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Exploitation of aromatic plants’ by-products for the development of novel cosmeceuticals and food supplements.

Periodic Reporting for period 2 - EXANDAS (Exploitation of aromatic plants’ by-products for the development of novel cosmeceuticals and food supplements.)

Reporting period: 2018-03-01 to 2020-02-29

Southeastern Europe is a region of rich biodiversity; several hundreds of different medicinal and aromatic plant species are collected and processed in the region and exported to other EU countries. Many of the collected wild or cultivated plants are subjected to steam distillation and/or hydro-distillation to obtain essential oils, creating a significant amount of aqueous water (hydrolat) and waste herbal residue. The plant material residue is considered waste and is disposed in landfills. These by-products represent a vast reservoir of valuable compounds, the exact composition of those wastes is not always known. Innovative approaches to extract, purify and process such substances are needed.

The main scientific and technological objectives of EXANDAS are:
- Application of emerging extraction and purification techniques for the development of novel natural products (extracts & enriched fractions), the optimization of their recovery and isolation of promising substances
- Unequivocal structure elucidation of chemical constituents through the utilization of advanced hyphenated techniques
- Development of validated analytical methodologies for the targeted quantitative analysis of specific bioactive ingredients in final products
- Assessment of the various biological activities of the extracts and pure compounds in order to reveal their potential activity.
- Scaling up of the extraction and purification processes and development of new products based on emerging technologies
- Formulation of final products and elaboration of stability and safety assessment
- Elaboration of a feasibility study for the development of several new commercial products (extracts, enriched fractions, pure compounds, cosmeceuticals and food supplements).

The project on 29/2/2020 was integrated and according to DOA it has achieved its objectives with relatively minor deviations. More specifically according to its objectives:
- Novel approaches have been tested and 12 protocols have been created for the development of extracts and enriched fractions.
- Isolation and structure elucidation of more than 90 pure compounds have been achieved with the use of advanced techniques.
- Biological assays have been performed concerning the antioxidant, anti-microbial, anti-whitening, antiaging, anti-inflammatory activity of the extracts and pure compounds.
- Formulation of final products has already been achieved for two finished new products.
- The researchers gained great transfer of knowledge through their secondments.
- Strong synergies have been created inside the consortium that will continue even after the end of the project, proving that EXANDAS is a great example of a RISE project!

The project objectives managed to be achieved within the time and resources that were available for the project. All the deliverables and milestones have been delivered although there was a delay due to the continuously progress of the project. The deviations from the work plan, the justification and the corrective actions were described in the first periodic report.
The responsible beneficiaries provided the consortium with the necessary by products and raw materials of plants with great interestsuch as R. damascene, L. vera, M. Officinalis, G. macrorrhizum, M. Camomilla, S. scardica etc. Mastic gum by-product named colophony and by-products of the mastic gum hydrodistillation were also collected. Collection of stems and leaves from Pistacia species was also performed. The by-products were transferred to the responsible participants in order to be investigated. A great number of extracts and enriched fractions were prepared using environmentally friendly technologies and extraction protocols were developed for each collected raw material. Depending on the raw material, “green” techniques such as Supercritical Fluid extraction, Microwave Assisted Solvent Extraction, Subcritical water extraction and highly cross-linked adsorptive resins were used. Additionally, the optimal extraction conditions for most of the raw material were also determined. All the preparations were forwarded for the initial assessment of their chemical content. Total Phenolic Content (TPC), Total Flavonoids Content (TFC) and HPTLC analysis using both general and specific reagents for the visualization of the chromatograms were performed. The results of that initial chemical profiling revealed the most promising extracts. These extracts were submitted to Centrifugal Partition Chromatography technology in order for the main components to be isolated. For the identification of the volatile components an analytical procedure on GC-MS was developed. The qualitative characterization of the extracts was also performed with analytical processes based on HPTLC. Antioxidant activities of extracts and enriched fractions were investigated using DPPH antioxidant assay and ABTS to measure the radical scavenging capacities of the samples. The extracts were also evaluated for their skin-whitening activity using an established tyrosinase assay. The evaluation of the anti-inflammatory activity, the cytotoxicity and their ability to inhibit enzymes involved in the degradation of elastin, collagen and hyaluronic acid was performed. In total, more than 150 preparations (extracts, enriched fractions, essential oils) have been prepared, 12 relative extraction protocols have been developed and more than 70 ingredients purified. Pure compounds were characterized with the use of chromatographic and spectroscopic features using LC-HRMS/MS combined with structural information deriving from 1D and 2D NMR experiments. Moreover, biotransformation of selected extracts using enzymes in several conditions and chemical transformations such as hydrolysis was carried out in order to obtain extracts enriched in bioactive compounds. In total, a library of extracts, enriched fractions and purified compounds was created. The pilot scale production of selected extracts was based on the laboratory scale extraction protocol. For the optimization of the methods, several parameters were taken under consideration. Furthermore, aiming to the smooth transition into industrial implementation all the respective steps of the process as well as the corresponding results were evaluated for their compliance with the demands from industry and regulatory authorities.
For the implementation of EXANDAS project specific aromatic and medicinal plants were selected. The selection was based on their economical importance for southern European countries, as well as on their therapeutic/bioactivity potential. More specifically by-products and wastes from the industrial exploitation of mastic gum, rose, mountain tea, lavender, geranium, sweet basil etc. were selected.

Overall EXANDAS implementation incorporated state of the art technologies for detailed chemical analysis of by-products obtained during the aforementioned aromatic/medicinal plants processing. EXANDAS introduced eco-friendly technologies for the production of natural “green” extracts, fraction and pure compounds. All products were evaluated extensively for their biological activity and safety using in vitro and cell-based assays in a successive and complementary manner. Most interesting ingredients were forwarded to scale up production, formulation and elaboration of a feasibility study for their commercial exploitation as novel active ingredients in cosmeceuticals and food supplements