CHEMICAL REACTIONS IN GAS, LIQUID AND SOLID PHASES: SYNTHESIS, PROPERTIES AND APPLICATION pptx

This volume includes information about kinetics and mechanism of chemical reactions in different phases: classification of polymers in reactivity toward nitrogen oxides polluted atmosphe

C HEMISTRY R ESEARCH AND A PPLICATIONS

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This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services If legal or any other expert assistance is required, the services of a competent person should be sought FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS

L IBRARY OF C ONGRESS C ATALOGING - IN -P UBLICATION D ATA

Chemical reactions in gas, liquid, and solid phases : synthesis, properties,

and application / editors, G.E Zaikov, R.M Kozlowski

p cm

Includes index

ISBN 978-1-61668-906-3 (eBook)

1 Polymers Biodegradation 2 Composite materials Biodegradation I

Zaikov, Gennadii Efremovich II Kozlowski, R (Ryszard)

C ONTENTS

Chapter 1 Classification of Polymers in Reactivity Toward Nitrogen Oxides 1 

E Ya Davydov, I S Gaponova, G B Pariiskii, T V Pokholok and G E Zaikov 

Chapter 2 Influence of the Initiation Rate of Radicals on the Kinetic

Characteristics of Quercetin and Dihydroquercetin in the Methyl

L I Mazaletskaya, N I Sheludchenko and L N Shishkina 

Chapter 3 An Antioxidant from Hindered Phenols Group Activates Cellulose

Hydrolysis By Celloviridin in a Wide Concentration Range,

E M Molochkina, Yu A Treschenkova, I A Krylov and E B Burlakova 

Chapter 4 a-Tocopherol as Modifier of the Lipid Structure of Plasma

Membranes In Vitro in a Wide Range of Concentrations

V V Belov, E L Maltseva and N P Palmina 

Chapter 5 Supercritical Carbon Dioxide Swelling of Polyheteroarylenes

Inga A Ronova, Lev N Nikitin, Gennadii F Tereschenko and Maria Bruma 

Chapter 6 Inhibition of 2-Hexenal Oxidation By Essential Oils of Ginger,

Marjoram, Juniper Berry, Black and White Pepper 65 

T A Misharina, M B Terenina, N I Krikunova and I B Medvedeva 

Chapter 7 The Organophosphorus Plant Growth Regulator Melaphen

I V Zhigacheva, E B Burlakova, T A Misharina, M B.Terenina,

N I Krikunova, I P Generozova and A G Shugaev 

Contents

vi

Chapter 8 Antioxidant Properties of Essential Oils from Clove Bud,

T A Misharina, M B Terenina, and N I Krikunova 

Chapter 9 Specific Properties of Some Biological Composite Materials 91 

N Barbakadze, E Gorb and S Gorb 

Chapter 10 Properties and Applications of Aminoxyl Radicals

E Ya Davydov, I S Gaponova, G B Pariiskii, T V Pokholok, and G E Zaikov  

Chapter 11 Synthesis of Flexible Manufacturings for Phosphoric Industry

Waste Utilization Based on the Cals-Concept 155 

A M Bessarabov, A V Kvasyuk and G E Zaikov 

Chapter 12 Practical Hints on the Application of Nanosilvers in Antibacterial

S Dadvar, A Oroume and A K Haghi 

Chapter 13 The Nanostructure and Yield Process of Cross-Linked Epoxy

Z M Amirshikhova, G V Kozlov, G M Magomedov and G E Zaikov 

Chapter 14 Nanostructures in Cross-Linking Epoxy Polymers and Their

Z M Amirshikhova, G V Kozlov, G M Magomedov and G E Zaikov  

Chapter 15 The Degradation Heterochain Polymers in The Presence of

E V Kalugina, N V Gaevoy,  

K Z Gumargalieva and G E Zaikov 

Chapter 16 Quantum-Chemical Calculation of Olefins of Cationic

Polymerization Branching in -, - and  Position on Relations to

V A Babkin, D S Andreev, T V Peresypkina and G E Zaikov  

Chapter 17 Thermodynamics for Catalase and Hydrogen Peroxide Interaction 227 

A A Turovsky, A R Kytsya, L I Bazylyak and G E Zaikov

Chapter 18 Dr Rer Nat Wolfgang Fritsche – Scientist and Organizer of

International Science (Secretary General Rtd of Gesellschaft Deutscher Chemiker, Honorary President of Federation

