Virginia Brighenti

Development of a new high-performance liquid chromatography method with diode array and electrospray ionization-mass spectrometry detection for the metabolite fingerprinting of bioactive compounds in Humulus lupulus L.

The study was aimed at developing a new analytical method for the metabolite fingerprinting of bioactive compounds in Humulus lupulus L. (hop), together with a simple extraction procedure. Different extraction techniques, including maceration, heat reflux extraction (HRE), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE), were compared in order to obtain a high yield of the target analytes. Dynamic maceration for 30min with MeOH-HCOOH (99:1, v/v) as the extraction solvent provided the best result in terms of recovery of secondary metabolites. The analysis of hop constituents, including prenylflavonoids and prenylphloroglucinols (bitter acids), was carried out by means of HPLC-UV/DAD, HPLC-ESI-MS and MS(2), using an ion trap mass analyzer. An Ascentis Express C18 column (150mm×3.0mm I.D., 2.7μm) was used for the HPLC analysis, with a mobile phase composed of 0.25% formic acid in both water and acetonitrile, under gradient elution. The method validation was performed to show compliance with ICH guidelines. The validated technique was successfully applied to the phytochemical analysis of ten commercial cultivars and twenty-three wild Italian hop genotypes, thus demonstrating to be a reliable and useful tool for the comprehensive multi-component analysis of hop secondary metabolites.

HPLC-UV/DAD and ESI-MSn analysis of flavonoids and antioxidant activity of an Algerian medicinal plant: Paronychia argentea Lam.

Paronychia argentea Lam., belonging to the Caryophyllaceae family, is a perennial plant widely distributed in Algeria. Even though this plant is used in the Algerian popular medicine, its phytochemical characterization is incomplete. In this study, the flavonoid profile and the in vitro antioxidant activity of the ethanolic extract, decoction and infusion of P. argentea aerial parts are reported. Flavonoids were analyzed by means of high-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry. Eleven compounds were identified and six of them, including isorhamnetin-3-O-dihexoside, quercetin-3-O-glucoside, quercetinmethylether-O-hexoside, quercetin, jaceosidin and isorhamnetin, were described in this plant for the first time. The ethanol extract showed the highest flavonoid content, followed by the decoction and the infusion (25.4 ± 0.8 mg/g of DM, 8.4 ± 0.5 mg/g of DM, 0.2 mg/g of DM, respectively), while the best antioxidant activity was shown by the decoction (RC0.5 = 178 μg/mL for reducing power, 72.4% of inhibition of lipid peroxidation, IC50 = 27.38μ g/mL for DPPH radical scavenging activity and 59.7% of inhibition of NO radical). These results showed that P. argentea decoction could be considered as a valuable source of flavonoids and antioxidants that might contribute to the valorization of the phytotherapeutic potential of this plant.

Metabolite profiling of flavonols and in vitro antioxidant activity of young shoots of wild Humulus lupulus L. (hop)

Graphical abstract Figure. No caption available. HighlightsA new HPLC method for the analysis of flavonols in young hop shoots was developed.Quercetin and kaempferol glycosides were the main phenolics in hop shoots.The content of flavonols is related to the in vitro antioxidant activity.Hop shoots represent a new source of bioactive antioxidant compounds. Abstract Humulus lupulus L., commonly named hop, is well‐known for its sedative and estrogenic activity. While hop cones are widely characterized, only few works have been carried out on the young shoots of this plant. In the light of this, the aim of this study was to identify for the first time the flavonoids present in young hop shoots and to compare the composition of samples harvested from different locations in Northern Italy with their antioxidant activity. The samples were extracted by means of dynamic maceration with methanol. The HPLC‐UV/DAD, HPLC‐ESI‐MS and MS2 analysis were carried out by using an Ascentis C18 column (250 × 4.6 mm I.D., 5 &mgr;m), with a mobile phase composed of 0.1 M formic acid in both water and acetonitrile, under gradient elution. Quercetin and kaempferol glycosides were the main compounds identified and quantified in hop shoot extracts. Total flavonols ranged from 2698 ± 185 to 517 ± 48 &mgr;g/g (fresh weight). The antioxidant activity was determined by means of the radical scavenging activity assay against diphenylpicrylhydrazyl (DPPH•) and by using a photochemiluscence assay with a Photochem® apparatus. The results showed that hop shoots represent a new source of flavonols; therefore, they can be useful for a possible incorporation in the diet as a functional food or applied in the nutraceutical ambit.

