antioxidanti 2

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ANTIOXIDANTS

Free radical reactions in biological systems –a concept approached in the researches carried out by professor

Cristofor I.Simionescu

Valentin I. PopaTechnical University of Iasi

vipopa@tuiasi.ro; vipopa15dece@yahoo.com

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• The aspects concerning induction, evolution, chemistry and inhibition following Bacterium tumefaciens- synonyms Agrobacterium tumefaciens (Rhizobium radiobacter), which is causal agent of crown gall disease (the formation of tumour) was studied by professor Simionescu and his coworkers (1956-1968).

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How the plants are infected? Crown gall is caused by the bacterium

Agrobacterium tumefaciens. This soil-borne bacterium enters the host plant through wounds caused by planting, cultivation, frost heaving, insects or nematodes. The bacteria, upon attaching to the plant cell walls, send DNA that causes production of plant growth hormones into the plant cell where it is incorporated into the plant cell chromosome. Affected cells begin to multiply at an uncontrolled rate, resulting in visible tumors within 2-4 weeks.

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Induction and evolution:• 1. Cr. I. Simionescu K voprosu o podavlenii protsessa rezvitiia rastitel’nîh opuholei-

Usvannih Bacterium tumefaciens Bul. Inst. Polit. Iaşi, Serie nouă, 1959, 5, 155-164.• 2. Cr. I. Simionescu, Elena Calistru, Natalia Simionescu Issledovanie nekotorâh himiceskih prevraşcenii vo vremia razvitiia

opuholei vîzvannîh Bacterium tumefaciens Rev. Roum. Chim., 1961, 6, 235-243. Chemistry:• 3. Cr. I. Simionescu, Margareta Grigoraş Contribuţii la chimismul tumorilor vegetale St. Cercet. Şt. Chim., 1956, 7, 145-148.• 4.Cr.I. Simionescu Considerations on some results obtained in the study of vegetable

cancer Buletinul Institutului Politehnic din Iasi, IV(VIII), Fasc. 3-4, 1958, 203--216• 5. Cr. I. Simionescu Cercetări asupra ligninei extrase dintr-o tumoare de fag St. Cercet. Şt. Chim., 1959, 10, 303-309.

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Chemistry:

• 6. Cr. I. Simionescu, Natalia Simionescu Cercetări privind transformările chimice într-o tumoare la

Prunus cerasifera var Mirabolana St. Cercet. Şt. Chim., 1961, 12, 234-239.• 7. Cr. I. Simionescu, Elena Calistru Studiul unor transformări chimice în timpul dezvoltării

tumorilor produse de Bacterium tumefaciens. II. Cercetarea cromatografică a zaharurilor

St. Cercet. Şt. Chim., 1961, 12, 227-234.• 8. Agata Cernătescu-Asandei, Cr. I. Simionescu Asupra procesului de lignificare în tumori Crown-Gall• St. Cercet. Şt. Chim., 1962, 13, 129-140. 9. Agata Cernătescu-Asandei, Margareta Grigoraş, Cr. I.

Simionescu Dynamics and Structure of Main Chemical Compounds in

Normal and Tumoral Tissues Cellulose Chem. Technol., 1968, 1, 75-88.

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Inhibition:

•10. Cr. I. Simionescu, Elena Calistru, Natalia Simionescu, Marta Hrihorov Acţiunea antioxidanţilor asupra procesului de dezvoltare a tumorilor vegetale St. Cercet. Şt. Chim., 1961, 12, 241-249.•11. Margareta Grigoraş, Natalia Simionescu, Cr. I. Simionescu Frânarea procesului de lignificare a ţesuturilor vegetale cu ajutorul antioxidanţilor St. Cercet. Chim., 1964, 3, 535-537.•12. Margareta Grigoraş, Natalia Simionescu, Cr. I. Simionescu Inhibition of Lignification Processes in Vegetable Tissues by Means of Antioxidants Rev. Roum. Chim., 1964, 9, 487-489.

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Modification of chemical composition

• Lignification has an important role in host defence against pathogen invasion.

-content of lignin in normal tissue: 26.74%; in tumor 31.29%

-cellulose content decreases from 43.14% to 37.77 %

• Biosynthesis of lignin take place by a radical mechanism involving laccase

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Concept of lignification using either cis- or trans-monolignol

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Dimerization of two dehydrogentated coniferyl alcohol monomers

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• The bacterium transfers parts of plasmid DNA to plant. The Ti plasmid causes the plant’s cells to multiply rapidly without going through apoptosis, resulting in tumour formation similar in nucleic acid and histology to human and animal cancers.