G E Zaikov  

Chapter 19 Professor Victor Manuel De Matos Lobo on His 70th Anniversary 249 

Gennady E Zaikov and Artur J M Valente  

Contents vii

Chapter 20 The Scientist Who Outstripped His Time 251 

Revaz Skhiladze and Tengiz Tsivtsivadze  

Chapter 21 Prof Dr Ryszard Michal Kozlowski: Half a Century in Science

Gennady Zaikov 

Chapter 22 The Second International Conference on Biodegradable Polymers

G E Zaikov, L L Madyuskina and M I Artsis  

“If you are sixty (or more)

and you are not feeling any pain in your body

when you are getting up in the morning,

it means that you have passed away (already)”

Russian proverb

This epigraph is very correct for the Russian Federation today because the average lifespan of Russian men in this country is 57 years (Russian women are living on ten years more) It is statistical data Russian scientists are an exception Particularly, the majority of contributors of this volume are older then 60 and the editor of volume is older then 75

We can explain this exception (phenomena) if we take in account the next Russian proverb: “All illnesses are from nerves and only a small amount from pleasure” We expect that readers immediately are thinking about sex as a part of pleasure It is only partly right Alcohol, tobacco and narcotics should also be included in the “pleasure” part Unfortunately, the Russian people are world champions for drinking The average Russian man (including ladies, children and even babies) drinks 18 liters of pure ethyl alcohol (calculation done in pure alcohol) per year It is twice more than twice the critical amount (9 liters)

Russian scientists are again an exception because the majority of them have pleasure only

in the case of communication with SCIENCE!

Now we should remember the Kazakh (people living in the Asian part of former USSR) proverb: “If sixty years are coming the mind (brain, memory) will go back (to childhood conditions)” We expect that this proverb is also not correct for Russian scientists As evidence of this opinion you can read the chapters of this volume where the most part of chapters were prepared by scientists from Russian Research Centers and from Research Centers of former Republics (now independent states) of the USSR

It is now the right time to remember English proverb: “Please eat one apple every day and you will not need a physician” (it is a reverse translation from Russian to English) We do not know exactly if it is enough to eat one apple a day to be permanently healthy or not We

do know that positive emotions are in favor for good health We expect that this book can (should) give only positive emotions to readers and we are waiting for the opinions of readers

in this case

So, we should stop about proverbs and start about chemical science and application This volume includes information about kinetics and mechanism of chemical reactions in different phases: classification of polymers in reactivity toward nitrogen oxides (polluted atmosphere), influence of the initiation rate of radicals on the kinetic characteristics of quercetin and dihydroquercetin in the methyl oleate oxidation, an antioxidant from hindered

Gennady E Zaikov and R M Kozlowski

x

phenols group activates cellulose hydrolysis by celloviridin in a wide concentration range, including ultralow doses, α-tocopherol as modifier of the lipid structure of plasma membranes

in vitro in a wide range of concentrations studied by spin-probes, supercritical carbon dioxide

swelling of polyheteroarylenes synthesized in N-methylpyrrolidone, inhibition of 2-hexenal oxidation by essential oils of ginger, marjoram, juniper berry, black and white pepper, specific properties of some biological composite materials, the organophosphorus plant growth regulator melaphen as adaptogen to low moisher, properties and applications of aminoxyl radicals in polymer chemistry, synthesis of flexible manufacturings for phosphoric industry waste utilization based on the cals-concept, practical hints on application of nanosilvers in antibacterial coating of textiles, the degradation heterochain polymers in the presence оf phosphorus stаbilizers, quantum-chemical calculation of olefins of cationic polymerization and antioxidant properties of essential oils

