale with bioreactor volumes of up to 2000 L.

Estimating the nonideality of eutectic systems containing thermally unstable substances.

Eutectic systems design requires an in-depth understanding of their solid-liquid equilibria (SLE). Modeling SLE in eutectic systems has as prerequisites, the melting properties and activity coefficients of components in the liquid phase. Thus, due to the unavailable melting properties of thermally unstable substances, it is impossible to estimate their activity coefficients from experimental SLE data and model the SLE phase diagram of their eutectic systems. Here, we evaluate the activity coefficients of thermally unstable constituents in the liquid phase, which were calculated independent of their melting properties by correlating the SLE data of their cocrystals. Differential scanning calorimetry and powder x-ray diffraction were employed to obtain the SLE phase diagram of three eutectic systems, i.e., tetramethylammonium chloride/catechol, tetraethylammonium chloride/catechol, and betaine/catechol systems, and identify the formation of nine cocrystals. The non-random, two-liquid equation was used to calculate the activity coefficients of the components in the liquid phase. The substantial negative deviation from ideality in the three studied systems indicated strong hydrogen bonding interactions in the liquid solution. Furthermore, modeling ion-ion interactions in eutectic systems containing ionic constituents is of utmost importance for understanding their nonideality.

Nonlinear liquid-liquid chromatography: Modeling a binary mixture separation.

In liquid-liquid chromatography (LLC), mixture components are separated due to their different distribution between the phases of a biphasic liquid system composed of three or four solvents. LLC separations are typically modeled assuming that only the solutes distribute between the two liquid phases and their distribution can be described with a concentration-independent distribution constant. With increasing solute concentration, the physicochemical properties of the biphasic system change, and the distribution of the solutes becomes a function of their concentration. However, the experimental determination of liquid-liquid equilibria in multicomponent systems is time-intensive, and its prediction using thermodynamic models is often not sufficiently accurate for process design purposes. Thus, in this work, we propose a simple approach to model and simulate LLC separations in the nonlinear (concentration-dependent) range of the solutes' distribution equilibria, namely cannabidiol (CBD) and cannabigerol (CBG). Using the inverse method, the distribution equilibrium equation parameters were estimated from pulse injection experiments of single solutes at concentrations ranging from 1 to 100 mg/mL and 1-50 mg/mL for CBD and CBG, respectively. The obtained parameters were then successfully used to predict the elution profiles of binary mixtures of different compositions at 40 mg/mL total cannabinoid concentration. The approach was demonstrated and validated for CBD and CBG as model compounds and n-hexane/methanol/water 10/7.5/2.5 (v/v/v) as the biphasic solvent system. It should be noted that the applicability of the proposed approach is system-dependent, and hence, it should be evaluated for each separation task individually.

Influence of Flow Rate, Particle Size, and Temperature on Espresso Extraction Kinetics.

Brewing espresso coffee (EC) is considered a craft and, by some, even an art. Therefore, in this study, we systematically investigated the influence of coffee grinding, water flow rate, and temperature on the extraction kinetics of representative EC components, employing a central composite experimental design. The extraction kinetics of trigonelline, caffeine, 5-caffeoylquinic acid (5-CQA), and Total Dissolved Solids (TDS) were determined by collecting and analyzing ten consecutive fractions during the EC brewing process. From the extraction kinetics, the component masses in the cup were calculated for Ristretto, Espresso, and Espresso Lungo. The analysis of the studied parameters revealed that flow rate had the strongest effect on the component mass in the cup. The intensity of the flow rate influence was more pronounced at finer grindings and higher water temperatures. Overall, the observed influences were minor compared to changes resulting from differences in total extracted EC mass.

Liquid-liquid chromatography isolation of Petasites hybridus sesquiterpenes and their LC-HR-MS/MS and NMR characterization.

Petasites hybridus L. (butterbur, Asteraceae) is a well-known medicinal plant traditionally used as a remedy for neurological, respiratory, cardiovascular, and gastrointestinal disorders. Eremophilane-type sesquiterpenes (petasins) are considered to be the major bioactive constituents of butterbur. However, efficient methods to isolate high-purity petasins in sufficient amounts for further analytical and biological testing are lacking. In this study, various sesquiterpenes were separated from a methanol rootstock extract of P. hybridus with liquid-liquid chromatography (LLC). The appropriate biphasic solvent system was selected using the predictive thermodynamic model COSMO-RS and shake-flask experiments. After the selection of the feed (extract) concentration and operating flow rate, a batch LLC experiment was performed with n-hexane/ethyl acetate/methanol/water 5/1/5/1 (v/v/v/v). For those LLC fractions containing petasin derivatives with purities < 95%, a preparative high-performance liquid chromatography purification step followed. All isolated compounds were identified by state-of-the-art spectroscopic methods, i.e., liquid chromatography coupled with high-resolution tandem mass spectrometry and nuclear magnetic resonance techniques. As a result, six compounds were obtained, namely 8ß-hydroxyeremophil-7(11)-en-12,8-olide, 2-[(angeloyl)oxy]eremophil-7(11)-en-12,8-olide, 8α/ß-H-eremophil-7(11)-en-12,8-olide, neopetasin, petasin, and isopetasin. The isolated petasins can be further used as reference materials for standardization and pharmacological evaluation.