• The mechanisms by which Agrobacterium inserts materials into human cells also by type IV system, is very similar to mechanisms used by animal pathogens to insert materials (usually Agrobacterium is an important topic of medical research as well).

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Inhibition of tumours• Tumour growth is inhibited by

antioxidants (see papers published by professor Simionescu et al.) and recent papers in which plyphenols are used in the model experiments based on different systems: vegetabale, human or microorganisms.

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• Prof. Simionescu et al. used

-hydroquinone, gallic acid and its derivatives propyl-, ethyl-, octyl- and dodecyl gallate, butylhydroxyanisol, norhydroxyguaiaretic acid to inhibit lignin biosynthesis in Solanum lycopersicum infected by crown-gall tumour.

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• Denham Harman Proposes The Free Radical Theory Of Aging. J Gerontol. 1956 Jul;11(3):298-300. Aging: a theory based on free radical and radiation chemistry.Institution: University of California – Berkeley

1956 Denham Harman proposed that free oxygen radicals produced in the body cause aging, and further stated that "reducing compounds" (what we would know call antioxidants) might represent a "chemical means of prolonging effective life" and that these molecules "might be of benefit in the field of cancer chemotherapy and nutrition."

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Polyphenols• Secondary metabolites (of the 8000 known

polyphenolic compounds, around 4000 are flavonoids)

Properties: antioxidants; prooxidants; anticancer

agents; apoptosis-inducing; antibacterial, antiparasite; anti-HIV activities; amelioration of cardiovascular diseases; improvement of endothelial function; modulation of gamma-glutamylcysteine synthase expression; improvement of health and survival on high –fat diet; colouring agents; chelating agents, modulators of plant development.

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Separation and characterization of compounds with biological activity

Raw material

Extraction

(II)

Chlorophyll and Carotenoid Pigments Extraction

(I)

Polyphenols

Fractionation

(I)Hemicelluloses

Fractionation

(II) CelluloseComposting

Bioremediation

Lignin

Acid hydrolysis/ enzymatic

Nano- and micro cellulose

NaOH solution

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• Valentin I.Popa -Hemicelluloses in pharmacy and medicine in Polysaccharides in medicinal and pharmaceutical application, Edited by Valentin I.Popa, Smithers, 2011

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• Valentin I.Popa, Lignin in biological systems in Polymeric biomaterials, 2 vol, Founding Editor: Severian Dumitriu, Editor: Valentin I.Popa, 2013, CRC Press

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Directions of studies:• Modulation agents of

carbohydrates metabolism in diabet.

• Regulation agents of physiological processes in plants, bioremediation;

• Modulation agents of metabolic processes in microorganisms and carbon sources as substrates

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Oxidized LDL platelet activation and atherosclerosis. LDL oxidation (A) and platelet activation (B) are two key events in atherogenesis, which lead to

the formation of the atherosclerotic lesion. These processes are interrelated, in that oxidized LDL can activate platelets (C), and activated platelets increase the

susceptibility of LDL to oxidation (D).

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• Liproteins and antioxidants.

LDL oxidation by macrophages is affected by both the LDL-associated antioxidants (vitamin E, carotenoids, polyphenols). and by the balance between cellular antioxidants [such as as glutathione (GSH)] and cellular oxygenase [such as NADPH- oxidase and lipoxygenases]. Oxidized LDL but not native LDL lead to the conversion of macrophages to cholesterol [cholesteryl ester and unesterified cholesterol]-laden foam cells

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Free radical attack of a polyunsaturated fatty acid group present in LDL

Catechin stabilization of the free radical protecting the polyunsaturated fatty acid

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Polyphenols are stimulators of mithocondrial biogenesis

• The mithocondria are the „power plant”of the cell, generating chemical energy by producing adenosine triphosphate (ATP), the body’s „energy currency”.

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Saccharomyces cerevisiae- model for antioxidants studies

• Yeast detects chemicals which damage DNA

Yeast is ideal in detecting cancerous chemicals because its DNA-damage response system is remarkably similar to human cells. It can also be used to detect other types of damage such as oxidative stress with high degree of accuracy.