The nanostructure and yield process of cross-linked epoxy polymers as well as nanostructures in cross-linking epoxy polymers and their influence on mechanical properties are discussed in this volume

Somebody asked Henry Ford: “Which car is better?” Ford answered: “A new one” No doubt a new car as well as new scientific information is better than old ones (only old cognac

is better than the new one) We took this idea into account in case preparation of our volume

So, this volume is a complete guide to the subject of kinetic and mechanisms of chemical reactions in gas, liquid and solid states The editors and contributors will be happy to receive from the readers some comments which we can use in our research in the future

Once, Agatha Christie was filling out a form There was a question: “What is your occupation” and she wrote “A married lady”

Of course Agatha Christie could write on any form whatever she wanted because everybody knew her

What is important for us is that in filling out any form we contributors could write with a clear conscience – “a scientist.”

Gennady E Zaikov

N.M Emanuel Institute of Biochemical Physics

Russian Academy of Sciences

4 Kosygin Str., Moscow 119334, Russia

Ryszard M Kozlowski

Institute for Engineering of Polymer Materials and Dyes

Torun, Poland

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G E Zaikov, R M Kozlowski, pp.1-10 ©2010 Nova Science Publishers, Inc

Chapter 1

E Ya Davydov, I S Gaponova, G B Pariiskii,

N.M Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia

The review is includies information about the kinetics and mechanism of interaction

of nitrogen oxides with carbon-chain polymers, rubbers, aliphatic polyamides and polyuretanes

Keywords: nitrogen oxides, reactivity, polymers, classification, kinetics, mechanism

The general review of influence of the pollutants on polymers has been presented by

Jellinek et al [1] Therein the characterization of reactivity of polymeric materials toward

aggressive gases is given The various polymers were used as films of 20 μ thickness The thickness is enough small to exclude in most cases the diffusion as the determining factor of the pollutant action The films were investigated under different conditions: 1) the pollutant action; 2) the oxygen action; 3) UV light action; 4) UV light and oxygen; 5) UV light, oxygen and pollutants For NO2, the exposure of samples was usually realized under the pressure of

15 mm Hg during 30 hours at 308 K However, in a case of nylon 66 and butyl rubber, the

NO2 pressure was lowered up to 1 mm Hg at during 30 min Polyisoprene and polybutadiene were exposed to NO2 during 5 min under a pressure of 1 mm Hg As a light source (λ > 290 nm), a mercury lamp was used The intrinsic viscosity of polymer solutions was measured before and after exposure of samples in the chosen conditions The rather high concentration

* N.M Emanuel Institute of Biochemical physics, Russian Academy of Sciences, 4 Kosygin Street, 119334 Moscow, Russia, Fax: (7-495)1374101, E-mail: pgb@sky.chph.ras.ru , chembio@sky.chph.ras.ru

E Ya Davydov, I S Gaponova, G B Pariiskii et al

2

of nitrogen dioxide in these experiences was used to be convinced that the certain effects can

be observed for a reasonable time

The polymers on the basis of their reactivity with respect to NO2 can be divided into two

main classes [1] The saturated polymers, for instance, polyethylene (PE) and polypropylene

(PP) belong to the first group, but nylon 66 is not included into this series The second group

covers elastomers Butyl rubber undergoes scissions of the main chain, and polybutadiene is

restrictedly cross-linked under the action of NO2 These elastomers have approximately the

same reactivity with respect to NO2 as to ozone All films exposed to NO2 become yellow,

and their IR spectra show that nitro groups enter into macromolecules In polyvinylchloride in

the presence of NO2, some decreasing the amount of chlorine along with the appearance of

nitro and nitrite groups are observed from IR spectra

It is the author’s opinion [1] that some estimations concerning influences of so low

concentration of nitrogen dioxides in an atmosphere (2⋅10−9 − 2⋅10−8 mol⋅l−1) on polymeric

materials can be obtained from the experiences with using concentrations of the gas of several

orders of magnitude higher The formulated assumption says that there is linear dependence

of the concentration effect of aggressive pollutants This means that the effect of aggressive

gases at low concentrations can be determined by the linear extrapolation of results obtained

under the influence of high concentrations The author pointed out that this procedure

contains an element of risk because scissions of macromolecules in some cases are not always

linearly decreased with the pressure reduction of the aggressive gas, but the rate of breaks can

change drastically at very low concentrations

The procedure of extrapolation was used for an estimation of the scission average number