Predicting the essential oil composition in supercritical carbon dioxide extracts from hop pellets using mathematical modeling.

Supercritical fluid extraction from hops (Humulus lupulus L.) can be used to extract essential oil for the flavoring of beer. With a special focus on the oil composition being linked to the hop aroma, the influence of pressure and temperature on the extraction kinetics of seven oil components (ß-myrcene, α-humulene, ß-caryophyllene, 2-methylbutyl isobutyrate, undecanone, linalool, and α-pinene) is analyzed and modeled in this article. Supercritical CO2 extraction from hop pellets was conducted at pressure-temperature combinations of 90/100/110 bar and 40/45/50 °C. The extract composition over time, analyzed by gas chromatography, was used for the parameterization of two existing mechanistic models: an internal-mass-transfer-control (IMTC), and a broken-and-intact-cells (BIC) model. The IMTC model was found to effectively describe most extraction kinetics and hence applied in this study. In contrast to previous studies, the IMTC model parameters were not only fitted to individual extraction curves from different experiments but also correlated to temperature and pressure as a further step towards model-based prediction. Using the parameterized model, the extract composition was predicted at 95 bar/48 °C, 105 bar/42 °C, and 105 bar/48 °C. Extraction yields were found to be higher at lower temperatures and higher pressures in general. The sensitivity towards pressure was observed to differ between components and to be particularly higher for ß-myrcene compared with α-humulene. Changes of the essential oil composition with a variation in pressure and temperature were predicted correctly by the model with a mean relative deviation from experimental data of 11.7% (min. 1.2%, max. 36.2%).

Value-Added Compounds with Antimicrobial, Antioxidant, and Enzyme-Inhibitory Effects from Post-Distillation and Post-Supercritical CO2 Extraction By-Products of Rosemary.

Hydrodistillation is the main technique to obtain essential oils from rosemary for the aroma industry. However, this technique is wasteful, producing numerous by-products (residual water, spent materials) that are usually discarded in the environment. Supercritical CO2 (SC-CO2) extraction is considered an alternative greener technology for producing aroma compounds. However, there have been no discussions about the spent plant material leftover. Therefore, this work investigated the chemical profile (GC-MS, LC-HRMS/MS) and multi-biological activity (antimicrobial, antioxidant, enzyme inhibitory) of several raw rosemary materials (essential oil, SC-CO2 extracts, solvent extracts) and by-products/waste materials (post-distillation residual water, spent plant material extracts, and post-supercritical CO2 spent plant material extracts). More than 55 volatile organic compounds (e.g., pinene, eucalyptol, borneol, camphor, caryophyllene, etc.) were identified in the rosemary essential oil and SC-CO2 extracts. The LC-HRMS/MS profiling of the solvent extracts revealed around 25 specialized metabolites (e.g., caffeic acid, rosmarinic acid, salvianolic acids, luteolin derivatives, rosmanol derivatives, carnosol derivatives, etc.). Minimum inhibitory concentrations of 15.6-62.5 mg/L were obtained for some rosemary extracts against Micrococcus luteus, Bacilus cereus, or Staphylococcus aureus MRSA. Evaluated in six different in vitro tests, the antioxidant potential revealed strong activity for the polyphenol-containing extracts. In contrast, the terpene-rich extracts were more potent in inhibiting various key enzymes (e.g., acetylcholinesterase, butyrylcholinesterase, tyrosinase, amylase, and glucosidase). The current work brings new insightful contributions to the continuously developing body of knowledge about the valorization of rosemary by-products as a low-cost source of high-added-value constituents in the food, pharmaceutical, and cosmeceutical industries.

Nonlinear liquid-liquid chromatography: Beyond a constant distribution coefficient.