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• Sirtuin or Sir2 proteins are regulate important biological pathways in eukaryotes.

• Sirtuins are implicated in influencing a wide range of cellular processes like aging, transcription, apoptosis, inflammation and stress resistance, as well as energy efficiency and alertness during low-calorie situations.

• Sirtuins can also control circadian clocks and mitochondrial biogenesis.

• The mithocondria are the „power plant”of the cell, generating chemical energy by producing adenosine triphosphate (ATP), the body’s „energy currency”.

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• Gallic and ascorbic acids, alcoholic extracts from grape seeds and spruce bark

• Absorption and metabolization of polyphenols

• Protection against oxidative stress determined by hydrogen peroxide and UV-irradiation

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Gallic acid; absorption and metabolization (a); survival ratio (b)

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Grape seeds extract; absorption and metabolization (a); survival ratio (b)

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Spruce bark extract; absorption and metabolization (a); survival ratio (b)

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• Many people take antioxidant products such as vitamins C and E or eat antioxidant-rich foods such as blueberries. Doing so may, however, be causing more harm than good, according to an idea proposed by Nobel Prize winner Prof James Watson.

• Chemotherapy killed cancer cells by causing reactive oxygen to form but its effect diminishes, Prof Watson said. The tumour cells fight back by releasing antioxidants to block the damage. “If you are going to cure it then you are going to have to get rid of the antioxidants.”

• He submitted a paper to the New England Journal of Medicine . It was refused the journal said “because we only publish facts not ideas”, Prof Watson said. “I guess they thought the idea was too controversial.”

• “Right now I am feeling slightly lonely,” he said.

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POLYPHENOLS ENCAPSULATION BY ELECTROSPINNING TO OBTAIN

SUBSTRATES WITH BIOLOGICAL ACTIVITY

Polyphenols (gallic, vanillic, syringic acids, catechin, spruce bark extract) were encapsulated in nanofibrous membranes, using biocompatible polymers: [poly (2-hydroxyethyl methacrylate (pHEMA), poly [(lactic acid)-co-(glycolic acid)] (PLGA)

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Electrospinning process

• This process involves the ejection of a charged polymer fluid onto an oppositely charged surface• Nanoscale fibers, highly porous meshes, tunable fiber diameter• Multiple polymers can be combined, stacked layer system• Controlled fiber morphology

Fib

er mat

Taylor con

e

PLGA fibers

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Encapsulation of polyphenols by electrospinning

pHEMA- 1% vanillic acidThe average fibers diameters

FTIR spectra of pHEMA, vanillic acid and vanillic acid5% loaded pHEMA nanofibers

pHEMA- 2.5% vanillic acid

pHEMA- 5% vanillic acid

Main interaction: hydrogen bonds

between polyphenols and polymer

Polyphenols Diameters of fibers (µm)

content

(% w/v) gallic acid vanillic acid syringic acid catechin

1 0.98±0.14 0.59±0.07 0.69±0.27 0.93±0.19

2.5 1.18±0.16 0.81±0.08 0.76±0.21 1.57±0.42

5 1.25±0.28 0.89±0.19 0.93±0.25 1.93±0.52

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Release study from pHEMA nanofibers

- PBS, 37ºC-Swelling pHEMA-Burst release

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Antioxidant activity of free and encapsulated polyphenols

initial time after 30 minutes of reaction

Mechanism DPPH

Violetantioxidant

Yellow

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Encapsulation of catechin in W/O emulsionDispersed phaseCatechin in distilled water (aqueous phase)• antioxidant, antiradical, anti-inflammatory, antibacterial, antiviral, and antiaging effect

Continuous phasePLGA and SPAN 80 in chloroform (oil phase)• Biodegradable• Biocompatible Catechin structure

Flow rate: 15 µL/minVoltage: 15 kVDistance :15 cm

Continuous phase

Dispersed phase

stirring 227 nm

75 nm

Core sheath structure

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The influence of Poly Lactic-co-Glycolic Acid (PLGA) concentration in chloroform on the fibers

morphology*

CH3

O

O

O

O

*

x y

PLGA

10 % w/v 15% w/v

17 % w/v 20% w/v

Flow rate:10 µL/minVoltage:15 kVDistance: 15 cmT=22ºC±1RH=40 %

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Cumulative release of catechin from fibers

Release medium: PBSTemperature: 37 ºC‘linear release kinetics’

Membrane of PLGAobtained in 30 min.

