RICERCA ITALIANA     

Natural products with antioxidant activity from plants

Università degli Studi di Napoli "Federico II"

Abstract

Some components in edible plants and in beverages are in the center of scientific debate for their potential positive effects for healt benefits. These compounds are named nutraceuticals and possess a wide range of biological activities and, parallely working with biosynthetic pathway, shown antioxidant and cytoprotective activity with a sensitive reduction of human chronic degenerative diseases.
The research project will be carried out in four phases and is focused to determination of new compounds with antioxidant activity starting from plants extracts such as : grapes, food plants and vegetable species, very common and few studied, coming from Valsesia and from Center of Italy. This project came together and coordinates the programs of four research units with different specific background in the field of botany, biology, chemistry and analysis of antioxidant activity that will be performed though specific tests.
The FIRST PHASE of this project involves an expert botanist for the selection, collection and taxonomic identification of vegetable species coming from Valsesia and from Center of Italy. Crude extracts of each species will be prepared and a preliminary screening will be performed. Perugia research unit will provide to literature screening for the antioxidant activity.
The SECOND PHASE of the project will concern the isolation and structural elucidation of bioactive components with positive results. Along with the study of Valsesia plants, the present project will provide to the isolation of bioactive components of grapes and the analysis of the compounds with antioxidant activity from food plants. In the THIRTH PHASE, all purified compounds will be submitted to test for the valuation of : antioxidant capability, LDL oxidation, DNA damage, HOC remotion, superoxide remotion, tromboxane B2 formation, leucotriene B4 formation, activity of the enzyme mieloperoxydase.
The FOURTH PHASE will consist in the develop clones with same characteristics of productivity as the spontaneous species, selected on the basis of phytochemical features. <<<

Principal Investigator

Franco ZOLLO Università degli Studi di NAPOLI "Federico II"

Research Objectives

The program is based on the chemical characterization of secondary metabolites with antioxidant activity from plants. Attention will be focused on grapes, on plants commonly used as a food and on plants, very common and few studied, coming from Valsesia and from central Italy.
The research units, participating to the project, possess a specific background in the fields of chemistry, biology and pharmaceutical botany and the project will be articulated in 4 phases as follows (each step is described in the section 2.4 of this research program) :


Fhase 1

Selection and collection of vegetal species from different geographical areas will include areas that differ greatly for climatic and geo-pedological characteristics of central Italy and Valsesia

Fhase 2.

- Chemical studies on active extracts in preliminary tests: isolation and structural characterization by spectroscopic techniques (NMR, IR, UV, MS).

- Extraction and structural characterization of polyphenolic metabolites from grapes with antioxidant activity: analysis of complex mixture of procyanidines from grapes by modern and sophisticated chromatographic methodologies.

Phases 3.

- Determination of antioxidant activities of pure metabolited isolated from different vegetal species.

Phases 4.

- Development of clones with the same characteristics of productivity as the spontaneous species, selected on the basis of phytochemical features. It will be establish the optimal growth, nutritional, and environmental conditions for each species and for the various phases of in vitro propagation. <<<

First Results

The predicted partial resuls are:

- Complete the collection of information concerning the traditional use and the presence of officinal plants in Valsesia

- Collection and taxonomic identification of the species coming from Valsesia, with the aid of an expert botanist operating in loco.

- Establishment of the herbarium.

- Preparation of extracts with increasing polarity solvents.

- Preliminary screening of biological activity on the raw extracts of the species collected in Valsesia and central Italy.

- Shipment of the biologically active fractions to the units in charge of the analysis, and identification of the active metabolites.The predicted partial resuls are:


- Isolation and structural characterization of compounds with antioxidant activity from food plants. Our attention will be devoted to food species used as spices or flavours such as Capsicum, Laurus nobilis, Foeniculum vulgaris, Juniperus communis and some species of Labiate family.

- Stereochemical determination of pure compounds by NMR (NOESY and ROESY experiments) and chemical approaches.Evaluation of antioxidant activity of metaboliets isolated and structural characterizated by research units involved in the phase 2.The predicted partial resuls are:


- Chemical analyses on the clones obtained to determine whether the original phytochemical characteristics have been maintained and to select the most productive clones.

- Isolation and identification of secondary metabolites by chromatographic and spectrometric techniques.

- Establishment of a germplasm bank for the aromatic and medicinal species of Valsesia. <<<

