Alginate-Based Carriers Loaded with Mulberry (Morus alba L.) Leaf Extract: A Promising Strategy for Prolonging 1-Deoxynojirimicyn (DNJ) Systemic Activity for the Nutraceutical Management of Hyperglycemic Conditions.

The iminosugar 1-deoxynojirimicyn (DNJ) contained in mulberry leaves has displayed systemic beneficial effects against disorders of carbohydrate metabolism. Nevertheless, its effect is impaired by the short half-life. Alginate-based carriers were developed to encapsulate a DNJ-rich mulberry extract: Ca-alginate beads, obtained by external gelation, and spray-dried alginate microparticles (SDMs). Mean size and distribution, morphology, drug loading, encapsulation efficiency, experimental yield, and release characteristics were determined for the two formulations. Ca-alginate beads and SDMs exhibited an encapsulation efficiency of about 54% and 98%, respectively, and a DNJ loading in the range of 0.43-0.63 µg/mg. The in vitro release study demonstrated the carriers' capability in controlling the DNJ release in acid and basic conditions (<50% in 5 h), due to electrostatic interactions, which were demonstrated by 1H-NMR relaxometry studies. Thus, alginate-based particles proved to be promising strategies for producing food supplements containing mulberry leaf extracts for the management of hyperglycemic state.

Multivariate Statistical Models for the Authentication of Traditional Balsamic Vinegar of Modena and Balsamic Vinegar of Modena on 1H-NMR Data: Comparison of Targeted and Untargeted Approaches.

This work aimed to compare targeted and untargeted approaches based on NMR data for the construction of classification models for Traditional Balsamic Vinegar of Modena (TBVM) and Balsamic Vinegar of Modena (BVM). Their complexity in terms of composition makes the authentication of these products difficult, which requires the employment of several time-consuming analytical methods. Here, 1H-NMR spectroscopy was selected as the analytical method for the analysis of TVBM and BVM due to its rapidity and efficacy in food authentication. 1H-NMR spectra of old (>12 years) and extra-old (>25 years) TVBM and BVM (>60 days) and aged (>3 years) BVM were acquired, and targeted and untargeted approaches were used for building unsupervised and supervised multivariate statistical modes. Targeted and untargeted approaches were based on quantitative results of peculiar compounds present in vinegar obtained through qNMR, and all spectral variables, respectively. Several classification models were employed, and linear discriminant analysis (LDA) demonstrated sensitivity and specificity percentages higher than 85% for both approaches. The most important discriminating variables were glucose, fructose, and 5-hydroxymethylfurfural. The untargeted approach proved to be the most promising strategy for the construction of LDA models of authentication for TVBM and BVM due to its easier applicability, rapidity, and slightly higher predictive performance. The proposed method for authenticating TBVM and BVM could be employed by Italian producers for safeguarding their valuable products.

Novel application of 1H NMR spectroscopy coupled with chemometrics for the authentication of dark chocolate.

The applicability of 1H NMR spectroscopy coupled with chemometric in the quality control of dark chocolate was investigated for the first time to detect cocoa-butter equivalents (CBEs) above the allowed limit by European regulation. Blends of chocolate-fats with CBEs in the range 0-50 % were prepared and analyzed by 1H NMR spectroscopy. Datasets composed of peaks' areas or spectral variables (fingerprinting) in glycerol region were tested for the creation of multivariate statistical models. Partial least-squares discriminant analysis (PLS-DA) and regression (PLS-R) methods were used to correctly identify the type of CBE and quantify its concentration respectively. The performances of the models created on the two datasets were evaluated in terms of chemometric indicators and compared. The robustness of models was investigated through the analysis of test sets and random permutation tests. Fingerprinting models revealed fruitful results in classifying and quantifying CBEs in blends demonstrating the applicability of NMR in chocolate quality control.

In vitro bioactivity evaluation of mulberry leaf extracts as nutraceuticals for the management of diabetes mellitus.