Sunder the action of aggressive gases at concentrations of 1 − 5ppm within 1 hour [1] This

value is given by the equation:

n

DP

DP

where DPn,0 and DPn,t are lengths of macrochains at t =0 and t correspondingly On the

basis of these estimations, it was concluded that aggressive gases, for instance NO2 and SO2,

slightly effect on vinyl polymers in concentrations really available in polluted air Even in a

combination with UV light, the deterioration of these polymers is hardly noticeable

However, nylon 66 is quite subjected to the action of small concentrations of NO2 with

essential degradation

I.1 INTERACTION OF CARBON-CHAIN POLYMERS WITH NO 2

Pioneering studies of the reaction of nitrogen dioxide with polyethylene (PE) and

polypropylene (PP) have been carried out by Ogihara et al [2, 3] Using IR spectroscopy,

they have found that nitrogen dioxide cannot abstract secondary and tertiary hydrogen atoms

from PE and PP at 298 K It can only add to the vinylene and vinylidene units that are formed

Classification of Polymers in Reactivity Toward Nitrogen Oxides 3

in the synthesis of the polymers These reactions resulted in dinitro compounds and nitro

NO2

O2N

C CH

NO2ONO

(I.3)

(I.4)

At T > 373 K, nitro, nitrite, nitrate, carbonyl and hydroxy groups are formed in these

polymers The following reaction mechanism at high temperatures was proposed:

This scheme allows rationalization of the accumulation of the nitro groups, which

proceeds at a constant rate and autoaccelerated formation of nitrates, alcohols and carbonyl

compounds

However, it provides no explanation for S-shaped dependence of the accumulation of

nitrites

The activation energies for the NO2 addition to the double bonds of PE are 8-16 kJ⋅mol−1

The activation energy for hydrogen abstraction is within of 56 and 68 kJ⋅mol−1 for PE and 60

kJ⋅mol−1 for PP

At room temperature and at NO2 concentrations of 5.4⋅10−4 – 5.4⋅10−3 mol⋅l−1, the

characteristics of PE, PP, polyacrylonitrile and polymethylmethacrylate (PMMA) are changed

only slightly even if they simultaneously undergo to a combined action of NO2, O2 and UV

radiation [4] Reactions of NO2 with polyvinylchloride and polyvinyl fluoride resulted in a

slight decrease in the content of chlorine and fluorine atoms, respectively [1, 4]

E Ya Davydov, I S Gaponova, G B Pariiskii et al

4

In the temperature range of 298−328 K nitrogen dioxide (7.8⋅10−3−3.4⋅10−2 mol⋅l−1) can

abstract tertiary hydrogen atoms from polystyrene (PS) molecules to introduce nitro and

nitrite groups into macromolecules in result of subsequent reactions [1] This process

proceeds at low rates and is accompanied by chain scissions [1, 5, 6] The number of chain

scissions on time α(t) was determined from intrinsic viscosity using the equation (I.1) The

experiments have been carried out at the temperature range of 298-328 K According to

Jellinek, the dependence of the decrease in the degree of polymerization of PS on the

exposure time in NO2 has three linear regions: initial, middle and final A decrease in the

apparent degradation rate was observed in the middle region of the dependence Presumably

this was related to the association of the macromolecules in solution, which is due to the

effect of polar groups and can affect the results of viscosimetric measurements Subsequent

increase in the apparent degradation rate was attributed to the consumption of these

nitrogen-containing groups and to a decrease in the degree of association of the macromolecules PS

films were also simultaneously exposed to NO2 (1.1·10−4 mol·l−1) and light (λ > 280 nm) [6]

