Isolation, characterization, activity and evaluation of natural and synthetic bioactive compounds

Referente: Prof.ssa Maria Giovanna Chini

Working group

  • Prof. Maria Iorizzi (Full Professor, CHIM/06);
  • Prof. Gabriella Saviano (Associate Professor, CHIM/03);
  • Prof. Claudio Craprari (Associate Professor, BIO/04);
  • Prof. Maria Giovanna Chini (Associate Professor, CHIM/06);
  • Dr. Vadym Samukha (Ph.D. student);
  • Dr. Francesca Fantasma (Ph.D. student).

Objectives and activities

Natural products derived from plants are the biggest resource for drug discovery in modern medicine and over 50% of currently available FDA-approved drugs are derived from natural products. The research for new synthetic drugs is a long, risky, and expensive process that frequently leads to unsuccessful results.

In recent years, much attention has been devoted to natural products of plant origin or from edible plants for their ‘drug-like properties’ to be used against human diseases. Many foods (fruit and vegetables) possess ‘nutraceutical’ value as they contain natural products that can protect and prevent several degenerative diseases. Also called secondary metabolites, they show interesting pharmacological properties in vitro and in vivo, such as anti-inflammatory, antioxidant, anticancer, antifungal and antibacterial activity. Drug discovery from natural sources involve a multifaceted approach combining phytochemical analysis, biological and molecular techniques. Along with the isolation and structural determination phase, nowadays great support to the target identification process is given by computational chemistry, which can highlight new targets, disclosing new therapeutic applications and shedding light on the molecular basis behind the observed side effects.

The project aims at the phytochemical analysis of selected plant extracts and the preparation of essential oils with the ultimate goal of identifying potential new drugs. Pharmacological evaluation and repositioning of isolated metabolites is planned with the aim of modulating the activity of selected protein targets involved in fungal infections and oxidative stress-related diseases. The further validation of their therapeutic potential will take place through in vitro analyses. The proposed research plan is well connected with the aims of the 2030 Agenda Goal N.3 “Ensure healthy lives and promote well-being for all at all ages”. In detail, this project aims to ensure health and well-being through the isolation and identification of secondary metabolites with structural diversity and then testing their potential activities in vitro. Our project enables a portfolio of methodologies and technological solutions for the assessment and treatment of diseases associated with fungal infections and oxidation processes.

Methodologies to be used to achieve the objectives and timetable of the activities

This scientific proposal is based on a combined in silico and experimental approach, following the general multi-stage workflow (Figure 1), aimed at characterizing agents, especially secondary active metabolites (SAMs), for the treatment of fungal infections and oxidative stress-related diseases.

Figure 1. General workflow of the project.

WP1: Selection of plant families and identification of secondary metabolites with chemical diversity

Task 1.2 Plant material extraction. The focus will be on plants rich in polyphenols. A specific extraction protocol will be developed for each species by using traditional extraction procedures or developing innovative extraction methods (“green extraction”, e.g., Accelerate Solvent or Microwave Assisted extraction).

Task 1.3 Qualitative analysis of the obtained extracts and structural determination of pure compounds. The extracts obtained from the different parts of the plants will be purified in the first step by e.g., Gel-filtration, chromatographic separations, HPLC, UHPLC. The structural determination of pure compounds will be the next step and will be carried out following a qualitative approach mainly using spectroscopic techniques such as NMR (Nuclear Magnetic Resonance) analysis. Mass spectrometry (MS) experiments will be combined (low and high resolution MS) with a wide range of ionization techniques (EI, ESI).
Part of the project will be devoted to the characterization, chemical variability and quality of essential oils (EOs) extracted from plant matrices by means of hydrodistillation, gas- chromatography (GC) and mass spectrometry MS.
If necessary, qualitative and/or quantitative approach, NMR analysis will be performed to obtain a fingerprint of the extracts.

WP2. Integrated approach for three-dimensional assessment of SAMs and target identification

Task 2.1 Computational methods for elucidating the structural features and In silico molecular profiling. If necessary, in silico methods coupled with NMR spectroscopy (e.g., the quantum mechanical (QM)/NMR combined approach) could be applied to resolve correct stereoassignment of SAMs.

Considering the experimental needs, IVS (Inverse Virtual Screening), a robust computational approach, could be applied in order to disclose a possible biological target, and it could also explain the selectivity or the presence of side effects of a compound.

WP3-4. From affinity to bioactivity: integration of the in vitro pharmacological protocols

Tasks 3-4. After the target recognition stage, if necessary, we will proceed in demonstrating the binding of the molecules with the receptor counterpart through NMR and/or MS techniques. As schematically reported in Fig 1, the first phase of evaluation of the biological activity will be performed through in vitro assays. As mentioned before, in vitro antifungal activity will be carried out by measuring the diameter (in mm) of mycelial radial growth on culture medium treated with purified or partially purified toxic compounds after in silico analysis. A dose-dependent fungal reduced growth is a specific marker of the compound toxicity.

WP5. Management, Dissemination and Training

Constant project management will be continuously provided by PI and personnel involved in the project. In addition to the classic dissemination modalities such as publication in high impact peer reviewed journals, the social media, will be used to share raw data and/or to promote the scientific results to reach a broader audience (Third Mission).

Expected results

The ground-breaking idea of the proposed project is based on the evaluation, characterization, valorization and enhancement of cultivars of the Apennine areas as potential ‘reservoirs’ of active secondary metabolites (SAMs) to be identified and/or repositioned in the pharmaceutical scenario. The main objective will be pursued through the extraction of selected plants, the determination of the chemical and pharmacological profile of all the SAMs responsible for the biological activity.
The achieving of the specific scientific objectives of this project (Table 1) will be accounted considering different stages grouped in work packages (WP1-WP4), following a multidisciplinary approach:

  • WP1. Rational selection of cultivar families rich in natural products (polyphenols) and identification of new and/or principal molecular entities from natural sources (Task 1).
  • WP2. Integrated approach for three-dimensional assignment of SAMs and target identification (Task 2).
  • WP3. Molecular binding assessment (Task 3).
  • WP4. From affinity to bioactivity: integration of the in vitro protocols (Task 4).
  • WP5. Management, dissemination and training.
Specific scientific objectives
Objective Specific scientific objectives of the project WPs
Objective 1 Collecting SAMs bioactive molecules from selected cultivars WP1
Objective 2 Molecular recognition and target identification of characterized SAMs from selected plan sources WP2-WP3
Objective 3 Assessment of the biological activity of «new» and «old» SAMs from selected plant sources WP4
Objective 4 Project management, dissemination of results (social media, specialized journals, meeting, congress) WP5


According to the reported activities (Table 1), accounting for the possible application of extracts and/or related SAMs in the food supplements, cosmetics, cosmeceuticals and nutraceuticals industry, their potential anti-oxidant, anti-ageing, and antifungal activity will be assessed by in silico, biophysical assays and in vitro approaches.

In summary, the expected results and their impact/application are related to:

  • optimization of the extraction conditions to obtain extract(s) with high phenolic content and antioxidant activity of selected cultivars of Apennine areas;
  • chemical profiling of the constituents;
  • identification of secondary active metabolites with potential health benefits; -quali/quantitative investigation of the constituents detected in the essential oil of selected plants;
  • antifungal activity of purified and/or partially purified plant extracts;
  • improvement of the local producers’ scientific, cultural, and economic relations.