BIOREFINERY AS A POSSIBILITY TO RECOVER

AROMATIC COMPOUNDS WITH BIOLOGICAL PROPERTIES

Valentin I. PopaTechnical University of Iasi

vipopa@ch.tuiasi.ro;vipopa15dece@yahoo.com

The life we receive is not short, but we make it so..Seneca

Benefits of renewable resourcesutilisation

1. Reduced dependence on imported fossil oil;

2. Reductions in greenhouse gas emissions;

3. Building on the existing innovation base to support new developments;

4. A bio-industry that is globally competitive;

5. The development of processes that use biotechnology to reduce energy consumption and the use of renewable materials;

6. Jobs and wealth creation;

7. The development of new, renewable materials;

8. New markets for the agriculture and forestry sectors, including access to high-value markets;

9. Underpinning a sustainable rural economy and infrastructure;

10. Sustainable development along the supply chain from feedstocks to products and their end-of-life disposal.

The US Department of Energy

defined the term biorefinery as:

“an overall concept of a processing plant where biomass feedstock is converted and extracted into a spectrum of valuable products” (US Department of Energy 1997)

Separation and characterization of compounds with biological activity of some plant species from spontaneous flora and by-

products resulted in agriculture and food industry

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

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.

Research and development focus on polyphenols is:

1. Polyphenols in plant-plant, plant-animal, plant-insects; plant-microbial and other aspects of ecological significance;

2. Polyphenols in everyday life (e.g. their properties on health effects in human nutrition and diseases prevention);

3. Advances in chemistry and biochemistry-chemical synthesis, biosynthesis, conformational analysis and mechanism of their action in biological systems;

4. Polyphenols as renewable sources of industrial chemicals.

Directions of studies:

• Modulation agents of carbohydrates metabolism in diabet.

• Regulation agents of physiological processes in plants;

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

Polyphenols as modulators of carbohydrate metabolism in diabet

• Extracts from grape seeds and spruce wood bark

Evolution of glicemy of Wistar rats: L1 animals with diabet induced with streptozocin.L2 animals with induced diabet and polyphenolic protection

Concentration of total cholesterol in serum (Ch-T) for Wistar rats. M –reference, M+P reference treated with polyphenols; DZ+P animals with diabet and polyphenols protection; DZ animals with diabet

Serum concentration of triglycerides for Wistar rats. M –reference, M+P reference treated with polyphenols; DZ+P animals with diabet and polyphenols protection; DZ animals with diabet

Serum low density lipoprotein concentration for Wistar rats. M –reference, M+P reference treated with polyphenols; DZ+P animals with diabet and polyphenols protection; DZ animals with diabet

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).

Index of atherogenicity

• Liporoteins 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

Hepatic catalase activity for Wistar rats. M –reference, M+P reference treated with polyphenols; DZ+P animals with diabet and polyphenols protection; DZ animals with diabet

Glutationperoxidase (G-Px) activity in the liver of Wistar rats. M –reference, M+P reference treated with polyphenols; DZ+P

animals with diabet and polyphenols protection; DZ animals with diabet

Superoxid dismutase (SOD) activity in liver of Wistar rats.M –reference, M+P reference treated with polyphenols; DZ+P

animals with diabet and polyphenols protection; DZ animals with diabet

Serum malonic aldehyde concentration for Wistar rats. M –reference, M+P reference treated with polyphenols; DZ+P animals with diabet and polyphenols protection; DZ animals with diabet

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

Catechin stabilization of the free radical protecting the polyunsaturated fatty acid

Miocard of diabetic rat (a) and miocard of diabetic rat protected with polyphenols (b)

Aorta of diabetic rat

Vascular lesions of diabetic rats

Liver of diabetic rat

Hyaline nodules and renal vascular lesions

Pancreatic fibrosis

• In case of animals protected with polyphenols, degenerative and inflamatory phenomena are associated with those of regeneration

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.

Saccharomyces cerevisiae- model for antioxidants studies

• Galic 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

Galic acid; absorption and metabolization (a); survival ratio

(b)

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

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

• 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."

• 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.

• One should never forget that the interpretation of “in vitro” antioxidant data to “in vivo” results requires great caution, or sometimes it is not possible at all.

• Bioactivity is no equal to positive health protective effect!