Liquid-liquid chromatography (LLC) is a technique in which the separation of mixture components is achieved due to their different distribution between the two phases of a pre-equilibrated biphasic solvent system. In this work, the LLC operation in the nonlinear range of the distribution isotherm was systematically examined for the first time. The influence of the feed concentration on the elution profiles of a model component (cannabidiol, CBD) was studied in three LLC units of different types and sizes ranging from ∼20 mL to ∼2 L. A series of pulse injections with CBD concentrations varying from 1 to 300 mg/mL was performed with n-hexane/methanol/water 5/4/1 (v/v/v) in descending mode (lower phase as the mobile phase). The elution profiles were simulated using the equilibrium-cell model and an anti-Langmuir-like equation for describing the CBD distribution equilibria. The distribution equilibria equation parameters were fitted to the CBD elution profiles using the peak fitting method. The model was validated and provided good predictions of the CBD elution profiles in the entire concentration range for all three LLC units.

Supercritical CO2 extraction of spices: A systematic study with focus on terpenes and piperamides from black pepper (Piper nigrum L.).

Black pepper (P. nigrum L.) is considered one of the most valuable spices and a promising candidate in natural product research. In this study, the influence of different combinations of pressures (100-300 bar) and temperatures (40-60 °C) on the supercritical CO2 (SC-CO2) recovery of several key compounds from black pepper was evaluated systematically. The extraction curves showed that terpenes were recovered in a short time under all studied conditions. In contrast, higher pressure values were required to extract piperamides efficiently. Furthermore, the differences in the extraction kinetics of piperine, piperettine, pellitorine, guineensine, and N-isobutyl-2,4,14-eicosatrienamide were linked with several structural features, such as the nature of the amine group or the terminal part of the fatty acid. The data from the isocratic experiments represented the starting point for designing a two-step pressure gradient SC-CO2 process in which one terpene-rich and one piperamide-rich product were successively obtained.

Influence of Cocatalysts (Ni, Co, and Cu) and Synthesis Method on the Photocatalytic Activity of Exfoliated Graphitic Carbon Nitride for Hydrogen Production.

Exfoliated graphitic carbon nitride (ex-g-CN) was synthesized and loaded with non-noble metals (Ni, Cu, and Co). The synthesized catalysts were tested for hydrogen production using a 300-W Xe lamp equipped with a 395 nm cutoff filter. A noncommercial double-walled quartz-glass reactor irradiated from the side was used with a 1 g/L catalyst in 20 mL of a 10 vol% triethanolamine aqueous solution. For preliminary screening, the metal-loaded ex-g-CN was synthesized using the incipient wetness impregnation method. The highest hydrogen production was observed on the Ni-loaded ex-g-CN, which was selected to assess the impact of the synthesis method on hydrogen production. Ni-loaded ex-g-CN was synthesized using different synthesis methods: incipient wetness impregnation, colloidal deposition, and precipitation deposition. The catalysts were characterized by X-ray powder diffraction, X-ray photoelectron spectroscopy, nitrogen adsorption using the Brunauer-Emmett-Teller method, and transmission electron microscopy. The Ni-loaded ex-g-CN synthesized using the colloidal method performed best with a hydrogen production rate of 43.6 µmol h-1 g-1. By contrast, the catalysts synthesized using the impregnation and precipitation methods were less active, with 28.2 and 10.1 µmol h-1 g-1, respectively. The hydrogen production performance of the suspended catalyst (440 µmol m-2 g-1) showed to be superior to that of the corresponding immobilized catalyst (236 µmol m-2 g-1).

Design of Deep Eutectic Systems: Plastic Crystalline Materials as Constituents.

Deep eutectic solvents (DESs) are a class of green and tunable solvents that can be formed by mixing constituents having very low melting entropies and enthalpies. As types of materials that meet these requirements, plastic crystalline materials (PCs) with highly symmetrical and disordered crystal structures can be envisaged as promising DES constituents. In this work, three PCs, namely, neopentyl alcohol, pivalic acid, and neopentyl glycol, were studied as DES constituents. The solid-plastic transitions and melting properties of the pure PCs were studied using differential scanning calorimetry. The solid-liquid equilibrium phase diagrams of four eutectic systems containing the three PCs, i.e., L-menthol/neopentyl alcohol, L-menthol/pivalic acid, L-menthol/neopentyl glycol, and choline chloride/neopentyl glycol, were measured. Despite showing near-ideal behavior, the four studied eutectic systems exhibited depressions at the eutectic points, relative to the melting temperatures of the pure constituents, that were similar to or even larger than those of strongly nonideal eutectic systems. These findings highlight that a DES can be formed when PCs are used as constituents, even if the eutectic system is ideal.

A review on the ethnobotany, phytochemistry, pharmacology and toxicology of butterbur species (Petasites L.).