No polymer degradation was observed in the initial stage during 10 h Then chain scission

occurred at a constant rate

An attempt to determine quantitative characteristics of the ageing of PS and

poly-t-butylmethacrylate (PTBMA) under the action of NO2 has been undertaken by Huber [7] The

samples were exposed to a stream of air containing NO2 (2.5⋅10−6 – 3.7⋅10−5 mol⋅l−1) at 300 K

and simultaneously irradiated with light (λ>290 nm) The number of chain scission per 10000

monomer units α(t) can be described by the empirical equation:

where P and Q are constants This equation describes an autocatalytic process At Q → 0,

degradation occurs at a constant rate Autocatalytic process is more pronounced for thin films

Degradation of thin PS films under the same conditions occurs slower than that of the

PTBMA films and its autocatalytic nature is more pronounced

The autocatalytic path of degradation of PTBMA was associated [7] with the

photo-induced formation of isobutylene, which reacts with NO2, thus initiating free-radical

degradation processes of macromolecules The IR spectrum of PS exposed toNO2 and light

exhibits two bands at 1686 and 3400 cm−1 corresponding to the carbonyl and hydroxyl

groups, respectively The formation of nitrogen-containing products has not been observed in

both PTBMA and PS The following reactions have been proposed [7] in PS:

~ CH2−C(Ph)H ~ + NO2 → HNO2 + ~ CH2−C•(Ph) ~ (R1 •) (I.13)

Classification of Polymers in Reactivity Toward Nitrogen Oxides 5

R1 + NO2

R1NO2

R1ONO

(I.16) (I.17)

It is believed that the decomposition of hydroperoxides exposed to NO2 and light leads to

autocatalytic degradation of PS

I.2 INTERACTION OF RUBBERS WITH NO 2

Rubbers are much more susceptible to NO2 than the polymers containing no double

bonds First, this is due to the ability of NO2 to add reversibly to carbon-carbon double bonds

to give nitroalkyl radicals (reaction (I.2)), thus initiating free radical conversions of

elastomers Second, nitrogen dioxide is able of abstracting hydrogen atoms in β−position to

the double bond to give allyl radicals, which then recombine with NO2 [8] Depending on the

structure of the alkene, the reaction resulting in the formation of the allyl radical can be either

weakly exothermic or weakly endothermic For instance, the strength of the weakest C−H

bond in the structure CH2=C(CH3)CH2−H is only 314 kJ·mol−1 [9]

The exposure of polyisoprene and polubutadiene to nitrogen dioxide leads to both

degradation and cross-linking of macromolecules, whereas butyl rubber (BR) (a copolymer of

36% isobutylene and 54% isoprene units) only undergoes degradation [10] The detailed

study of the ageing BR exposed to NO2 (5.2⋅10−7 – 5.2⋅10−5 mol⋅l−1) alone, an NO2−O2

mixture and an NO2−O2 mixture plus UV light (λ > 280 nm) at 298−358 K has been

performed by Jellinek et al.[11, 12] IR spectra before and after the exposure of samples show

that the band at 1540 cm−1 of ~ C=C ~ bonds disappears, and the new band at 1550 cm−1

arises The latter belongs to nitro groups appearing as a result of addition to double bonds by

the reaction (I.2)

The chain scission process in BR causes by the following scheme:

E Ya Davydov, I S Gaponova, G B Pariiskii et al

6

] ][NO R [ ]

where n ' is a number of isoprene units in BR After integration of (I.23) taking into account

a stationary concentration of R•, the following equation for the degradation degree is derived:

] NO [ (

]

[

] NO [ ] ' [

2320

2031

k k n

t n

where [n ' ]0 and [n ]0are the initial concentrations of isoprene units and all units The

amount of double bonds remains practically constant because only a small number of those

are destroyed Really, only 1/50 of macromolecules of BR are subjected to scissions Taking

into account low concentrations of NO2, the linear dependence on time is obtained:

t

k exp

=

where kexpis the experimentally determined constant This constant is represented by the

following Arrhenius equation: kexp = 3 8 ⋅ 10−2e−7450/RT, h−1

The degradation of BR in a polluted atmosphere runs in three directions: 1) the action of