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After 14 days in PBS

After one month in PBS

Fibers before immersion in

PBS After 3 days in PBS

After 1 days in PBS

Degradability of PLGA fibers after release measurement

Table 1 GPC analysisPLGAL/G:75:25

Mw (Dalton)

Mn (Dalton)

Mw/Mn

(Polydispersity)

Diameter of fibers (µm)

Initial 85.2 48.9 1.74 0.85±0.23

After 2 weeks

75.7 41.9 1.80 1.93±0.56

After one month

73.3 41.8 1.75 -

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A549 cell culture

A549 originates from an explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male.

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DCF-

DCFH-DA

DCFH

DCFH-DA

esterase

• DCFH/DA diffuses through the cell membrane where it is enzymatically deacetylated by intracellular esterases to the more hydrophilic nonfluorescent reduced dye dichlorofluorescein.

• In the presence of reactive oxygen metabolites, DCFH is rapidly oxidized to DCF.

• DCF, excited with 485-495 nm and emission at 517- 528 nm.

2′,7′- Dichlorodihydrofluorescein diacetate (DCFH/DA) assay

100 µm

A 549 cells

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DCF 30 minutes

0100200300400500600700800

20µg/mlMWCNT

100 50 25 12.5 6.25 3.12 1.5 0.78

Concentration of catechin (%)

Flu

ores

cen

ce 4

85n

m

[MW

-bla

nk

s]

no catechin 0.25% catechin 0.5% catechin

DCF 10 minutes

0100200300400500600700800

20µg/mlMWCNT

100 50 25 12.5 6.25 3.12 1.5 0.78

Concentration of catechin (%)

Flu

ores

cen

ce 4

85n

m

[MW

-bla

nk

s]

no catechin 0.25% catechin in PLGA fibers 0.5% catechin in PLGA fibers

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DCF 60 minutes

0100200300400500600700800

20µg/mlMWCNT

100 50 25 12.5 6.25 3.12 1.5 0.78

Concentration of catechin (%)

Flu

ores

cen

ce

485n

m [

MW

- b

lan

ks]

no catechin 0.25% catechin in PLGA fibers 0.5% catechin in PLGA fibers

DCF 120 minutes

0100200300400500600700800

20µg/mlMWCNT

100 50 25 12.5 6.25 3.12 1.5 0.78

Concentration of catechin (%)

Flu

ores

cen

ce 4

85

nm

[M

W-b

lan

ks]

no catechin 0.25% catechin in PLGA fibers 0.5% catechin in PLGA fibers

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ROS reduction in percentage for 10 minutes

020406080

100120140

20µg/m

l MW

CNT 100 50 25

12.5

6.25

3.12 1.5 0.7

8

% of conditioned buffer

Flu

ores

cenc

e 48

5 nm

[M

W-b

lank

s]

no catechin 0.25% catechin 0.5% catechin

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Vanillic acid 0.5 mg/mL Spruce bark extract 0.1 mg/mL

MW-blanks Polyphenols 10min

050

100150200250300350

20µg/mlMWCNT

100 50 25 12.5 6.25 3.12 1.5 0.78

% of conditioned buffer

Fluore

scence

485nm

[M

W-bla

nks]

MW-blanks Polyphenols 10min

050

100150200250300350400

20µg/mlMWCNT

100 50 25 12.5 6.25 3.12 1.5 0.78

% of conditioned buffer

Fluore

scen

ce 48

5nm

[MW-

blank

s]

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CONCLUSIONS• The antioxidant properties of

polyphenols was proposed by prof. Simionescu and his coworkers who demonstrated their capacity to inhibit vegetal tumour development.

• Polyphenolic compounds can be obtained from secondary resources or from cultivated plants using biorefining.

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CONCLUSIONS• There are a lot of polyphenols with different

structure and composition depending on raw material.

• Polyphenols are characterized by many properties (antioxidants, antifungal and antibacterials, preventing and inhibiting agents of cancer, protection agents of vascular system) which recommend their utilization in different biological systems.

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CONCLUSIONS• The biological properties of polyphenols

can be studied using different model systems: plant, animal and human cells and microorganisms (yeast).

