Suspect screening analysis to improve untargeted and targeted UHPLC-qToF approaches: the biodegradability of a proton pump inhibitor medicine and a natural medical device.

Suspect screening and untargeted analysis using UHPLC-qToF are two advanced analytical approaches now used to achieve an extensive chemical profile of samples, which are then typically confirmed through targeted analysis. These techniques can detect a large number of chemical features simultaneously and are currently being introduced into the study of contaminants of emerging concern (CECs) and into the study of the extent of human chemical exposure (the exposome). Here is described the use of these techniques to characterize chemical mixtures derived from the OECD 301F ready biodegradability test (RBT) of a chemical and natural formulation currently used to treat reflux disease and functional dyspepsia. Untargeted analysis clearly evidenced a different behavior between formulations containing only natural products with respect to that containing synthetic and non-naturally occurring substances. Suspect screening analysis improved the untargeted analysis of the omeprazole-based medicine, leading to the tentative identification of a number of omeprazole-derived transformation products, thereby enabling their preliminary quali-quantitative evaluation. Targeted analysis was then performed to confirm the preliminary data gained from the suspect screening approach. The validation of the analytical method for the quantitative determination of omeprazole and its major metabolite, omeprazole sulphide, has provided robust data to evaluate the behavior of omeprazole during the OECD 301F test. Using advanced analytical approaches, the RBT performed on the two products under investigation confirmed that omeprazole is not readily biodegradable, while the medical device made of natural substances has proven to be readily biodegradable.

Natural complex substances: From molecules to the molecular complexes. Analytical and technological advances for their definition and differentiation from the corresponding synthetic substances.

Natural complex substances (NCSs) are a heterogeneous family of substances that are notably used as ingredients in several products classified as food supplements, medical devices, cosmetics and traditional medicines, according to the correspondent regulatory framework. The compositions of NCSs vary widely and hundreds to thousands of compounds can be present at the same time. A key concept is that NCSs are much more than the simple sum of the compounds that constitute them, in fact some emerging phenomena are the result of the supramolecular interaction of the constituents of the system. Therefore, close attention should be paid to produce and characterize these systems. Today many natural compounds are produced by chemical synthesis and are intentionally added to NCSs, or to formulated natural products, to enhance their properties, lowering their production costs. Market analysis shows a tendency of people to use products made with NCSs and, currently, products made with ingredients of natural origin only are not conveniently distinguishable from those containing compounds of synthetic origin. Furthermore, the uncertainty of the current European regulatory framework does not allow consumers to correctly differentiate and identify products containing only ingredients of natural origin. The high demand for specific and effective NCSs and their high-cost offer on the market, create the conditions to economically motivated sophistications, characterized by the addition of a cheap material to a more expensive one, just to increase profit. This type of practice can concern both the addition of less valuable natural materials and the addition of pure artificial compounds with the same structure as those naturally present. In this scenario, it becomes essential for producers of natural products to have advanced analytical techniques to evaluate the effective naturalness of NCSs. In fact, synthetically obtained compounds are not identical to their naturally occurring counterparts, due to the isotopic composition or chirality, as well as the presence of different trace metabolites (since pure substances in nature do not exist). For this reason, in this review, the main analytical tests that can be performed to differentiate natural compounds from their synthetic counterparts will be highlighted and the main analytical technologies will be described. At the same time, the main fingerprint techniques useful for characterizing the complexity of the NCSs, also allowing their identification and quali-quantitative evaluation, will be described. Furthermore, NCSs can be produced through different manufacturing processes, not all of which are on the same level of quality. In this review the most suitable technologies for green processes that operate according to physical extraction principles will be presented, as according to the authors they are the ones that come closest to creating more life-cycle compatible NCSs and that are well suited to the European green deal, a strategy with the aim of transforming the EU into a sustainable and resource-efficient society by 2050.

Ready Biodegradability study and insights with ultra-high-performance liquid chromatograph coupled to a quadrupole time of flight of a Metformin-based drug and of Metarecod, a natural substance-based medical device.