NO2 alone, 2) the action of O2, 3) the combined (synergetic) action of these gases The

general scheme of the process can be represented as follows:

Classification of Polymers in Reactivity Toward Nitrogen Oxides 7

~CH2C(CH3)=CHCH2CH2~ + O2 → ~CH2C(CH3)=CHCH(OO•)CH2~ + HO2 • (I.36)

~CH2C(CH3)=CHCH(OO•)CH2~ + RH → ~CH2C(CH3)=CHCH(OOH)CH2~ + R (I.37)

~CH2C(CH3)=CHCH(OOH)CH2~ + NO2 → ~CH2C(CH3)=C(NO2)CH(OOH)CH2~ (I.38)

BR is not sensitive to UV light (λ > 290 nm) alone Probably, UV light in the presence of

NO2 effects on nitro groups of macromolecules

I 3 INTERACTION OF NITROGEN DIOXIDE WITH ALIPHATIC

Polymers containing amide and urethane groups form a particular class of materials

sensitive to NO2 Jellinek et al [13, 14] showed that exposure of nylon-66 films of different

morphology to NO2 (10−5 − 2.6⋅10−1 mol⋅l−1) causes main-chain scission in the polymers The

degradation of nylon is a diffusion-controlled reaction Its rate and depth depend essentially

on the degree of crystalline of samples and on the size of crystallites The degradation is

accelerated in the presence of air and UV light in addition to NO2 The following mechanism

for the polymer degradation under the action of NO2 was proposed:

The degradation process is inhibited by small amounts of benzaldehyde or benzoic acid

It is believed that these compounds block the amide groups and that only a few of them, not

involved in hydrogen bonding, enter into the reaction:

E Ya Davydov, I S Gaponova, G B Pariiskii et al

of 10−3 mol⋅l−1 The number of chain scissions in the sol fraction (the degree of degradation) increases initially, then decreases and eventually increases again; however, the final degradation rate is lower than the initial one Exposure of the polyurethane films to NO2 is accompanied by release of CO2 The IR spectra of the films allow assessment of the consumption of NH bonds (ν = 3300 cm−1)

The reaction mechanism proposed [15, 16] involves the abstraction of hydrogen atoms from two types of structures, namely, a carbamate structure (A) and a tertiary amide structure (B):

Classification of Polymers in Reactivity Toward Nitrogen Oxides 9

According to the Jellinek, recombination of R1• and R2• radicals leads to cross-linking of the polymer chains, while decomposition of R1• radicals results in the degradation of macromolecules and the CO2 release Energetically, the decomposition of the R1• radicals seems to be improbable since this reaction results in the formation of terminal macroradical

R3•and a nitrene, which is a very reactive species On the other hand, the R1• decomposition reaction involving cleavage of C−C or C−O is more bonds produce no alkoxycarbonyl macroradicals R3•, which can undergo decarboxylation [16] Therefore, the ageing of polyurethanes in an NO2 atmosphere can be represented as follows [17]:

where i = 1−4

This scheme expresses the degradation accompanied by cross-linking of macromolecules, the consumption of NH groups of the polymer as well as the release of carbon dioxide upon degradation

The investigations performed earlier characterize in general the reactivity of polymers of different classes in their reactions with nitrogen dioxide However, mechanisms of free radical processes proposed on basis of the results considered are enough formal As a rule they take account of changing molecular weights and the composition of final molecular products of the nitration In connection with this, the study of structures of free radicals forming in primary and intermediate stages of polymer conversions attracts an especial interest Such researches allow drawing conclusions on the mechanism of initiation of free radical conversions dependent on nature of functional groups of macromolecules As is shown by ESR measurements, different stable nitrogen containing macroradicals are formed

on exposure of polymers to NO2 [17] The analysis of the radical composition from ESR spectra gives an opportunity for estimation of the polymer stability by the quite simple method [18]