• The polyphenols can be immobilized by electrospining. The resulted membrane showed no loss of antioxidant capacity and can be used to study biological activity for tissue engineering applications.

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• Daniela Jitaru

• Ioana Ignat

• Mihaela Danaila• Roxana Ghitescu-Sciex program-

cooperation Romania-Switzerland

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Thank you for your attention!

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• The grape polyphenolic extract affects the duration of fermentative process, depending on concentration used.

• For small quantities of vegetal extract (10-70 mg/L) added on fermentative media the duration of pre-fermentation phase is the same as in case of control samples for the two yeast strains used. At 140 mg/L vegetal extract used the duration is greater with 33.3% and at quantities between 740-4240 mg/L extract by 160%.

• The duration of pre-fermentative phase does not depend on the yeast strain used.

Pre-fermentation phase for S. ellipsoideus (A2B) and S. oviformis (7.2)

NATURAL POLYPHENOLIC COMPOUNDS – MODULATORS OF YEAST FERMENTATIVE

ACTIVITIES

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• For yeast strains used, the duration of tumultuous and post-fermentative phase is identical with the duration of control samples for the extract added between 10 – 1240 mg/L (120 hours).

• In case of Saccharomyces ellipsoideus (A2B) the duration of this period is greater with 2 h for 1740 mg/L extract and 12 h for 4240 mg/L extract added on fermentation media, comparative with the same quantity of extract used on Saccharomyces oviformis (7.2.) yeast.

Tumultuous and post-fermentative phase for Saccharomyces ellipsoideus (A2B) and

Saccharomyces oviformis (7.2.)

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• For the wines obtained using both strains, in case of adding 10 to 30 mg/L vegetal extract in the grape juice, the color intensity was close

to the control sample, at a difference of only 10%. • For quantities, exceeding 30 mg/L the color intensity was higher compared with

reference sample. • Also, the color intensity was different in the case of the two yeast strains, Saccharomyces ellipsoideus (A2B) strain showing an increase up to

10% in case of the maximum extract dose, in comparison to the same dose used in the Saccharomyces oviformis (7.2) strain.

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

martor 10mg/L

20mg/L

25mg/L

30mg/L

35mg/L

70mg/L

140mg/L

740mg/L

1240mg/L

1740mg/L

4240mg/L

vegetal extract (mg/L)

colo

r in

ten

sity

S. ellipsoideus A2B S. oviformis 7.2

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• The reduction of pH and the temperature level for quantities from 10 to 70 mg/L sustain the existing data on the polyphenolic compounds degradation through acids forms intermediates and utilization of these structures in yeasts carbon

metabolism.

pH evolution in grape juice inoculated with S. oviformis (7.2.)

Temperature evolution in grape juice inoculated with S. oviformis (7.2.)

pH evolution in grape juice inoculated with S. ellipsoideus

Temperature evolution in grape juice inoculated

with S. ellipsoideus

The inhibitor effect of grape seed extract on yeasts showed through slight increase of the pH and temperature decrease, for the samples containing from 140 to 4240 mg/L vegetal extract.

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• The experimental data points to the role of natural polyphenolic compounds as modulators for the alcoholic fermentative processes, a role that is strictly dependent of the relation between the extract dose, fermentation medium and yeast strain.

• The mechanism of interaction can be exclusively physical, involving the establishment of weak and reversible interactions mainly between anthocyanins and yeast walls by absorption.

• Various yeast metabolites, such as pyruvic acid, and acetaldehyde, were shown to react with different classes of phenolics, suggesting that they offer an important way of stabilizing pigments during the maturation and ageing of wine.

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• Depending on the used dosage, the polyphenolic compounds obtained from the grape seeds influence the alcoholic fermentative process and therefore the quality of the obtained wines, by promoting the accumulation of higher quantities of total and non-tannins polyphenolic compounds and modifying their sensorial and organoleptic properties.

• In addition, a high quantity of biomass is formed by the use of the vegetal extract as a carbon and energy source by the yeasts, which is a fact shown also by both pH and temperature in dynamic evolution.

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Yeast Interactions with Anthocyanins

• Stimulation effect of anthocyanins on yeast biomass accumulation and on glucose consumption

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Carotenoids production by the yeast Rhodotorula:

Use of polyphenols as a carbon source• Stimulatory effect of

phenol on b-carotene ratio in cultivation of Rhodotorula glutinis K-501 which had been isolated from soil.

