Scientific Evidence

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 com-plex 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 avail-able 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 gallo-tannins 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 gallotan-nins among witch-hazel extract (WHE) samples.

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.

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 1H 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.

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.

Natural substances, particularly medicinal plants and their extracts, are still today intended as source for new Active Pharmaceutical Ingredients (APIs). Alternatively they can be validly employed to prepare medicines, food supplements or medical devices. The most adopted analytical approach used to verify quality of natural substances like medicinal plants is based still today on the traditional quantitative determination of marker compounds and/or active ingredients, besides the acquisition of a fingerprint by TLC, NIR, HPLC, GC.

Here a new analytical approach based on untargeted metabolomic fingerprinting by means of Mass Spectrometry (MS) to verify the quality of grinTuss adulti syrup, a complex products based on medicinal plants, is proposed. Recently, untargeted metabolomic has been successfully applied to assess quality of natural substances, plant extracts, as well as corresponding formulated products, being the complexity a resource but not necessarily a limit. The untargeted metabolomic fingerprinting includes the monitoring of the main constituents, giving weighted relevance to the most abundant ones, but also considering minor components, that might be notable in view of an integrated – often synergistic – effect on the biological system.

Two different years of production were investigated. The collected samples were analyzed by Flow Injection ElectroSpray Ionization Mass Spectrometry Analysis (FIA-ESI-MS) and a suitable data processing procedure was developed to transform the MS spectra into robust fingerprints. Multivariate Statistical Process Control (MSPC) was applied in order to obtain multivariate control charts that were validated to prove the effectiveness of the proposed method.

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.

A hypothesis is presented to explain the bioactivity of natural extracts, often higher than that of the synthetic bioactive components. This view is different from that based on synergy of the different chemical entities present in the natural substrate. It is based on the occurrence of molecular interactions originating from the high complexity of the natural matrix, following the rules of supramolecular chemistry. The formation of complexes between the active compound and other molecules (or oligoelements) present in the natural environment (leading to conformations more effective for interaction with the receptor) may be justified by 1 H and 13C nuclear magnetic resonance (NMR) data. Examples are presented and discussed, suggesting that differences observed between the spectra of the pure compounds and those due to the same molecular species in the natural extracts may depend on some interactions with other molecular species or interactions with oligoelements.

In order to explore new possibilities to optimize the quality control of herbal products, a non-conventional method of analysis of complex systems, the fractal analysis, applied to ElectroSpray Ionisation (ESI) mass spectrometry, was evaluated. The ESI spectra obtained with herbal commercial products were converted into bitmap images and submitted to fractal analysis using the so-called ìbox countingî method. The fractal dimension (DB ) so obtained permitted to classify single plant extracts. Also the ESI spectrum obtained with a mixture of plant extracts contained in a commercial herbal product provided a fractal pattern;statistical analysis on several replicates obtained with different batches indicated that DB tends to display a normal distribution around a mean value, which might be suggested as a typical reference for that product. DB of ESI spectra of plant extracts which underwent thermal treatment indicates that this measure maybe useful also to evaluate the changes occurring with aging of the product. In conclusion, DB might be proposed as a new promising technique of investigation to be used, coupled to mass spectrometry, as a summary measurement of complexity of the overall phytochemical composition and stability of a herbal product. ¿ 2013 Trade Science Inc. - INDIA

The use of phytochemical preparations has shown a massive growth and consequently the related quality control is an important topic. Considering the possible interactions of the active molecules, arising to synergic phe- nomena, the use of the classical approach, based on the evaluation of the level of one or more active compounds can be limitative.Recently a method, based on the direct infusion of commercial botanical dietary supplements in an ESI source operating in positive and negative ion modes has been proposed. The results so obtained were highly promising, allowing the presence of specific plant extracts in commercial products to be deter- mined. In order to evaluate its validity, the data so obtained have been compared with those achievable by a more consolidated technique, as LC- MS is. For this aim Sedivitax gocce (a commercial product composed by extracts of Passiflora incarnata, Eschscholtzia californica, Melissa of icinalis and Valeriana of icinalis) has been considered. Either the plant extracts or Sedivitax samples produced in different years have been analyzed by ESI (±) with direct infusion and LC-MS. The data obtained were elaborated with different statistical methods. The results suggest that mass spectrometry linked to statistical methods can be a quick method to assess the overall stability of a botanical dietary supplement, and can be pro- posed as a promising perspective in quality control. ¿ 2012 Trade Science Inc. - INDIA

Five commercial botanical dietary supplements have been directly analyzed by mass spectrometry by laser irradiation with and without the presence of matrix (in MALDI and LDI conditions). The data so obtained allowed to obtain the characterization of the different samples on the basis of metabolite specific for the different botanic species contained. Furthermore, in the case of positive ion analysis the presence of polysaccharides is put in evidence by using suitable matrices(DHB,super-DHB). If the MALDI experiments are carried out with 1,5-diaminonaphtalene as matrix with basic character, negative ion spectra of good quality are obtained, with the detection of an high number of metabolites, so leading to a clear characterization of the different dietary supplements.  2012 Trade Science Inc. - INDIA