E Ya Davydov, I S Gaponova, G B Pariiskii et al

10

[1] Jellinek H H D Degradation and Stabilization of Polymers New York: Elsevier,

1978

[2] Ogihara T Bull Chem Soc Jap 1963, 31, 58-

[3] Ogihara T., Tsuchiya S., Kuratani K Bull Chem Soc Jap 1965, 38, 978-

[4] Jellinek H H D The 2nd International Symposium on Degradation and Stabilization of

Polymers (Abstracts of Reports), Dubrovnik, 1978

[5] Jellinek H H D., Toyoshima Y J Polym Sci A-1 1967, 5, 3214-

[6] Jellinek H H D., Flajsman F J Polym Sci A-1 1969, 7, 1153-

[7] Huber A Diss Doktor Naturwiss Stutgart: Fakultät Chemie der Universität Stutgart,

1988

[8] Giamalva D H., Kenion G B., Church D F., Pryor W A J Am Chem Soc 1987,

108, 7059-7063

[9] Rånby B., Rabek J F Photodegradation, Photo-oxidation and Photostabilization of

Polymers London: Wiley, 1975

[10] Jellinek H H D., Flajsman F., Kryman F J J Appl Polym Sci 1969, 13, 107-

[11] Jellinek H H D., Flajsman F J Polym Sci A-1 1970, 8, 711-

[12] Jellinek H H D., Hrdlovič P J Polym Sci A-1 1971, 9, 1219-

[13] Jellinek H H D., Chandhuri A J Polym Sci A-1 1972, 10, 1773-

[14] Jellinek H H D., Yokata A R., Itoh Y Polym J 1973, 4, 601

[15] Jellinek H H D., Wang A T J W J Polym Sci., Polym Chem Ad 1973, 11, 3227-

[16] [Jellinek H H D., Martin F H., Wegener H J Appl Polym Sci 1974, 18, 1773- [17] Pariiskii G B., Gaponova I S., Davydov E Ya Russ Chem Rev 2000, 69, 985-999 [18] Zaikov G.E Success in chemistry and biochemistry, New York, Nova Science

Publishers, 2009

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G E Zaikov, R M Kozlowski, pp.11-20 ©2010 Nova Science Publishers, Inc

Chapter 2

Emanuel Institute of Biochemical Physics of Russian Academy of Sciences,

Moscow, Russia

The antioxidant activity of quercetin (Q) and dihydroquercetin (QH2) is studied by the methyl oleate autooxidation model at 323 K in thin layers, and also the antiradical activity of these substances is investigated by the initiated oxidation of methyl oleate at

333 K It is shown that the rate constant of the inhibition for Q is equal 1.7×105 dm-3 mol-1

s-1, that is 2.2 times greater than k ing for QH2 (7.9×104 dm3 mol-1 s-1) Under the autooxidation condition the inhibitory effectiveness of Q is also 1.6 times greater than that for QH2 The existence of the direct correlation between the induction period of the methyl oleate autooxidation in the presence of flavonoids and the Q and QH2

concentrations indicates their primary interaction with peroxyl radicals

Keywords: autooxidation, initiated oxidation, kinetics, flavonoids, methyl oleate

Flavonoids are the compounds within the group of the vitamin P and of interest as the biologically active substances with a wide spectrum of action Biological activity of flavonoids is associated with their ability to inhibit of oxidation processes by the reaction

* Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, 4 Kosygin st., 119334, Moscow, Russia e-mail: lim@sky.chph.ras.ru

L I Mazaletskaya, N I Sheludchenko and L N Shishkina 12

with reactive oxygen species, as well as free radicals [1-6] In addition to the use of flavonoids as drugs and the component of the biologically active additives for the stabilization of the food products, cosmetics, preparations, etc, from the air oxygen oxidation

is of the great practical interest, because the number permitted to be used for this purpose synthetic antioxidants is extremely limited