There is an increasing need for new options to treat diabetes mellitus at its early stage and natural remedies have been recently reassessed as potential candidates owing to their low-cost and effectiveness. Genus Morus plants contain many active compounds with hypoglycaemic, hypolipidemic, and antioxidant effects. Current research on mulberry chemical composition and bioactivity has been generally carried out only on Asian cultivation, where this plant has been traditionally used in the form of leaf infusion for decades. In this work, twelve Italian mulberry cultivars were fully characterised to fill this gap of knowledge, since a strong correlation among composition, genetics and growing area was proven. Antiglycative and hypoglycaemic effects of leaf extracts were evaluated using different in vitro models. The results indicate that the inhibitory effect on carbohydrate digestive enzymes was likely mediated by 1-deoxynojirimycin, kaempferol, quercetin, and chlorogenic acid, acting in a synergistic way. Besides, the combined antiglycative and carbonyl trapping capacities, tested here for the first time, may help in preventing long-term complications related to AGEs in diabetic patients.

Chemical characterization of non-psychoactive Cannabis sativa L. extracts, in vitro antiproliferative activity and induction of apoptosis in chronic myelogenous leukaemia cancer cells.

In this study, extracts from non-psychoactive Cannabis sativa L. varieties were characterized by means of ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) and their antiproliferative activity was assessed in vitro. The human chronic myelogenous leukaemia cell line K562 was chosen to investigate the mechanism of cell death. The effect on the cell cycle and cell death was analysed by flow cytometry. Proteins related to apoptosis were studied by western blotting. Mechanical properties of cells were assessed using the Micropipette Aspiration Technique (MAT). The results indicated that the cannabidiol (CBD)-rich extract inhibited cell proliferation of K562 cell line in a dose-dependent manner and induced apoptosis via caspase 3 and 7 activation. A significant decrease in the mitochondrial membrane potential was detected, together with the release of cytochrome c into the cytosol. The main apoptotic markers were not involved in the mechanism of cell death. The extract was also able to modify the mechanical properties of cells. Thus, this hemp extract and its pure component CBD deserve further investigation for a possible application against myeloproliferative diseases, also in association with other anticancer drugs.

A Novel qNMR Application for the Quantification of Vegetable Oils Used as Adulterants in Essential Oils.

Essential oils (EOs) are more and more frequently adulterated due to their wide usage and large profit, for this reason accurate and precise authentication techniques are essential. This work aims at the application of qNMR as a versatile tool for the quantification of vegetable oils potentially usable as adulterants or diluents in EOs. This approach is based on the quantification of both 1H and 13C glycerol backbone signals, which are actually present in each vegetable oil containing triglycerides. For the validation, binary mixtures of rosemary EO and corn oil (0.8-50%) were prepared. To verify the general feasibility of this technique, other different mixtures including lavender, citronella, orange and peanut, almond, sunflower, and soy seed oils were analyzed. The results showed that the efficacy of this approach does not depend on the specific combination of EO and vegetable oil, ensuring its versatility. The method was able to determine the adulterant, with a mean accuracy of 91.81 and 89.77% for calculations made on 1H and 13C spectra, respectively. The high precision and accuracy here observed, make 1H-qNMR competitive with other well-established techniques. Considering the current importance of quality control of EOs to avoid fraudulent practices, this work can be considered pioneering and promising.

Separation and non-separation methods for the analysis of cannabinoids in Cannabis sativa L.

Cannabis sativa L. is a plant known all over the world, due to its history, bioactivity and also social impact. It is chemically complex with an astonishing ability in the biosynthesis of many secondary metabolites belonging to different chemical classes. Among them, cannabinoids are the most investigated ones, given their pharmacological relevance. In order to monitor the composition of the plant material and ensure the efficacy and safety of its derived products, extraction and analysis of cannabinoids play a crucial role. In this context, in addition to a conventional separation method based on HPLC with UV/DAD detection, a new strategy based on a non-separation procedure, such as 13C-qNMR, may offer several advantages, such as reduced solvent consumption and simultaneous acquisition of the quali/quantitative data related to many analytes. In the light of all the above, the aim of this work is to compare the efficiency of the above-mentioned analytical techniques for the study of the main cannabinoids in different samples of cannabis inflorescences, belonging to fibre-type, recreational and medical varieties. The 13C-qNMR method here proposed for the first time for the quantification of both psychoactive and non-psychoactive cannabinoids in different cannabis varieties provided reliable results in comparison to the more common and consolidated HPLC technique.