• The biosynthesis of carotenoids started in the growth phase and continued even after stationary or death phase.

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• It was observed that the cell mass and composition of carotenoids produced by R. glutinis K-501 was dependent upon the addition of phenol into medium.

• The ratio of b-carotene increased and that of torularhodin decreased with increasing phenol concentration.

• The b-carotene content increased up to 35% when phenol was added to culture media at 500 ppm.

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• Inhibition effect of grape seed extract on b-carotene ratio in cultivation of Rhodotorula 9.3.

• Stimulation effect of polyphenols on yeast biomass accumulation.

The HPLC chromatogram of yeast carotenoids extract

Yeast biomass accumulation in the presence of polyphenols

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• The study of the influence of polyphenols on Sacchaoromyces cerevisae development have practical implication in the field of alcoholic fermentation or in biomass obtaining when acid hydrolysis products are used as substrates.

• At the same time, this strain represent a model recommended to be used with the aim to obtain information about role played by the polyphenols in its lifespan when the microorganism is cultivated in the presence of different stress factors.

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1. Lignins as antibacterials

2. Lignins as antioxidants and photoprotectors

3. Lignins in reduction of carcinogenesis

4. Anti-HIV properties of lignins

5. Lignin as spermicide

Lignin

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Conversion of native lignin into lignophenol derivatives and control of their functionality

and hydrophilicity

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Lignins as antibacterials/Escherichia coli

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Influence of different lignin samples on pathogenic bacteria sorption (HL-hydrolysis lignin, Curan-

commercial kraft lignin-Borregaard Ltd)

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The influence of lignin on phytopatogenic

microorganisms

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Lignins as antioxidants and photoprotectors

• Inhibitory effect of different lignin solutions on haemolysis induced by AAPH. [2,2’-azobis (2-amidopropane) dihydrochloride] a peroxyl radical initiator. LG-lignosulfonates, BG –lignin from bagasse, SE lignin from steam explosion and CU- Curan a commercial lignin.

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Haemolysis and photohaemolysis of CPZ (chlorpromazine a photohaemolytic compound) in the

presence and absence of different lignins

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Relative ABTS-radical scavenging activity of lignin samples

• The ABTS+* [ABTS - 2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulphonate)] cation radicals were generated by an enzymatic system consisting of peroxidase

and hydrogen peroxide.

• He-hemp, Si-sisal, Ab-abaca, Ju-jute, Fl-1-flax, SW-Ls-1- lignosulfonate from softwood (Boresperse 3A),SW-Kr-1- kraft from softwood (Indulin AT), SW-Ls-2- lignosulfonate from softwood (Wafex P), Fl-2- soda flax (Bioplast), Fl-ox-soda flax oxidised, SW-Kr-2- kraft from softwood (Curan 100), SW-SF-1( soda from softwood (precipitated at high pH), SW-Kr-3-kraft from softwood, HW-organosolv (Alcell) from mixed hardwoods, SW-SF-2- soda softwood (precipitated at low pH), SW-Kr-4-kraft (Curan 2711P

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Relative chain-breaking antioxidant effect of lignin in lipid peroxidation

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Precipitated lignin 3mg/mL; melatonin 1µM; quercetin 1µM; commercial lignin 3 mg/mL.

SampleSuperoxide aninon Hydroxyl radical

Precipitated lignin 51.44 ±1.29 33.68±0.91

Commercial lignin 47.15±2.04 27.81±1.30

Melatonin 79.06±0.32 53.89±1.07

Quercetin 71.46±0.85 53.07±1.13

Inhibition percentages of superoxide anion and hydroxyl radical generation

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Antimutagenic activity of modified kraft spruce lignin against 4-nitroquinoline-N-

oxide

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LIGNIN

Macrophages are specialized phagocytic cells that attack foreign substances, infectious

microbes and cancer cells through destruction and ingestion

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Concluzii• Polyphenolic compounds can be

obtained from secondary resources or from cultivated plants using biorefining

• There are a lot of polyphenols with different structure and composition depending on isolation raw material

• Polyphenols are characterized by variable properties (antioxidants, antifungal and antibacterials, preventing and inhibiting agents of cancer, protection agents of vascular system, colorants and chelatants) which recommend their utilization in different biological systems