ETHNOPHARMACOLOGICAL RELEVANCE: Petasites (butterbur, Asteraceae) species have been used since Ancient times in the traditional medicine of Asian and European countries to treat central nervous system (migraine), respiratory (asthma, allergic rhinitis, bronchitis, spastic cough), cardiovascular (hypertension), gastrointestinal (ulcers) and genitourinary (dysmenorrhea) disorders. AIM OF THE REVIEW: This study summarized and discussed the traditional uses, phytochemical, pharmacological and toxicological aspects of Petasites genus. MATERIALS AND METHODS: A systematic search of Petasites in online databases (Scopus, PubMed, ScienceDirect, Google Scholar) was performed, with the aim to find the phytochemical, toxicological and bioactivity studies. The Global Biodiversity Information Facility, Plants of the World Online, World Flora Online and The Plant List databases were used to describe the taxonomy and geographical distribution. RESULTS: The detailed phytochemistry of the potentially active compounds of Petasites genus (e.g. sesquiterpenes, pyrrolizidine alkaloids, polyphenols and essential oils components) was presented. The bioactivity studies (cell-free, cell-based, animal, and clinical) including the traditional uses of Petasites (e.g. anti-spasmolytic, hypotensive, anti-asthmatic activities) were addressed and followed by discussion of the main pharmacokinetical and toxicological issues related to the administration of butterbur-based formulations. CONCLUSIONS: This review provides a complete overview of the Petasites geographical distribution, traditional use, phytochemistry, bioactivity, and toxicity. More than 200 different sesquiterpenes (eremophilanes, furanoeremophilanes, bakkenolides), 50 phenolic compounds (phenolic acids, flavonoids, lignans) and volatile compounds (monoterpenes, sesquiterpenes) have been reported within the genus. Considering the phytochemical complexity and the polypharmacological potential, there is a growing research interest to extend the current therapeutical applications of Petasites preparations (anti-migraine, anti-allergic) to other human ailments, such as central nervous system, cardiovascular, malignant or microbial diseases. This research pathway is extremely important, especially in the recent context of the pandemic situation, when there is an imperious need for novel drug candidates.

Carbon Dioxide Solubility in Nonionic Deep Eutectic Solvents Containing Phenolic Alcohols.

Deep eutectic solvents (DES) are a new class of green solvents that have shown unique properties in several process applications. This study evaluates nonionic DES containing phenolic alcohols as solvents for carbon dioxide (CO2) capture applications. Potential phenolic alcohols and the molar ratio between DES constituents were preselected for experimental investigations based on the conductor-like screening model for realistic solvation (COSMO-RS). CO2 solubility was experimentally determined in two different DES, namely, L-menthol/thymol in 1:2 molar ratio and thymol/2,6-xylenol in 1:1 molar ratio, at various temperatures and pressures. CO2 solubility in the studied systems was higher than that reported in the literature for ionic DES and ionic liquids. This study demonstrates that nonionic DES containing phenolic alcohols can be excellent, inexpensive, and simple solvents for CO2 capture.

Separation of minor cannabinoids from hemp extract with trapping multiple dual mode liquid-liquid chromatography.

Aside from Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), other less common cannabinoids have recently gained an increasing popularity, mostly due to their promising biological potential. However, time-saving and cost-effective methods for their preparative purification are missing. In this study, trapping multiple dual mode (MDM), a flow-reversal liquid-liquid chromatography (LLC) operating mode, was used for the separation of different minor cannabinoids from a hemp extract. Separation task specific biphasic solvent systems were selected for the purification of the target constituents, as follows: n-hexane/methanol/water 10/6.5/3.5 for cannabielsoin (CBE); n-hexane/methanol/water 10/7/3 for cannabidivarin (CBDV) and cannabigerol (CBG); n-hexane/methanol/water 10/8/2 for cannabinol (CBN) and n-hexane/methanol/water 10/9/1 for cannabichromene (CBC) and cannabicylol (CBL). For each separation task, the concentration of the hemp extract in the feed stream and mobile phase flow rate were selected by shake-flask and stationary phase retention experiments, respectively. For the determination of the trapping MDM operating parameters, the short-cut method was implemented and followed by equilibrium-cell model-based simulations. The trapping MDM allowed the separation of the targeted cannabinoids with purities of 93-99%, yields of 73-95%, solvent consumption 2-4-fold lower and productivities almost double than those obtained using batch separation.

Coinfection and Interference Phenomena Are the Results of Multiple Thermodynamic Competitive Interactions.

