wood bark as valuable raw material for compounds with biological activity

Wood bark as valuable raw material for compounds with biological activity

Valentin I.PopaGheorghe Asachi Technical University of Iasi

Faculty of Chemical Engineering and Environmental Protection

Blvd. Mangeron No.71, Iasi, 700050, Romaniae-mail [email protected]; [email protected]

mailto:[email protected]

mailto:[email protected]

Structure of the bark• Bark is a highly

heterogeneous and chemically complex section of woody biomass. It is usually divided into the living inner bark and dead outer bark, representing 10-15 % of the total weight of the tree.

Component Softwoods HardwoodsWood Bark Wood Bark

Lignin 25-30 40-55 18-25 40-50Polysaccharides 66-72 30-48 74-80 32-45Extractives 2-9 2.-25 2-5 5-10Ash 0.2-0.6 Up to 20 0.2-0.6 Up to 20

Composition by mass of lignin, polysaccharide, extractive and ash in woods and barks. The non-extractive components are based on extractive-free material. Taken from (USDA, 1971)

• Bark contains useful products waiting for the right economic conditions or the development of satisfactory commercial processes

Solvent Typical substances removed in whole or part

Petroleum etherether, benzene,

chloroform

Alcohol, acetone, aqueous alcohol, aqueous acetone

Hot or cold water

Aqueous alkali

Acid hydrolysis

Terpenes and their derivatives, fats, waxes, free and wax acids and alcohols, sterols, resins.

Simple polyphenols and their glycosides, tannins, mono- and disaccharides (sugars).

Disaccharides, starch, gums, pectins, tannins, mucilages.

Phlobaphenes, phenolic acids, some bark lignin and hemicelluloses, suberin fragments.

Simple sugars and uronic acids derived from holocellulose, leaves residue of “lignin.”

Fractionation of bark

Chemical composition of barkChemical compounds,%

Softwood bark

Hardwood bark

Alcohol-benzene extractCold water extract

Hot water extractExtract with NaOH,1 %CelluloseLigninPentosansTannin

4.45-7.503.20-5.10

6.00-7.7031.40-50.0030.60-35.0032.70-39.7011.30-12.70

1.90-3.00

2.70-5.506.30-7.40

16.00-16.5028.40-29.0031.60-41.7019.00-30.0021.00-24.500 -

• C.I.Simionescu, V.I.Popa et al., Holzforschung und Holzverwertung,40(6), 136 (1988)

biochemicaldegradation

burningadditives

compost fodderlignocelluloses

SEPARATION

BARK

EXTRACTION OF SECONDARY COMPOUNDS

Rough mixture ofhemicelluloses + polyphenols

Hcell-OH

PF-OHHcell-OH

softwoods24-26%hardwoods15-20%

-furfural- galacotse- glucose- arabinose- xylose

PF-OH

phenolsubstitute(adhesives)

softwoods10-12%

hardwoods4-6%

- C6 phenols- C6-C1 phenolic acids- C6-C2 acetophenone- C6-C3 coumarone- C6-C1-C6 xanthone- C6-C3-C6 stilbens-(C6-C3)2 lignans

PF O CH2 CHO

CH2OH PF OH+

PF O CH2 CHO

CH2OH PF OH+

O CH2PF CH CH2 O

OH

PF OH prepolymer crosslinking

Obtaining phenolformaldehyderesins with vegetal alkaline extracts

Compound, g R1 R2 R3 R4 R5PhenolExtract from:hardwood barksoftwood barkFormaldehyde, 37 %ParaformaldehydeSodium hydroxide solution, 40 %

150

55

200 9

17

150

60

180 8

17

150

40

181 6

17

150

65207 12

16

150

70215 10

17

Shear strength of plywood glued (N/mm2) withmodified phenolformaldehyde resins

Plywood made of:

Fenoplac R1 R2 R3 R4 R5

3 veneerminimummaximum5 veneerminimummaximum

1.5 -

2.1-

1.82.6

2.73.0

1.82.4

2.22.8

1.62.6

--

2.42.8

2.73.0

2.42.6

2.52.8

Influence of addition of the alkaline extract from beech bark on the properties of wood fiber boards (Transversal, L-longitudinal)

Degree of resin substitution, %

Strength, kg/cm2

Density, Kg/cm3

Water absorption, %

Swelling, %

0

10

20

30

40

T 340L 340T 665L 568T 350L424T 430L 386T 505L 446

1000

1150

1100

1090

1090

30.00

13.70

23.30

23.68

23.90

17.5

12.5

12.8

11.5

15.2

Physico-mechanical properties of wood fiber board obtained at industrial level (compared

with standard level)Characteristics First quality Second

qualityExperimental values

Apparent density,kg/m3

Water absorption (after 24 h immersion), %Thickness swelling (after 24 h immersion), %Static bending strength, daN/cm2Internal transversal cohesion, daN/cm2

1000(+10%,-5 %)