10 misconceptions about antioxidants

1. Antioxidants cure any disease

2. Antioxidants increase mortality

3. The more the better

4. At high doses, antioxidants become pro-oxidants

5. Any antioxidants will do

6. Theoretically, antioxidants cannot behave as such

7. Antioxidants status measure health

8. Once used, antioxidants are inactive

9. Natural antioxidants are superior

10.Antioxidants drugs do not work

• The Court of Justice of the European Communities (EC) defines medicinal products as those designed “to cover products whose pharmacological properties have been scientifically observed and which genuinely designed to make a medical diagnosis or to restore, correct or modifiy physiological functions”.

Separation and characterization of compounds with biological activity of some plant species from spontaneous flora

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

HEMICELLULOSES

• Xylitol- xylose reducing- sweetener

• Carboxymethylxylan- packages• Xylan sulfate (antitumoral, dyslipidemic

treatment)• Biofilms (xyloglucan/chitosan)-immobilisation

of streptomycin, antioxidants, antifungal, antimicrobials, colorings, nutrients etc.)

Arabinoxylans emulsions, thickenings, immunotherapy

-4-O-metilglucuronoxilan-antitumoral

Advantages to be used in pharamcy, cosmetics and medicine:

- accessibility- nontoxicity- can be chemically modified- are biodegradable- are biocompatible

Benefic effects on health:-Improve the lipid and mineral metabolism -Improve the functions of colon protecting it

against cancer-Decrease the risk of cardiovscular diseaseExamples:-regeneration of tissues (particularly

cartilages)-support for controlled drug delivery -gels for cells immobilization

- implants

- films

- microparticles

- inhalation and injections systems

- obtaining viscous liquids

- foods additives, thickening, emulsion, gel forming, adhesives, absorption agents

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

Cellulose and derivatives

• A. Blood purification- Hemodialysis (regenerated cellulose or

cellulose acetate) - Plasmapheresis -plasma separation

(membranes of cellulose acetate)- Separation of viruses (membranes

based on regenerated cellulose from copper-ammonia solution)

- Hemostaza (oxidized cellulose)

• B. Systems for controlled drugs delivery

-ethylcellulose

-microcrystalline cellulose

-carboxymethylcellulose

-hydroxyethyl-, hydroxypropyl- and hydroxypropylmethylcellulose

• Biomaterials based on cellulose and its derivatives

have been also used as:

• (1) diffusion-controlling membranes and membrane carriers for enzyme immobilization in biosensors

• (2) coating materials for drugs and drug-releasing scaffolds ;

• (3) in vitro hollow fibers perfusion systems and in other biomedical applications.

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

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

Lignins as antibacterials/Escherichia coli

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

commercial kraft lignin-Borregaard Ltd)

The influence of lignin on phytopatogenic

microorganisms

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.

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

presence and absence of different lignins

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

Relative chain-breaking antioxidant effect of lignin in lipid peroxidation

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

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

oxide

LIGNIN

Macrophages are specialized phagocytic cells that attack foreign substances, infectious

microbes and cancer cells through destruction and ingestion

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

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, foods, pharamceutical, medical and cosmetics fields.

• The observed positive effects require their standaridization and the establishment a correlation between structure-function and properties.

• Biorefining allow to separate also the other compounds (hemicelluloses, cellulose and lignin with valuable biological activities, determining the increase the efficiency of natural resources utilization.

These results have been obtained by the following PhD students:

• Irina Volf

• Ioana Ignat

• Daniela Jitaru (Cibotariu)• Mihaela Danaila

• Roxana Ghitescu

• Oana Bujor

THANK YOU VERY MUCH FOR YOUR ATTENTION!

NCF-0, NCF-5 and NCF-10 corresponded to pure PCL nanofiber meshes with GTP content of 0%, 5% and 10%, respectively, whereas CF-0, CF-5 and CF-10 stood for PCL/MWCNTs composite nanofiber

meshes with GTP content of 0%, 5% and 10%, respectively.

Electrospining of polylactic acid/tea polyphenols/antibacterial/Results of improved shake flask tests after shaking for 1h

against Escherichia coli (A, C, E) and Staphylococcus aureus(B, D, F) for PLA/TP composite films with different blend ratios;

(A, B) 100/0, (C, D) 90/10, and (E, F) 80/20