Timescale

24 months

National and international background

One of the main goals of the chemistry of naturally occurring compounds is screening for promising biologically active substance from plant origin. Many plants, commonly used as vegetable foods, have been employed during the past centuries, as spices, foods or for traditional medicine. The major contribution of plants to human diet is due to the large amount of vitamins, folic acid and minerals. However they contain other secondary metabolites, called nutraceuticals, that have recently been at the center of an intense scientific debate (1, 2). Edible plants are rich in phytochemicals such as terpenoids, flavonoids, alkaloids, pigments, polyphenolic, fitosterols, plyunsaturated fatty acids which may play a important role for healt benefits. Mediterranean diet includes large amounts of food plants (fruits, vegetables, nuts, seeds, wine, olive oil) (3) and the prevention of the oxidative reactions has been associated with lower risk factors for coronary heart desease (CHD) such as lower blood plasma cholesterol levels (4).
A sensitive reduction of human chronic degenerative diseases was also observed as in cancer or Alzehimer's desease, Parkinson’s desease, autoimmune pathology, multiple sclerosis. (5,6). Epidemiological studies have suggested a positive association with antioxidant compounds in foods and beverages such as polyphenols. From a chemical point of view polyphenols are classified in two groups: the flavonoids and the nonflavonoids. The flavonoid family includes the flavonols myricetin, quercetin,
kaempferol and isorhamnetin, which exist both as free form and sugar cojugates; the flavan-3-ols, catechin, epicathecin and the anthocyanins such as malvidin-3-glucoside. The non-flavonoids encompass gallic acid; hydroxycinnamates, including p-cumaric acid, caffeic acid and caftaric acid; and the stilbenes, trans-resveratrol and cis-resveratrol. Condensed tannins, known as procyanidins, are the main components of grapes and are oligomers or polymers of catechin and epicatechin subunits linked each other throug
carbon-carbon bonds 4-6 or 4-8. Grapes contain catechins and procyanidins in seeds and skin and they are the major components of red wine along with resveratrol; epigallocatechin gallate is detectable in green tea (6, 9). Catechins and procyanidins are known for the antioxidant activity. Studies have shown that trans-resveratrol, isolated from grapes and wine, is a potent phytoestrogen (8).The interest for antioxidant molecules arises from the observation of their role in the modulation of free radical production. A current topic of great relevance for this study in which the etiology of many diseases is based is the role of the uncontrolled production of free radicals: the participation of free radicals as mediators of tissue damage has been, in fact, recognized in many pathophysiological processes such as inflammation, atherosclerosis, ischemia, tumors, damage induced by ionizing radiations, aging, skin photoreactions. However yet, it is known that the production of free radicals does not only involve pathology: their production concerns several metabolic processes and can constitute a useful process (in biological terms), as in the case of phagocytes, whose mechanism of defense against noxious agents involves the production of high amounts of superoxide anion. Free radicals can become damaging every time their formation exceeds the capability of antioxidant systems to rapidly dispose of them.
It needs to be underlined how the antioxidant molecules are endowed with potential estrogenic activity. On the grounds of recent research, phytoestrogens, appear to contribute to the healthful properties of plants, due to their ability to selectively interact with alpha and beta estrogen receptors It is thus necessary to study the molecules that have been isolated from the plants in order to study this action as well. Most Italian medicinal plants are collected in the wild, a practice which, together with the substitution of local varieties with highly productive modern varieties, can result in the rapid simplification of ecosystems, reducing their biodiversity. In vitro propagation of aromatic and medicinal species represents a potential means of maintaining ex-situ ecotypes with peculiar characteristics (9) and of producing bioactive molecules on a large scale (10), favouring the development of new areas of production in Italian agriculture.
The area of Valsesia constitutes an uncontaminated habitat, in that imported plants are scarce, and it is a good example of a habitat in which biodiversity has been maintained (11). The native flora of Valsesia includes different species belonging to the following families: Lamiaceae [Thymus pulegioides L., Thymus polytrichus Kerner, Thymus froelichianus Opiz, Thymus praecox Opiz, Thymus alpestris Tausch, Mentha longifolia (L.) Hudson, Mentha acquatica L., and Nepeta cataria L.]; Valerianaceae (Valeriana officinalis L., Valeriana collina Wallroth, and Valeriana celtica L.); Asteraceae [Achillea millefolium L., Achillea distans Walldst, Achillea stricta (Koch) Schleicher ex Gremli, Achillea macrophylla L., and Achillea moschata Wulf.]; and Gentianaceae [Gentiana purpurea L., Gentiana kochiana Perr. et Song, Gentianella ramosa (Hegetschw.) Holub] (12). Some of these species have been used as aromatic herbs and for extracting essential oils and aromatic compounds for use in pharmacology and herbal medicine.
Food plants such as Capsicum annuum L. (red pepper) in sweet or hot forms (13, 14) and from plants belonging to the family Liliaceae as garlic (A .sativum L), onion (A. cepa L.), leek (A. porrum L.), shallot (A .ascalonicum L.) (15-19)are used as foods or spices. Several research publications and thousands of years of traditional use have attributed to their organic extracts interesting pharmacological activities, e.g. antimicrobial, platelet antiaggregating and cancer preventing. Recent studies resulted in the identification of a number of organic compounds , whose stereostructure have been determined mainly by the aid of non-destructive spectroscopy methods (mono-and bi-dimentional NMR experiments) and by Mass spectrometry, thus preserving the required amounts of natural products for the pharmacological screening. From different species of Allium and from Capsicum seeds we have isolated new saponins and sapogenins with antimicrobial and cytotoxic activities (14,16-18) with several glycosides and phenolics (13,15,19). Many of them are tested for platelet anti-aggregating and antioxidant activity (13). Lamiacee is a plant family widely distributed throughout the temperate zone. There are about thirty genera in the Mediterranean area and there is great within species variability. Some Lamiacee have been used for ages in folk medicine, cosmetics and nutrition. It is well known that the secondary metabolites may be subject to considerable variation in the living plant, depending on environmental and ontogenetic factors (20). There is high secondary metabolite variability between morphologically similar plants and this could be taken into account in determining the biological activity. It could be beneficial to link a fingerprint of the secondary metabolites, or at least the amount of the more biologically active compounds, to the morphology of each plant. It can be speculated that the biological activity of morphologically similar plants is not the same if the secondary metabolites are not similar and the chemical compounds of plants such as Lamiacee with high secondary metabolite variability need to be accurately determined. The biological activities of the Lamiacee that have been most studied are the antimicrobial and radical scavenger activities, of which the radical scavenging ones have been the less studied. Rosemary (21), sage(22,23), Satureja (24), basil (25,26), mint (27), oregano (28) and thyme (29) have been studied extensively for their antioxidative properties; otherwise the biological activities of other genera such as Galeopsis have not been studied at all.
These observations have prompted us to propose the present project that will be aimed to the isolation and characterization of all secondary metabolites from plants, and than submit them to antioxidant activity tests. <<<