Development of a new extraction technique and HPLC method for the analysis of non-psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp)

&NA; The present work was aimed at the development and validation of a new, efficient and reliable technique for the analysis of the main non‐psychoactive cannabinoids in fibre‐type Cannabis sativa L. (hemp) inflorescences belonging to different varieties. This study was designed to identify samples with a high content of bioactive compounds, with a view to underscoring the importance of quality control in derived products as well. Different extraction methods, including dynamic maceration (DM), ultrasound‐assisted extraction (UAE), microwave‐assisted extraction (MAE) and supercritical‐fluid extraction (SFE) were applied and compared in order to obtain a high yield of the target analytes from hemp. Dynamic maceration for 45 min with ethanol (EtOH) at room temperature proved to be the most suitable technique for the extraction of cannabinoids in hemp samples. The analysis of the target analytes in hemp extracts was carried out by developing a new reversed‐phase high‐performance liquid chromatography (HPLC) method coupled with diode array (UV/DAD) and electrospray ionization‐mass spectrometry (ESI–MS) detection, by using an ion trap mass analyser. An Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 &mgr;m) was selected for the HPLC analysis, with a mobile phase composed of 0.1% formic acid in both water and acetonitrile, under gradient elution. The application of the fused‐core technology allowed us to obtain a significant improvement of the HPLC performance compared with that of conventional particulate stationary phases, with a shorter analysis time and a remarkable reduction of solvent usage. The analytical method optimized in this study was fully validated to show compliance with international requirements. Furthermore, it was applied to the characterization of nine hemp samples and six hemp‐based pharmaceutical products. As such, it was demonstrated to be a very useful tool for the analysis of cannabinoids in both the plant material and its derivatives for pharmaceutical and nutraceutical applications. Graphical abstract Figure. No caption available. HighlightsA new HPLC method for non‐psychoactive cannabinoids analysis in hemp is proposed.The fused‐core stationary phase remarkably improved the HPLC performance.The extraction procedure was optimized to obtain a high yield.The method was fully validated in agreement with ICH guidelines.Hemp inflorescences and derivatives were analysed for the content in cannabinoids.

Metabolite fingerprinting of Punica granatum L. (pomegranate) polyphenols by means of high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection

The present study was aimed at the development of a new analytical method for the comprehensive multi-component analysis of polyphenols in Punica granatum L. (pomegranate) juice and peel. While pomegranate juice was directly analysed after simple centrifugation, different extraction techniques, including maceration, heat reflux extraction, ultrasound-assisted extraction and microwave-assisted extraction, were compared in order to obtain a high yield of the target analytes from pomegranate peel. Dynamic maceration with a mixture of water and ethanol 80:20 (v/v) with 0.1% of hydrochloric acid as the extraction solvent provided the best result in terms of recovery of pomegranate secondary metabolites. The quali- and quantitative analysis of pomegranate polyphenols was performed by high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection. The application of fused-core column technology allowed us to obtain an improvement of the chromatographic performance in comparison with that of conventional particulate stationary phases, thus enabling a good separation of all constituents in a shorter time and with low solvent usage. The analytical method was completely validated to show compliance with the International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use guidelines and successfully applied to the characterisation of commercial and experimental pomegranate samples, thus demonstrating its efficiency as a tool for the fingerprinting of this plant material. The quantitative data collected were submitted to principal component analysis, in order to highlight the possible presence of pomegranate samples with high content of secondary metabolites. From the statistical analysis, four experimental samples showed a notable content of bioactive compounds in the peels, while commercial ones still represent the best source of healthy juice.

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 β-myrcene and β-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.

Analytical methods for the study of bioactive compounds from medicinally used Echinacea species

Echinacea purpurea (L.) Moench, Echinacea angustifolia DC. var. angustifolia and Echinacea pallida (Nutt.) Nutt. are frequently used as medicinal plants and their preparations are among the most widely used herbal medicines. The extracts from these species have shown a highly complex chemical composition, including polar compounds (caffeic acid derivatives, CADs), non-polar compounds (alkylamides and acetylenic secondary metabolites; essential oil) and high molecular weight constituents (polysaccharides and glycoproteins). All these chemical classes of compounds have demonstrated to possess interesting biological activities. In the light of all the above, this paper is focused on the analytical techniques, including sample preparation tools and chromatographic procedures, for the chemical analysis of bioactive compounds in medicinally used Echinacea species. Since sample preparation is considered to be a crucial step in the development of analytical methods for the determination of constituents present in herbal preparations, the strength and weakness of different extraction techniques are discussed. As regards the analysis of compounds present in Echinacea plant material and derivatives, the application of different techniques, mainly HPLC, HPLC-ESI-MS, HPLC-ESI-MS/MS, HPCE, HPTLC and GC, is discussed in detail. The strength, weakness and applicability of the different separation tools are stated.

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

AbstractHumulus 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 abstractGraphical abstract related to the extraction and analytical methods applied in this work for the analysis of bioactive compounds in Humulus lupulus L. (hop) cones