Determination of 1-Deoxynojirimycin (1-DNJ) in Leaves of Italian or Italy-Adapted Cultivars of Mulberry (Morus sp.pl.) by HPLC-MS.

Recently, 1-DNJ has been widely studied by scientists for its capacity to inhibit α-glucosidase and reduce postprandial blood glucose and fat accumulation. To the best of our knowledge, this is the first analytical determination of 1-DNJ in Morus sp.pl. leaves carried out on Italian crops, and it could be used as a reference to assess the quality of the plant material in comparison to Far Eastern Asia cultivations. The effects of two thermal treatments were compared to test the incidence of the drying process on the 1-DNJ extractability. In addition, two harvesting seasons in the same year (2017) and two subsequent harvesting years (2017-2018) were considered. The amount of 1-DNJ herein found was comparable to that reported in the scientific literature for Asian cultivations. The increase in 1-DNJ along the summer and the higher level of this compound in the apical leaves also complies with previous findings. However, a strong implication for the climatic conditions in the different years and a significant interaction between climate and genotypes suggest exploring very carefully the agronomic practices and selecting cultivars according to different environmental conditions with a view to standardize the 1-DNJ amount in leaves.

Novel Strategy for the Recognition of Adulterant Vegetable Oils in Essential Oils Commonly Used in Food Industries by Applying 13C NMR Spectroscopy.

Essential oils (EOs) are valuable products commonly employed in the food industry and intensively studied as biopreservatives for the extension of food shelf-life. Unfortunately, EOs might be counterfeit to increase industrial profits. Among the possible adulterants, vegetable oils (VOs) must be considered for their characteristics and low costs. We aimed to apply nuclear magnetic resonance (NMR) spectroscopy for the detection and identification of VOs in mixtures with EOs. This innovative strategy is based on comparing the peak area ratio matrices of characteristic VO 13C NMR fatty acid signals with those of adulterated EOs. The identification of the VOs was achieved by calculating the matrix similarity at different confidence levels. The strategy demonstrated the capacity to efficiently recognize the presence of adulteration and the type of VO adulterant in mixtures. Thus, the method was applied to 20 commercial EOs, and VOs were detected and then identified in four samples.

Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy coupled with chemometric analysis for detection and quantification of adulteration in lavender and citronella essential oils.

INTRODUCTION: The growing consumer interest in "naturals" led to an increased application of essential oils (EOs). The market outbreak induced the intensification of EO adulterations, which could affect their quality. OBJECTIVES: Nowadays, little is known about the illegal practice of adulteration of EOs with vegetable oils. Therefore, the application of mid-infrared spectroscopy coupled with chemometrics was proposed for the detection of EO counterfeits. MATERIALS AND METHODS: Two EOs, three seed oils, and their mixtures were selected to build the adulteration model. EO-adulterant mixtures for model calibration and validation were analyzed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. The spectral data were analyzed with principal component analysis (PCA) and partial least-squares (PLS) regression. RESULTS: PCA allowed the discrimination of the EO and adulterant percentages by explaining 97.47% of the total spectral variance with two principal components. A PLS regression model was generated with three factors explaining 97.73% and 99.69% of the total variance in X and Y, respectively. The root mean square error of calibration and the root mean square error of cross-validation were 0.918 and 1.049, respectively. The root mean square error of prediction value obtained from the external validation set was 1.588 and the coefficients of determination R2 CAL and R2 CV were 0.997 and 0.996, respectively. CONCLUSIONS: The results highlighted the robustness of the developed method in quantifying counterfeits in the range from 0 to 50% of adulterants, disregarding the type of EO and adulterant employed. The present work offers a research advance and makes an important impact in phytochemistry, revealing an easily applicable method for EO quality assessment.

HR-1 H NMR spectroscopy and multivariate statistical analysis to determine the composition of herbal mixtures for infusions.