Drugs are indispensable products with incontrovertible benefits to human health and lifestyle. However, due to their overuse and improper disposal, unwanted residues of active pharmaceutical ingredients (APIs) have been found in different compartments of the environment and now are considered as contaminants of emerging concern (CECs). Therefore, they are very likely to have a boomerang effect on human health, because they can enter into the food cycle. In the current legislation framework, one of the tests first used to evaluate biodegradation of APIs as well as chemical compounds is the ready biodegradability test (RBT). This test can be performed according to a series of protocols prepared by Organization for Economic Co-operation and Development (OECD) and usually is carried out on pure compounds. RBTs, largely used due to their relatively low cost, perceived standardization, and straightforward implementation and interpretation, are known to have a number of well-documented limitations. In this work, following a recently reported approach, we propose to improve the evaluation of the RBT results applying advanced analytical techniques based on mass spectrometry, not only to the APIs but also to complex formulated products, as the biodegradability can potentially be affected by the formulation. We evaluated the ready biodegradability of two therapeutic products, Product A-a drug based on Metformin-and Product B-Metarecod a natural substance-based medical device-through the acquisition of the fingerprint by ultra-high-performance chromatograph coupled to a quadrupole time of flight (UHPLC-qToF) of samples coming from the RBT OECD 301F. Untargeted and targeted evaluation confirmed the different behavior of the two products during the respirometry-manometric test, which showed a difficulty of the Metformin-based drug to come back in the life cycle, whereas Metarecod resulted ready biodegradable. The positive results of this research will hopefully be useful in the future for a better evaluation of the risk/benefit ratio of APIs extended to the environment.

Mass spectrometry-based metabolomic analysis as a tool for quality control of natural complex products.

Metabolomics is an area of intriguing and growing interest. Since the late 1990s, when the first Omic applications appeared to study metabolite's pool ("metabolome"), to understand new aspects of the global regulation of cellular metabolism in biology, there have been many evolutions. Currently, there are many applications in different fields such as clinical, medical, agricultural, and food. In our opinion, it is clear that developments in metabolomics analysis have also been driven by advances in mass spectrometry (MS) technology. As natural complex products (NCPs) are increasingly used around the world as medicines, food supplements, and substance-based medical devices, their analysis using metabolomic approaches will help to bring more and more rigor to scientific studies and industrial production monitoring. This review is intended to emphasize the importance of metabolomics as a powerful tool for studying NCPs, by which significant advantages can be obtained in terms of elucidation of their composition, biological effects, and quality control. The different approaches of metabolomic analysis, the main and basic techniques of multivariate statistical analysis are also briefly illustrated, to allow an overview of the workflow associated with the metabolomic studies of NCPs. Therefore, various articles and reviews are illustrated and commented as examples of the application of MS-based metabolomics to NCPs.

Metabolomic fingerprint of Hamamelis virginiana L. gallotannins by suspect screening analysis with UHPLC-qToF and their semiquantitative evaluation.

The evolution of the regulatory framework for medical devices in the EU (Reg 2017/745) has opened the study of complex systems emerging properties. This makes necessary to identify new analytical approaches able of characterizing complex natural substrates as completely as possible. Therefore, omics approaches and advanced analytical methods for the determination of metabolite classes appear to be at the forefront to meet this need. In this perspective, a new approach based on the suspect screening was developed to detect gallotannins. Gallotannins are a class of phenols with a polymeric nature; thus, there are no pure analytical standards available for all possible structures and their quali-quantitative determination in complex natural substrates can be a challenge. A new UHPLC-qToF method was developed and used to create an "in-house tannin database" with a dual purpose: (1) as a classic list of suspects and (2) to identify core fragments common to gallotannins to have another list of putative suspects based on the common fragment. The method was validated. The application of the method to a "system of molecules" extracted from the leaves of Hamamelis virginiana L. (Witch-hazel) allowed to the characterization of a total of 29 phenols by a suspect screening approach. Therefore, 15 gallotannins were putatively annotated while another 3 were confidently identified. All the gallotannins were semiquantified according to external regression curves of gallic acid and hamamelitannin based on core fragments at m/z 125.0244 and m/z 169.0142, the building blocks of the polymers. This new method provides a practical fit-to-purpose approach for the quali-quantitative screening evaluation of gallotannins, useful for creating multivariate control charts applicable in process development of complex natural systems or in quality control. The approach is innovative, and after specific checks, it can in principle be suitable for metabolomic fingerprint analysis of gallotannins among witch-hazel extract (WHE) samples.

