FITOCHIMICA - BIOTECNOLOGIE FARMACEUTICHE APPLICATE ALLE PIANTE OFFICINALI

Learning Objectives

• Phytochemistry

  The course has the goal of giving the main information about biosynthesis and accumulation of secondary metabolites in vegetable species together with their classification, their role in plants, and their use ad chemotaxonomic markers.

  Particular attention will be given to the main extraction techniques to be applied in the treatment of vegetable matrices; a series of case studies and practical examples will be given during the class section. Hints on analytical techniques normally used in the determination of secondary metabolites in vegetable species will be also given.

• Pharmaceututical biotechnology applied to medicinal plants

  The course aims to provide students with the basic knowledge concerning pharmaceutical biotechnology applied to plant organisms. Particular attention will be devoted to medicinal and aromatic plants, to the production of bioactive compounds of pharmaceutical interest, and to the knowledge of the main innovative biotechnological processes for the production of transgenic medicinal plants. The course will cover topics ranging from the chemistry of secondary in vitro metabolites to their isolation and characterization, molecular biology, and genetic engineering.

Course Structure

• Phytochemistry
  
  The course consists mainly in frontal lessons; a series of case studies will be also discussed in class
• Pharmaceututical biotechnology applied to medicinal plants

  Two modules:

  Module I, 1 ECTS

  Module II, 5 ECTS

Detailed Course Content

• Phytochemistry

  SECTION I – 3CFU

  • SECONDARY METABOLISM – INTRODUCTION primary and secondary metablites; role of secondary metabolites in vegetable species; factors influencing the content of secondary metabolites in plants
  • THE THREEE SECONDARY METABOLIC CYCLES: acetate, mevalonate, shikimate pathway; building blocks and products of plant secondary metabolism
  • SPECIAL FOCUS ON PLANT SECONDARY METABOLISM: carotenes, carotenoids, apocarotenoids: the metabolic pool of saffron; phenols and polyphenols: the different subclasses, from phenylpropanoids to anthocyanins; terpenes and terpenoids; essential oils, alkaloids

  SECTION II – 3 CFU

  • HOW TO DEAL WITH A VEGETABLE MATRIX extraction techniques for volatile and non volatile secondary metabolites; cascade extraction, hydrodistillation, non conventional extraction techniques
  • ANALYTICAL TECHNIQUES normally used for the determination of volatiles and non volatiles in complex vegetable matrices; chemotaxonomy
  • CASE STUDIES AND OPEN DISCUSSIONS
• Pharmaceututical biotechnology applied to medicinal plants
  • Module I - 1CFU
  • INTRODUCTION TO VEGETABLE BIOTECHNOLOGIES. Definition, history, evolution.
  • PLANT TISSUE CULTURE. Historical background of plant tissue culture. Laboratory organization, culture chambers, culture media, plant growth regulators (auxins, cytokinins, giberellins, abscissic acid, ethylene). Crops in liquid soils. Crops on solid soils: agar and support systems for plant crops.
  • STERILIZATION TECHNIQUES. Filtration, dry sterilization, steam sterilization, use of UV rays. Sterilization of instrumentation and culture media, sterilization of plant tissues. Vertical and horizontal laminar flow hoods. Indessage: techniques, main phases, virus identification, serological tests (ELISA), bioassays, virus-free plant production.
  • CHOICE OF THE PLANT TISSUE. Seasonal moment, type and size of the explant, explant from specific tissues. Advantages and disadvantages of individual explants.
  • Module II -5CFU
  • PROPAGATION TECHNIQUES AND MICROPROPAGATION. Traditional biotechnologies: selection, hybridization, crossbreeding and domestication for the improvement of medicinal crops. Innovative biotechnologies: in vitro cultures of plant cells, tissues and organs - related techniques, micropropagation, genetic engineering or recombinant DNA technology - transgenic plants and organisms, cryopreservation of germplasm. Micropropagation: culture of meristems and apices, culture of nodal segments. Elimination of pathogens from the mother plant: chemotherapy, thermotherapy. Micropropagation stages: 0, I - septic culture implantation, II - sprout proliferation, III - pre-transplantation and rooting, IV - transfer to the external environment. Advantages and disadvantages of micropropagation.
  • CELL CULTURE. Types of suspension cultures: discontinuous, continuous (open or closed), semi-continuous, culture of immobilized cells. Cell growth curve: latency phase, acceleration phase, exponential phase, deceleration phase, stationary phase. Synchronized cultures: synchronization by selection and by induction. Induction methods: cold treatment, starvation, use of inhibitors, colchicine method. Control of cell growth: viability and cell growth. Cell growth: cell count, fresh weight (fw), packed volume cells (pcv), dry weight (dw).
  • PRODUCTION OF SECONDARY METABOLITES IN VITRO. Advantages and disadvantages. Selection of highly productive cell lines. Influence of the presence in the culture medium of: sugars, nitrate, nitrogen, phosphate, PGR, supply of precursors, beta-cyclodextrins. Optimization of the culture environment: temperature, lighting, pH, agitation and ventilation. Elicitation and elicitors. Removal of the product in situ. Immobilization. Matrices. Advantages and disadvantages. Examples of production of active ingredients and substances of pharmaceutical and pharmaceutical-technological interest.
  • ISOLATION, CULTURE AND FUSION OF PROTOPLASTS. History and definitions: protoplast and cell wall. Protoplasts isolation: mechanical and enzymatic methods. Protoplast isolation steps: choice of the type of explant, choice of the appropriate enzymatic mixture, removal of the epidermis, plasmolysis of the tissues, enzymatic digestion, suspension and washing protoplasts, estimation of the number and vitality of the isolated protoplasts, protoplasts culture. Factors influencing the growth and development of protoplasts. Cell wall formation. Protoplast fusion and somatic hybridization. History and definitions. Types of hybrids: somatic, asymmetric, cybrids. Stages of somatic hybridization: fusion of protoplasts (spontaneous or induced), selection of hybrid cells, identification of hybrid plants. Direct and induced fusion. Methods: treatment with NaNO3, treatment with high pH Ca++ ions, polyethylene glycol (PEG) method, electrofusion. Identification and selection of hybrid cells: use of resistance markers, use of visual characteristics and vital dyes. Examples. Applications of somatic hybridization.
    GENETIC ENGINEERING TECHNIQUES. PGM and OGM: history and definitions. Molecular vectors, plasmids, restriction enzymes, DNA ligases, polymerase chain reaction and DNA amplification (PCR). First and second generation PGM. Transformation of plants. Procedural steps for the production of PGM: isolation and characterization of a gene, preparation of the construct, transformation, analysis and selection of transgenic lines, introgression of the transgene in more productive lines, field tests. Transformation methods. Direct transformation: electroporation of protoplasts, microinjection. Transformation “in planta”: biolistic methods, methods of Agrobacterium tumefaciens and rizhogenes. Advantages and limitations of each method. Construction of a cloning vector based on the plasmid Ti: system of the binary vector, system of the cointegrative vector. Triparental mating. Analysis and selection of transgenic cell lines. Selection methods: antibiotic resistance, herbicide resistance, introduction of reporter genes. Advantages and problems. Gene transfer mediated by Agrobacterium rhizogenes: hairy roots. Examples of products obtained from cultivation of hairy roots.
  • OGM – EUROPEAN AND NATIONAL LEGISLATION. Directives on: releases in the environment, food and feed, traceability and labeling, transboundary movements of GMOs. Precautionary principle. Examples of commercialized products and mechanisms of action of the introduced exogenous genes.
  • CONSERVATION OF GERMOPLASMA AND CRIOCONSERVATION. Regulatory framework and conventions on the protection of biodiversity. In situ and ex situ conservation. Networks of germplasm banks. Germplasm collection. Germplasm transfer. Germplasm treatment before storage. Packaging and storage. Cryopreservation. Stages of cryopreservation. Germination. Germplasm management.
  • ORGANOGENESIS. Direct organogenesis, indirect organogenesis. Caulogenesis, rhizogenesis. Phases of organogenesis: explant, de-differentiation, competence, induction, determination, differentiation, organ. Applications to the propagation of medicinal plants and to the obtainment of secondary metabolites of pharmaceutical and pharmaceutical-technological interest.
  • EMBRYOGENESIS. Zygotic (sexual) and non-zygotic (somatic) embryogenesis. Protocols of embryogenic culture. Origin of non-zygotic embryos. Induction of embryogenic cells and use of PGR. Embryo development and maturation. Quiescence and dormancy. Germination of the embryo and development of the plant. Applications of embryogenic cultures. Synthetic seed technology and encapsulation.