INTRODUCTION: The ever-growing diffusion and consumption of herbal teas, due to their sensory attributes and well-known health benefits exposes them to the real risk of adulteration, especially in the case of commercial mixtures already minced for infusion. Therefore, novel and suitable tools for the control of these valuable products are increasingly required. OBJECTIVES: This work provides new insights for the authenticity study of infusions. The main objective was verifying the potential of proton nuclear magnetic resonance (1 H-NMR) combined with partial least square (PLS) regression to build highly predictive models, useful for the determination of the real amounts of herbs in mixtures, by the simple analysis of the related infusion. MATERIALS AND METHODS: Peppermint, fennel, lemon balm, and passiflora were chosen to set-up an experimental plan according to a central composite design (CCD). One-dimensional nuclear Overhauser effect spectroscopy (1D-NOESY) spectra were properly pretreated and then analysed by chemometrics to extract significant information from the raw data. RESULTS: Venetian-blind cross-validation and different chemometric indicators (RMSEC, RMSECV, RMSEP, R2 CAL , R2 CV, R2 PRED ) were used to establish the best model, which include four factors explaining 88.70 and 83.77% of the total variance in X and Y, respectively. CONCLUSIONS: These promising results have laid the basis for further development of the method, to extend its applicability and make it more scalable. This tool could replace expensive separative techniques and protect the rights of consumers with particular attention to safety issues and quality assurance.

New Insights into Bioactive Compounds from the Medicinal Plant Spathodea campanulata P. Beauv. and Their Activity against Helicobacter pylori.

The medicinal plant Spathodea campanulata P. Beauv. (Bignoniaceae) has been traditionally applied for the prevention and treatment of diseases of the kidney and urinary system, the skin, the gastrointestinal tract, and inflammation in general. The present work shows for the first time how chemical components from this plant inhibit Helicobacter pylori growth by urease inhibition and modulation of virulence factors. The crude extract and the main fractions of S. campanulata bark were tested on H. pylori isolated strains and the active ones were further fractionated. Fractions and sub-fractions of the plant crude extract were characterized by ultra-high-performance liquid chromatographic tandem high resolution-mass spectrometry detection (UHPLC-HRMS). Several phenolics and triterpenoids were identified. Among the sub-fractions obtained, SB2 showed the capacity to inhibit H. pylori urease in a heterologous bacterial model. One additional sub-fraction (SE3) was able to simultaneously modulate the expression of two adhesins (HopZ and BabA) and one cytotoxin (CagA). The flavonol kaempferol was identified as the most interesting compound that deserves further investigation as a new hit for its capacity to modulate H. pylori virulence factors.

Propolis Affects Pseudomonas aeruginosa Growth, Biofilm Formation, eDNA Release and Phenazine Production: Potential Involvement of Polyphenols.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for a wide range of clinical conditions, from mild infections to life-threatening nosocomial biofilm-associated diseases, which are particularly severe in susceptible individuals. The aim of this in vitro study was to assess the effects of an Albanian propolis on several virulence-related factors of P. aeruginosa, such as growth ability, biofilm formation, extracellular DNA (eDNA) release and phenazine production. To this end, propolis was processed using three different solvents and the extracted polyphenolic compounds were identified by means of high performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) analysis. As assessed by a bioluminescence-based assay, among the three propolis extracts, the ethanol (EtOH) extract was the most effective in inhibiting both microbial growth and biofilm formation, followed by propylene glycol (PG) and polyethylene glycol 400 (PEG 400) propolis extracts. Furthermore, Pseudomonas exposure to propolis EtOH extract caused a decrease in eDNA release and phenazine production. Finally, caffeic acid phenethyl ester (CAPE) and quercetin decreased upon propolis EtOH extract exposure to bacteria. Overall, our data add new insights on the anti-microbial properties of a natural compound, such as propolis against P. aeruginosa. The potential implications of these findings will be discussed.

The Effect of Polyphenols on Pomegranate Fruit Susceptibility to Pilidiella granati Provides Insights into Disease Tolerance Mechanisms.