Biological, physical and chemical interaction between one (or more) microorganisms and a host organism, causing host cell damage, represents an infection. Infection of a plant, animal or microorganism with a virus can prevent infection with another virus. This phenomenon is known as viral interference. Viral interference is shown to result from two types of interactions, one taking place at the cell surface and the other intracellularly. Various viruses use different receptors to enter the same host cell, but various strains of one virus use the same receptor. The rate of virus-receptor binding can vary between different viruses attacking the same host, allowing interference or coinfection. The outcome of the virus-virus-host competition is determined by the Gibbs energies of binding and growth of the competing viruses and host. The virus with a more negative Gibbs energy of binding to the host cell receptor will enter the host first, while the virus characterized by a more negative Gibbs energy of growth will overtake the host metabolic machine and dominate. Once in the host cell, the multiplication machinery is shared by the competing viruses. Their potential to utilize it depends on the Gibbs energy of growth. Thus, the virus with a more negative Gibbs energy of growth will dominate. Therefore, the outcome can be interference or coinfection, depending on both the attachment kinetics (susceptibility) and the intracellular multiplication machinery (permittivity). The ratios of the Gibbs energies of binding and growth of the competing viruses determine the outcome of the competition. Based on this, a predictive model of virus-virus competition is proposed.

Insights into the Phytochemical and Multifunctional Biological Profile of Spices from the Genus Piper.

Piper spices represent an inexhaustible reservoir of bioactive compounds that may act as drug leads in natural product research. The aim of this study was to investigate a series of methanolic fruit extracts obtained from P. nigrum (black, green, white and red), P. longum and P. retrofractum in comparative phytochemical and multi-directional biological (antimicrobial, antioxidant, anti-enzymatic and anti-melanogenic) assays. The metabolite profiling revealed the presence of 17 piperamides, with a total content of 247.75-591.42 mg piperine equivalents/g. Among the 22 tested microorganism strains, Piper spices were significantly active (MIC < 0.1 mg/mL) against the anaerobes Actinomyces israelii and Fusobacterium nucleatum. The antioxidant and anti-enzymatic activities were evidenced in DPPH (10.64-82.44 mg TE/g) and ABTS (14.20-77.60 mg TE/g) radical scavenging, CUPRAC (39.94-140.52 mg TE/g), FRAP (16.05-77.00 mg TE/g), chelating (0-34.80 mg EDTAE/g), anti-acetylcholinesterase (0-2.27 mg GALAE/g), anti-butyrylcholinesterase (0.60-3.11 mg GALAE/g), anti-amylase (0.62-1.11 mmol ACAE/g) and anti-glucosidase (0-1.22 mmol ACAE/g) assays. Several Piper extracts (10 µg/mL) inhibited both melanin synthesis (to 32.05-60.65% of αMSH+ cells) and release (38.06-45.78% of αMSH+ cells) in αMSH-stimulated B16F10 cells, partly explained by their tyrosinase inhibitory properties. Our study uncovers differences between Piper spices and sheds light on their potential use as nutraceuticals or cosmeceuticals for the management of different diseases linked to bacterial infections, Alzheimer's dementia, type 2 diabetes mellitus or hyperpigmentation.

Influence of the Post-Harvest Storage Time on the Multi-Biological Potential, Phenolic and Pyrrolizidine Alkaloid Content of Comfrey (Symphytum officinale L.) Roots Collected from Different European Regions.

Comfrey (Symphytum officinale L.) roots are well-known bioactive ingredients included in various cosmeceutical and pharmaceutical preparations. In this study, the influence of the post-harvest storage on the chemico-biological potential of roots collected from different European regions and stored for up to six months was investigated. Total phenolic content (TPC) and total phenolic acid content (TPAC) were spectrophotometrically estimated, whereas the levels of individual phenolic and pyrrolizidine alkaloidal markers were determined by HPLC-DAD and HPLC-MS/MS, respectively. The changes in the biological potential was tracked via antioxidant (DPPH, ABTS, CUPRAC, and FRAP) and anti-enzymatic (cholinesterase, tyrosinase, glucosidase, and amylase) assays. TPC and TPAC varied from 6.48-16.57 mg GAE/g d.w. root and from 2.67-9.03 mg CAE/g, respectively. The concentration of the four phenolics (rosmarinic acid, globoidnan A, globoidnan B, rabdosiin) and six pyrrolizidine alkaloids generally showed maximum values at 1-3 months, after which their levels significantly decreased. With respect to the bioassays, the samples showed a wide range of antioxidant and anti-enzymatic effects; however, a direct storage time-bioactivity relationship was not observed. Similar conclusions were also revealed by the multivariate and correlation analyses. Our study could improve the current knowledge of the shelf-life properties of comfrey-based products and enhance their industrial exploitation.