Dottorato in Scienze Farmaceutiche e Biomolecolari

Contacts

Supervisor

Curriculum vitae

Phd thesis

Due to the increased interest of consumers in products more natural for own health-care, in the last years a widespread use of dietary supplement was been recorded. Often the figure of dietary supplements was been associated with that of “health supplements”, or rather substances that correct specific deficiency diseases. This new generation of supplements are products that can be considered as something between a drug and food, including vitamins, minerals, herbals and botanicals, amino-acids, enzymes and many other compounds.

In contrast to pharmaceuticals, dietary supplements completely bypass the stringent and regulatory process of quality/safety control, and may be sold with very little regulation and outside of health environment such as pharmacy, herbalists or hospitals.

All products categorized as “dietary supplement” always carry a table/panel, a dosage information and a claim. The first one lists the contents, amount of each active ingredients per serving and other excipients. However, in the most of cases, labels are prudently formulated to avoid any clash with the few rules issued, with the consequences to obtain vague and misleading tables, reporting also irrational advertisements or unreal quantification. Indeed, due to the miss of a real legislation, the chemical characterization of dietary supplements is object of controversial debates. Actually, very often the dietary supplements market is characterized by claims on the content of bioactives present in the raw material that sometimes are far from being reliable. In most of the cases, wrong titrations occur when the identification of the main bioactive molecules is based on simple chemical characterization or on wrong and amateur protocols.

Finally, the dietary supplements are supported by assumed claims of specific health benefits. Contrary to what can be ensured, in the major of the cases, there is no sufficient scientific evidence to justify these claims. Indeed, many claims about dietary supplements are not based on serious research, without study on adverse effects or possible interaction with traditional medicines and/or other dietary supplements.

Nevertheless, beyond these negative aspects of dietary supplements, if the development of a new supplement preparation is based on a rigorous scientific method, the evidences show that their intaking may be beneficial for overall health and for managing some health conditions. On the other hand, the scientific approach is not the only necessary condition for the development of a good product. Indeed, it is necessary that the solid scientific research be combined with a good quality control policy.

About this last point, due to the fact that in the most of cases dietary supplements are made with plant extracts, the quality control should verify not only the absence of toxic and dangerous substances (such as aflatoxins, pesticides, heavy metals, etc...) but also provide for proper geographic identification and phytochemical characterization of raw materials. Therefore, basically, working with food supplements should be more difficult than working with medicines, because at the traditional chemical-control should be put beside biochemical and biomolecular assays in order to prove the correct origin and composition of raw materials used for processing. Unfortunately, currently it is the exact opposite.

In this contest, the topic of my doctoral thesis was focused on the quality control of food and dietary supplements using advances analytical methods coupled with biomolecular and biochemical techniques.

My main project involved the combination of the genetic and chemical approaches in order to provide an unequivocal identification of raw materials to support the identification when the simple evaluation of morphological characteristic is not possible. Object of this study was a particular Mediterranean raw material: pistachio (Pistacia vera). Here, spectrophotometric assays (Folin-Ciocalteu, pH jump and DMAC) were combined with either HPLC-DAD-MS/MS or GC-MS-FID analysis in order to investigate the phytochemical diversity within six pistachio verities of commercial interest (Bronte, Larnaka, Mateur, Mawardi, Kern and Kerman). Moreover, DNA fingerprinting coupled with RFLP analysis was assessed to determine the differences in non-transcribing regions (ITS and NTS).

Due to the presence of a large amount of antioxidants in the skin of pistachios, these particular waste products of manufacturing industry were also employed in further studies in order to evaluate its biological activities, with the aim to use it as innovative component for a novel dietary supplement. In particular, were assessed the antioxidative (AOA) and antiproliferative (APA) activities, and their potential mechanism of action was researched along with qRT-PCR and enzymatic studies.

Another interesting aspect of the dietary supplement market is the overestimation of bioactive compounds. During my doctorate career, I proven that the most of the supplements made by Boswellia are object of chemical-overestimation. Indeed, is frequently reported in the label of these products a concentration of boswellic acids equal to or higher than 70% or AKBA contents of 30%, in both Boswellia sacra and Boswellia serrata gum resin extracts. Due to the importance of these extracts in the treatment of several diseases, such as inflammation and osteoarthritis, asthma and age related disorder, the qualitative and quantitative determination of the bioactive boswellic acids is crucial. By the use of only HPLC-DAD-MS/MS analytical instrumentation, we provided a solid guideline for the chemical standardization and validation of all dietary supplements containing Boswellia gum resin extracts.

The now widespread concept that the intaking of food supplements is synonymous of safety and lack of pharmacological complications, is obviously wrong. Phytochemicals content in these preparations are bioactive compounds, with different chemical structure, belonging to different classes and having pharmacological properties. The same instrumentation used for the chemical quality control may be applicate also to monitor the possible desired and adverse effects both in vitro and in vivo models after the intaking of dietary supplements. Regard this last point, during my last PhD year I had the opportunity to collaborate with zoologist group of the department of Life Sciences and Systems Biology. Here we evaluate how the administration of two simple Volatile Organic Compounds (VOCs) of Origanum vulgar plants (thymol and carvacrol) can affect the behaviour changes in what that can be considered the best model system for neurotheological studies: the ants. HPLC-MS/MS technology was instrumental for the identification and quantification of the biogenic amines (such as dopamine, tyramine, serotonin, etc…), that were the main responsible compounds involved in ant behaviour changes.

In conclusion, the results of this doctorate period demonstrated that only analytical methods based on gas and/or liquid chromatography coupled to mass spectrometry allow the precise identification and quantification of bioactive compounds in different plant and animal extracts. Moreover, coupling this methodology with biomolecular and biochemical methods could be the best approach both to obtained precise information useful for the unequivocal identification of plant species, to have a correct phytochemical quantification in plant extracts and, finally, to study their potential biologic affect in vitro and in vivo model systems.