Pilidiella granati, also known as Coniella granati, is the etiological agent of pomegranate fruit dry rot. This fungal pathogen is also well-known as responsible for both plant collar rot and leaf spot. Because of its aggressiveness and the worldwide diffusion of pomegranate crops, the selection of cultivars less susceptible to this pathogen might represent an interesting preventive control measure. In the present investigation, the role of polyphenols in the susceptibility to P. granati of the two royalties-free pomegranate cultivars Wonderful and Mollar de Elche was compared. Pomegranate fruit were artificially inoculated and lesion diameters were monitored. Furthermore, pathogen DNA was quantified at 12-72 h post-inoculation within fruit rind by a real time PCR assay setup herein, and host total RNA was used in expression assays of genes involved in host-pathogen interaction. Similarly, protein extracts were employed to assess the specific activity of enzymes implicated in defense mechanisms. Pomegranate phenolic compounds were evaluated by HPLC-ESI-MS and MS2. All these data highlighted 'Wonderful' as less susceptible to P. granati than 'Mollar de Elche'. In the first cultivar, the fungal growth seemed controlled by the activation of the phenylpropanoid pathway, the production of ROS, and the alteration of fungal cell wall. Furthermore, antifungal compounds seemed to accumulate in 'Wonderful' fruit following inoculation. These data suggest that pomegranate polyphenols have a protective effect against P. granati infection and their content might represent a relevant parameter in the selection of the most suitable cultivars to reduce the economic losses caused by this pathogen.

Use of 1H NMR to Detect the Percentage of Pure Fruit Juices in Blends.

The consumption of high-nutritional-value juice blends is increasing worldwide and, considering the large market volume, fraud and adulteration represent an ongoing problem. Therefore, advanced anti-fraud tools are needed. This study aims to verify the potential of 1H NMR combined with partial least squares regression (PLS) to determine the relative percentage of pure fruit juices in commercial blends. Apple, orange, pineapple, and pomegranate juices were selected to set up an experimental plan and then mixed in different proportions according to a central composite design (CCD). NOESY (nuclear Overhauser enhancement spectroscopy) experiments that suppress the water signal were used. Considering the high complexity of the spectra, it was necessary to pretreat and then analyze by chemometric tools the large amount of information contained in the raw data. PLS analysis was performed using venetian-blind internal cross-validation, and the model was established using different chemometric indicators (RMSEC, RMSECV, RMSEP, R2CAL, R2CV, R2PRED). PLS produced the best model, using five factors explaining 94.51 and 88.62% of the total variance in X and Y, respectively. The present work shows the feasibility and advantages of using 1H NMR spectral data in combination with multivariate analysis to develop and optimize calibration models potentially useful for detecting fruit juice adulteration.

Use of 13C-qNMR Spectroscopy for the Analysis of Non-Psychoactive Cannabinoids in Fibre-Type Cannabis sativa L. (Hemp).

Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The discovery of the presence of many biologically-active metabolites (cannabinoids) in fibre-type Cannabis (hemp) has recently given rise to the valorisation of this variety. In this context, the present study was aimed at the multi-component analysis and determination of the main non-psychoactive cannabinoids (cannabidiol, cannabidiolic acid, cannabigerol and cannabigerolic acid) in female inflorescences of different hemp varieties by means of 13C quantitative nuclear magnetic resonance spectroscopy (qNMR). The method proposed here for the first time for the determination of cannabinoids provided reliable results in a competitive time with respect to the more consolidated HPLC technique. In fact, it gave sufficiently precise and sensitive results, with LOQ values lower than 750 µg/mL, which is easily achievable with concentrated extracts, without affecting the quality of 13C-qNMR spectra. In conclusion, this method can be considered as a promising and appropriate tool for the comprehensive chemical analysis of bioactive cannabinoids in hemp and other derived products in order to ensure their quality, efficacy and safety.

Identification and determination of bioactive phenylpropanoid glycosides of Aloysia polystachya (Griseb. et Moldenke) by HPLC-MS.

Aloysia polystachya (Griseb. et Moldenke) has not been deeply investigated in past years and currently data about its chemical composition are limited. Phenolic compounds characterization can be very difficult in vegetable matrices, owing to bonds to sugar moieties or conjugation, giving rise to complex structures. In this work, methanolic extracts of Aloysia polystachya leaves were analyzed by HPLC-ESI-MS, the favourite technique for the separation and quantification of their polyphenols. To assess the complete characterization and quantification of the phenylpropanoid fraction, three different MS techniques have been coupled to HPLC: ion trap mass spectrometry (Ion Trap LC/MS), quadrupole-time of flight high resolution mass spectrometry (Q-TOF HRMS) and triple-quadrupole (TQ LC/MS) for the quantification. Eleven phenylpropanoid glycosides were identified and quantified and, among them, the compounds forsythoside A, plantainoside C, purpureaside D, martynoside and its two isomers were detected for the first time to the best of our knowledge. The results presented here could be helpful to assess the quality of this plant and could further contribute to the chemotaxonomy of the genus.