30

18

400

8.38

1000(+10%,-5 %)

40

25

300

8.38

991-1021

12.55-17.29

13.80-15.60

327-424

8.63-12.80

Bark

Extraction (II)

ResinsExtraction

(I)

Polyphenols

Fractionation(I) Hemicelluloses

Fractionation(II) Cellulose

CompostingBioremediation

Lignin

Acid hydrolysis/ enzymatic

Nano- and micro cellulose

NaOH solution

Polyphenols• Secondary metabolites (more than 8000

compounds) 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

Polyphenols were tested in:• Seed germination• Plant cultivation• Bioremediation• Plant grafting• Tissue plant culture• Microorganism cultivation (carotenes

pigments obtaining, mutagenesis)• Modulation of sugars metabolism

(diabete and alcoholic fermentation) O.C. Bujor, I. A. Talmaciu, I. Volf, and V. I. Popa Biorefining to recover aromatic compounds with biological

properties, Tappi J.,14 (3) 187-193 (2015)

17

POLYPHENOLS ENCAPSULATION BY ELECTROSPINNING TO OBTAIN SUBSTRATES

WITH BIOLOGICAL ACTIVITY

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

Roxana-Elena Ghitescu, Ana-Maria Popa, Valentin I. Popa, Rene M. Rossi, Giuseppino Fortunato

Encapsulation of polyphenols into pHEMA e-spun fibers and determination of their antioxidant activities

International Journal of Pharmaceutics, 494, 278–287(2015)

• The immobilized polyphenols were tested with very good results to inhibit the reactive oxygen species produced by carbon nanotubes in the cells A549 originated from an explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male.

19

DCF 60 minutes

0100200300400500600700800

20µg/mlMWCNT

100 50 25 12.5 6.25 3.12 1.5 0.78

Concentration of catechin (%)

Fluo

resc

ence

48

5nm

[MW

- bl

anks

]

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 (%)

Fluo

resc

ence

485

nm

[MW

-bla

nks]

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

Hemicelluloses based products

21

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

Filtratediscarded

sodium chlorite oxidation

Residue (Holocellulose)

Extractive-free wood bark

Filtrate

(Pectic substancesand water-soluble

polysaccharides)* Yield – 12. 5 % Filtrate

(Mixture of pectic substances and acidic xylan)

Residue

Filtrate (Xylan, “glucan” and water-soluble galacto-glucomann)

Residue

Residue

Filtrate(Mainly glucomannan)

Yield – 2.5%

Residue (pure cellulose)

Yield – 30.3%

Three successive preparations with aqueous barium hydroxidePure glucomannan (alkali-soluble)Yield – 2.0%; Percent composition:Galactose -4; Glucose – 25; Mannose – 71.

*All yields reported in this scheme are based on dry extractive- free bark

Hot ammonium oxalate solution

10% (w/w) sodium carbonate solution

24% (w/w) Potassium hydroxide

Yield – 4.3%

Yield – 8.5%

17% (w/w)sodium hydroxide + 4% boric acid

Fig.1 Fractionation scheme for Extraction of Hemicelluloses from Bark of Engelmann Spruce

24% Potassium hydroxide Extract Uronic acid – 7.3; Galactose – 8.8; Glucose – 29.4;Mannose – 3.3; Arabinose – 8.8; Xylose – 42.5

Composition:(relative percent)

Precipitate Yield – 2.2 %

Filtrate (see fig. 3)

Uronic acid – 3.3; Galactose – 11.4; Glucose – 6.64;Mannose – 10.8; Arabinose – Trace; Xylose – 27.9


Insoluble Portion Yield – 0.3%

Soluble Portion Yield – 1.8%

Uronic acid – 2; Galactose – 10; Glucose –38;Mannose – 40; Arabinose – Trace; Xylose – 10


Uronic acid – 2; Galactose – 11; Glucose – 57;Mannose – 2; Arabinose – Trace; Xylose – 28


Three successivePrecipitations with Fehling’s solution

Aqueous Barium hydroxide (5%)

(This fraction consist mainly of the water-soluble galactoglucomannan ) (This fraction consist mainly of the heteropolymeric “glucan”)

Fig.2 Fractionation scheme for Extraction of Hemicelluloses from Bark of Engelmann Spruce: Resolution of 24% Potassium hydroxide Extract

Filtrate remaining after addition of Fehling’s Solution to Potassium hydroxide extract (See fig. 2)Yield – 6.0%

Uronic acid – 7.9; Galactose – 9.1; Glucose – 25.1; Mannose – Nil; Arabinose – 14.3; Xylose – 43.6


Insoluble PortionYield – 0.5%

Soluble PortionYield – 4.8%

Uronic acid – 10; Galactose – 12; Glucose – 15; Mannose – Nil; Arabinose – 33; Xylose – 30


Uronic acid – 7; Galactose – 20; Glucose – 25.1; Mannose – Nil; Arabinose – 10; Xylose -56