Efficacy of a vegetal mixture composed of Zingiber officinale, Echinacea purpurea, and Centella asiatica in a mouse model of neuroinflammation: In vivo and ex vivo analysis.

Experimental evidence suggests that neuroinflammation is a key pathological event of many diseases affecting the nervous system. It has been well recognized that these devastating illnesses (e.g., Alzheimer's, Parkinson's, depression, and chronic pain) are multifactorial, involving many pathogenic mechanisms, reason why pharmacological treatments are unsatisfactory. The purpose of this study was to evaluate the efficacy of a vegetal mixture capable of offering a multiple approach required to manage the multifactoriality of neuroinflammation. A mixture composed of Zingiber officinale (150 mg kg-1), Echinacea purpurea (20 mg kg-1), and Centella asiatica (200 mg kg-1) was tested in a mouse model of systemic neuroinflammation induced by lipopolysaccharide (LPS, 1 mg kg-1). Repeated treatment with the vegetal mixture was able to completely counteract thermal and mechanical allodynia as reported by the Cold plate and von Frey tests, respectively, and to reduce the motor impairments as demonstrated by the Rota rod test. Moreover, the mixture was capable of neutralizing the memory loss in the Passive avoidance test and reducing depressive-like behavior in the Porsolt test, while no efficacy was shown in decreasing anhedonia as demonstrated by the Sucrose preference test. Finally, LPS stimulation caused a significant increase in the activation of glial cells, of the central complement proteins and of inflammatory cytokines in selected regions of the central nervous system (CNS), which were rebalanced in animals treated with the vegetal mixture. In conclusion, the vegetal mixture tested thwarted the plethora of symptoms evoked by LPS, thus being a potential candidate for future investigations in the context of neuroinflammation.

Evidence of noncovalent complexes in some natural extracts: Ceylon tea and mate extracts.

Considering the high complexity of natural extracts, because of the presence of organic molecules of different chemical nature, the possibility of formation of noncovalent complexes should be taken into account. In a previous investigation, the formation of bimolecular complexes between caffeine and catechins in green tea extracts (GTE) has been experimentally proven by means of mass spectrometric and 1 H nuclear magnetic resonance experiments. The same approaches have been employed in the present study to evaluate the presence of bimolecular complexes in Ceylon tea and mate extracts. The obtained results show that in the case of Ceylon tea extracts, protonated theaflavin is detectable, together with theaflavin/caffein complexes, while caffeine/catechin complexes, already detected in green tea, are still present but at lower concentration. This aspect is evidenced by the comparison of precursor ion scans performed on protonated caffeine for the two extracts. The spectra obtained in these conditions for GTE and Ceylon tea show that the complexes of caffeine with epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG), highy abundant in the case of GTE (signal-to-chemical noise ratio in the range 50-100), are negligible (signal-to-chemical noise ratio in the range 2-3) in the case of Ceylon tea. Mate extracts show the formation of bimolecular complexes involving caffeine but not catechins, and chlorogenic acid becomes responsible for other complex formation. Under positive ion and negative ion conditions, accurate mass measurements allow the identification of malealdehyde, chlorogenic acid, caffeine, two isomers of dicaffeoylquinic acid, rutin, and kaempferol-3-O-rutinoside. These data indicate that the formation of complexes in natural extracts is a common behavior, and their presence must be considered in the description of natural extracts and, consequently, in their biological activity.

Experimental Evidence of the Presence of Bimolecular Caffeine/Catechin Complexes in Green Tea Extracts.

A hypothesis on the peculiar pharmacological behavior of biologically active natural compounds is based on the occurrence of molecular interactions originating from the high complexity of the natural matrix, following the rules of supramolecular chemistry. In this context, some investigations were performed to establish unequivocally the presence of caffeine/catechin complexes in green tea extracts (GTEs). 1H NMR spectroscopy was utilized to compare profiles from GTEs with caffeine/catechin mixtures in different molar ratios, showing that peaks related to caffeine in GTEs are generally upfield shifted compared to those of free caffeine. On the other hand, ESIMS experiments performed on GTE, by means of precursor ion scan and neutral loss scan experiments, proved unequivocally the presence of caffeine/catechin complexes. Further investigations were performed by an LC-MS method operating at high-resolution conditions. The reconstructed ion chromatograms of the exact mass ions corresponding to caffeine/catechin species have been obtained, showing the presence of complexes of caffeine with gallate-type catechins. Furthermore, this last approach evidenced the presence of the same complex with different structures, consequently exhibiting different retention times. Both MSE and product ion MS/MS methods confirm the nature of caffeine/catechin complexes of the detected ions, showing the formation of protonated caffeine.