Textbook Information

• Phytochemistry

  SECONDARY METABOLISM

  Alessandro Bruni, Biologia Farmaceutica, Edizioni Pearson, Capitolo 7 (pagg. 127-171); per approfondire Paul M. Dewick, Chimica biosintesi e bioattività delle sostanze naturali, Casa Editrice Piccin

  EXTRACTION AND MAIN ANALYTICAL TECHNIQUES APPLIED TO THE STUDY OF COMPLEX VEGETABLE MATRICES

  Marco D’ischia, La Chimica organica in laboratorio, Casa Editrice Piccin:

  Capitolo 2.5 (pag. 98-105); Capitolo 3.1 (pag. 111-120); Capitolo 3.2 (pag. 121-132); Capitolo 3.7 (pag. 152-163); capitolo 3.8 (pag. 164-182); Capitolo 4 (pag. 197-199); Capitolo 4.3 (pag. 219-238); Capitolo 6.2 (pag. 384-395); Capitolo 8.4 (pag. 639-653).

• Pharmaceututical biotechnology applied to medicinal plants
  • Notes taken during class
  • Internet sites indicated from the teacher
  • Teaching material available on Studium for the current academic year
  • Trigiano R.N., Gray D.J. “La Coltura dei Tessuti Vegetali” Edagricole, Bologna, Italia.
  • Gianluigi Bacchetta, Piero Belletti, Salvatore Brullo, Luisa Cagelli, Valentina Carasso, Josè, Luis Casas, Claudio Cervelli, M. Carmen Escribà, Giuseppe Fenu, Fabio Gorian, Jaime Güemes1, Efisio Mattana, Massimo Nepi, Ettore Pacini, Pietro Pavone, Beti Piotto, Cristiano, Pontecorvo, Aranxta Prada, Gianfranco Venora, Lorenzo Vietto, Myriam Virevaire. APAT - Agenzia per la protezione dell’ambiente e per i servizi tecnici. Manuale per la raccolta, studio, conservazione e gestione ex situ del germoplasma
  • Chawla H.S. “Introduction to plant biotechnology”, Springer, Netherlands.
  • Sala F., Cella R. “Colture Vegetali.Maffei M. "Metabolismo e Prodotti Secondari delle Piante" 1999, UTET Libreria Srl, Torino, Italia.
  • Glick B.R., Pastrernak J.J “Biotecnologia Molecolare - Principi e Applicazioni del DNA Ricombinante” 2003, Zanichelli.