New Methods for the Comprehensive Analysis of Bioactive Compounds in Cannabis sativa L. (hemp).

Cannabis sativa L. is a dioecious plant belonging to the Cannabaceae family. The main phytochemicals that are found in this plant are represented by cannabinoids, flavones, and terpenes. Some biological activities of cannabinoids are known to be enhanced by the presence of terpenes and flavonoids in the extracts, due to a synergistic action. In the light of all the above, the present study was aimed at the multi-component analysis of the bioactive compounds present in fibre-type C. sativa (hemp) inflorescences of different varieties by means of innovative HPLC and GC methods. In particular, the profiling of non-psychoactive cannabinoids was carried out by means of HPLC-UV/DAD, ESI-MS, and MS². The content of prenylated flavones in hemp extracts, including cannflavins A and B, was also evaluated by HPLC. The study on Cannabis volatile compounds was performed by developing a new method based on headspace solid-phase microextraction (HS-SPME) coupled with GC-MS and GC-FID. Cannabidiolic acid (CBDA) and cannabidiol (CBD) were found to be the most abundant cannabinoids in the hemp samples analysed, while ß-myrcene and ß-caryophyllene were the major terpenes. As regards flavonoids, cannflavin A was observed to be the main compound in almost all the samples. The methods developed in this work are suitable for the comprehensive chemical analysis of both hemp plant material and related pharmaceutical or nutraceutical products in order to ensure their quality, efficacy, and safety.

Nuclear magnetic resonance and high-performance liquid chromatography techniques for the characterization of bioactive compounds from Humulus lupulus L. (hop).

Humulus lupulus L. (hop) represents one of the most cultivated crops, it being a key ingredient in the brewing process. Many health-related properties have been described for hop extracts, making this plant gain more interest in the field of pharmaceutical and nutraceutical research. Among the analytical tools available for the phytochemical characterization of plant extracts, quantitative nuclear magnetic resonance (qNMR) represents a new and powerful technique. In this ambit, the present study was aimed at the development of a new, simple, and efficient qNMR method for the metabolite fingerprinting of bioactive compounds in hop cones, taking advantage of the novel ERETIC 2 tool. To the best of our knowledge, this is the first attempt to apply this method to complex matrices of natural origin, such as hop extracts. The qNMR method set up in this study was applied to the quantification of both prenylflavonoids and bitter acids in eight hop cultivars. The performance of this analytical method was compared with that of HPLC-UV/DAD, which represents the most frequently used technique in the field of natural product analysis. The quantitative data obtained for hop samples by means of the two aforementioned techniques highlighted that the amount of bioactive compounds was slightly higher when qNMR was applied, although the order of magnitude of the values was the same. The accuracy of qNMR was comparable to that of the chromatographic method, thus proving to be a reliable tool for the analysis of these secondary metabolites in hop extracts. Graphical abstract Graphical abstract related to the extraction and analytical methods applied in this work for the analysis of bioactive compounds in Humulus lupulus L. (hop) cones.

Novel 2D-NMR Approach for the Classification of Balsamic Vinegars of Modena.

The aim of this work is to evaluate the possibility of using 2D-NMR for the construction of classification models for balsamic vinegars of Modena. The goal was to obtain an indirect indicator of authenticity and a quality control tool. The spectral data were analyzed by chemometric methods, aiming to discriminate the samples in relation to their origin. Application of general discriminant analysis (GDA) revealed a good discrimination; the two obtained models explained 83.9% and 97.3% of the total variance with a predictive capacity of 98.6% and 98.4%, respectively. The signals of 5-HMF, ß-glucose, 2,3-butanediol, 6-acetyl glucose, and different aliphatic signals of sugars were the most significant variables. These results are very promising for giving an important contribution in quality control and characterization of such very valuable foods.