Efficacy of AdipoDren® in Reducing Interleukin-1-Induced Lymphatic Endothelial Hyperpermeability.

Lymphatic leakage can be seen as a detrimental phenomenon associated with fluid retention and deposition as well as gain of weight. Moreover, lymphatic dysfunction is associated with an inflammatory environment and can be a substrate for other health conditions. A number of treatments can ameliorate lymphatic vasculature: natural substances have been used as treatment options particularly suitable for their consolidated effectiveness and safety profile. Here we report the protective effect of AdipoDren®, an association of a series of plant-derived natural complexes, on lymphatic endothelium permeability promoted by interleukin-1 beta (IL-1ß) and the associated molecular mechanisms. AdipoDren® demonstrated a protective effect on dermal lymphatic endothelial cell permeability increased by IL-1ß. Reduced permeability was due to the maintenance of tight junctions and cell-cell localisation of occludin and zonula occludens-1 (ZO-1). Moreover, AdipoDren® reduced the expression of the inflammatory key element cyclooxygenase-2 (COX-2), while not altering the levels of endothelial and inducible nitric oxide synthases (eNOS and iNOS). The upregulation of antioxidant enzymatic systems (catalase and superoxide dismutase-1, SOD-1) and the downregulation of pro-oxidant markers (p22 phox subunit of NADPH oxidase) were also evident. In conclusion, AdipoDren® would be useful to ameliorate conditions of altered lymphatic vasculature and to support the physiological functionality of the lymphatic endothelium.

An integrated approach to the evaluation of a metabolomic fingerprint for a phytocomplex. Focus on artichoke [Cynara cardunculus subsp. scolymus] leaf.

The availability of reliable herbal formulations is essential in order to assure the maximal activity and to limit unwanted side-effects. The correct concentration of declared components of herbal products is a matter of health legislation and regulation, but is still a topic under debate in the field of quality control assessment. In the present work specific constituents of artichoke leaf extracts, considered as a test herbal product, were measured by standard spectrophotometric and HPLC methods (for quantitative determination of some components only), and results were correlated with the ESI-MS (showing the full metabolomic fingerprint). Phytocomplex stability over time was also investigated in batches submitted to different storage conditions. The results indicated excellent agreement between the two approaches in the measurement of total caffeoylquinic acids and chlorogenic acid contents, but the metabolomic ESI-MS method approach provides a more complete evaluation and monitoring of the composition of a herbal product, without focusing only on a single/few compound measurements. Therefore, the ESI-MS method can be proposed for the evaluation of the quality of complex matrices, such as those in a phytocomplex. Another aspect lies in the possibility to obtain a broad-spectrum stability control of herbal formulations, requiring minimal sample pre-processing procedures.

St. John's Wort seed and feverfew flower extracts relieve painful diabetic neuropathy in a rat model of diabetes.

Painful diabetic peripheral neuropathy (DPN) is a common complication of diabetes and the few approved therapies for the management of pain have limited efficacy and side effects. With the aim to explore and develop new pharmacological treatments, we investigated the antihyperalgesic properties of St. John's Wort (SJW) and feverfew in streptozotocin (STZ)-diabetic rats. Acute administration of a SJW seed extract reversed mechanical hyperalgesia with a prolonged effect. A SJW extract obtained from the aerial portion of the plant and a feverfew flower extract partially relieved neuropathic pain whereas a feverfew leaf extract was ineffective. The antihyperalgesic efficacy of these herbal drugs was comparable to that of clinically used antihyperalgesic drugs (carbamazepine, lamotrigine, l-acetyl-levocarnitine). Further examinations of SJW and feverfew composition revealed that hyperforin and hypericin might be responsible for the antihyperalgesic properties of SJW whereas the efficacy of feverfew seems to be related to the presence of parthenolide. Rats undergoing treatment with SJW and feverfew did not show any behavioral side effect or sign of altered locomotor activity. Our results suggest that SJW and feverfew extracts may become new therapeutic perspectives for painful DPN.