Soluble PortionYield – 4.5%

Small precipitate(Discarded)

Uronic acid – 7; Galactose –6; Glucose – 12; Mannose – Nil; Arabinose – 10; Xylose – 65


Aqueous barium hydroxide

CTA-OH + aqueous Sodium hydroxide

(This fraction consist mainly of the acidic arabinoxylan)

Fig.3 Fractionation scheme for Extraction of Hemicelluloses from Bark of Engelmann Spruce: Resolution of 24% Potassium hydroxide Extract (continued)

Reactions:• Acid hydrolysis• Enzymatic hydrolysis• Esterification• Etherification• Enzymatic modification (treatment

with laccase of hemicelluloses from annual plants allow obtaining gels)

Directions to use hemicelluloses: • ethanol-fermentation C6• polyols-2,3 butylene glycol-aerobic

fermentation• lactic, acetic, butyric acids-fermentation• fodder yeast (50 % proteins; 2-7 % fats-

vitamins) Candida utilis• xylitol- xylose reducing- sweetener• furfural-furfurilic alcohol,furan resins,

poly(amide) -4,6

Derivatives of de xylan:-acetates; -butyrates;-benzoates- extrusion agents for fatty acids -carboxymethylxylan- (surfactants, flocculants,

adhesives for paper coating);-eating packages;-xylan sulfate- (antiHIV, antitumor, antioxidant,

anticoagulant, antimicrobial, decrease of cholesterol);

-biofilms (xyloglucan/chitosan) –immobilisation of streptomicyn, antioxidants, antifungal and, antimicrobials agents, dyes, nutrients, packagings).

-arabinoxylans- emulsifying agents; thickening, food stabilisers, immunotherapy agents;

-4-O-methylglucuronoxylan-antitumor.

Advantages to use hemicelluloses in pharmacy, cosmetics and medicine:

- are accessible-are not toxic-can be chemically and enzymatically modified-are biodegradable-are biocompatible

Health benefic effects:-they improve lipids and minerals

metabolism;-they improve the function of colon and

assure protection against cancer;-they reduce the risk of heart diseases Examples:-regeneration of tissues-support for controlled delivery of drugs--gels for cells immobilisation

Cellulose based products

Nanocellulose-supermaterial• Eco-friendly• Lightweight• Ductile• Stronger than steel and Kevlar• The super-material is theoretically derived from plant

matter that has been reduced to small bit and pieces, and then purified by a homogenizer to remove non-cellulose components like lignin. The remaining cellulose fibers are finally separated and processed into a thick substrate that boasts of long polymers or crystallized structures. This ultimately results in what is termed as nanocrystalline cellulose or nanocellulose ‘paste’, an incredible material with flexibility, malleability, super-strength as well as low-impact credentials.

Uses of nanocellulose• Composites• Paper and boards• Food• Hygiene and absorbent products• Emulsion and dispersion• Oil recovery• Medical , cosmetic and phramaceutical

• V.I.PopaNanotechnology and nanocelluloseCeluloză şi Hârtie, 63 (4), 14-23 (2014)• V.I.PopaObtaining of nanocellulose (I)Celuloză şi Hârtie, 64 (1), 3-10 (2015)• V.I.Popa Obtaining nanocellulose (II)Celuloză şi Hârtie, in press

Nanofibrile

Schematic representation of (a) the homogenizer and (b) the

microfluidizer

Procedure for individualizing cellulose nanofibers by ultrasonication

Nanocellulose

1) High-strength yet lightweight body armor -

2) Low-impact (fuel efficient) yet super-durable vehicles

3) Medical usages

4) Bendable battery systems

5) Flexible electronic displays • -

6) Bio-fuel can be a by-product when ‘growing’ nanocellulose

• Sugars resulted in the hydrolysis pretreatments colud be used by fermentation to obtain biofuels or other valuable bioproducts, thus contributing to the efficiency of the porocess of nanocellulose fabrication.

Lignina

Lignin based products

Biological properties of lignin

1. Lignins as antibacterials2. Lignins as antioxidants and

photoprotectors 3. Lignins in reduction of

carcinogenesis4. Anti-HIV properties of lignins5. Lignin as spermicide

48

• Valentin I.Popa, Lignin in biological systems

in Polymeric Biomaterials, 2 vol, Founding Editor: Severian Dumitriu, Editor: Valentin I.Popa, 2013, CRC Press

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

Lignins as antibacterials/Escherichia coli

Influence of different lignin samples on pathogenic bacteria sorption (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 The are specialized phagocytic cells that attack foreign substances, infectious

microbes and cancer cells through destruction and ingestion

http://en.wikipedia.org/wiki/Microbes

CONCLUSION• Wood bark contains useful products

waiting for the right economic conditions or the development of satisfactory commercial processes.

• By applying the biorefining concept wood bark could be used to obtain compounds of high interest in the biological field.

Thank you for your attention!

Questions