Many investigations are made to examine the antioxidant activity of flavonoids and their content in food product consistent by using the different methods Advantages and disadvantages of these methods are discussed in detail in review [7]

The analysis of the available literature data shows that the parameters characterizing the antioxidant activity as well as the rate constant of the antiradical activity of flavonoids obtained under different conditions substantially differ and depend on many factors, including the chemical structure of flavonoids and the oxidation model Thus, one would expect that the antioxidant activity of the two closest to the structure of flavonoids - quercetin (Q) and dihydroquercetin (QH2), the molecule structure of which is the presence or absence of the C2-

C3 double bond, will be little different, because their rate constants with oxygen radicals are close and equal 1.7×105 dm3 mol-1 s-1 and 1.5×105 dm3 mol-1 s-1, correspondingly [8] However, the effectiveness of Q is more 2 times greater than that for QH2, when it is determined by the NADPH- and CCl4-dependent lipid peroxidation in microsomal membranes of the rat liver8 On the contrary, the greatest antioxidant activity has QH2 that follows from the results of the comparative tests presented in the paper [9]

anion-As shown, the antiradical activity of flavonoids significantly changes depending on the oxidizing substrate and the oxidation conditions So, the rate constants of their interaction with peroxyl radicals obtained in the initiated oxidation reaction of diphenylmethane10 significantly higher than that obtained by methyl linoleate oxidation in the homogenous and micellar solutions [11] Besides, as noted in Ref 10 - 11, flavonoids did not behave as the classical antioxidants and the constant of their antiradical activity depends on the initial concentrations of both flavonoid and the oxidation substrate

To use flavonoids as stabilizers from the spontaneous oxidation of foods, cosmetics and medicinal agents, it is necessary to establish the regularities of their antioxidant action under the autooxidation conditions, which simulates the natural aging process taking place under the air oxygen action As known, it is the side-reactions of antioxidants (InH) play the most substantial role under the degenerate branching chain in the autooxidation process, they proceed with the participation of the initial molecules of antioxidants and products of their oxidative conversion and lead to loss of the effectiveness of the antioxidant action For this reason, there may exist substantial differences in regularities of the antioxidant action of these substances between the autooxidation reactions and reactions with the constant rate of the free radical initiation So, for example, the deviation from linearity is observed for the dependence

of the induction period on the initial concentration for the natural antioxidant α-tocopherol (TP) under the autooxidation conditions, and, in some cases, this dependence can be extremal12 On the contrary, the linear relation between these parameters was detected by both the initial oxidation and autooxidation for the hindered phenols, radicals of which are practically not consumed in side-reactions [13,14]

The aim of our research was to study the influence of the quercetin and dihydroquercetin concentrations on the effectiveness of their inhibitory action and the antiradical activity depending on the oxidation conditions of the same substrate – methyl oleate

Influence of the Initiation Rate of Radicals on the Kinetic Characteristics… 13

Autooxidation of methyl oleate is carried out by the atmospheric oxygen in a thin layer at

323 K The course of oxidation was followed by the accumulation of hydroperoxides (ROOH), the concentration of which was determined by iodometric titration The error of the ROOH concentration determination did not exceed 1.5% The antioxidant effectiveness was

evaluated by τ values, which were graphically determined from the kinetic curves of the

ROOH accumulation during the methyl oleate autooxidation in the absence and presence of InH As duration of the oxidation induction period (τ), we took the interval from zero to the perpendicular dropped on the X axis from point of intersection of the linear plots of kinetic curve of the ROOH accumulation: the initial oxidation rate within the induction period and the maximal rate of the ROOH accumulation

The initiated oxidation of methyl oleate in a mixture (1:1) with an inert solvent (chlorobenzene) was performed by the air oxygen at 333 K The reaction mixture containing methyl oleate, chlorobenzene and the initiator – dinitrile of azoizobutyric acid, placed in oxidative cell equipped with a magnetic mixer The reaction mixture was thermostated at 333

K, and then InH was injected and measured the kinetics of oxygen uptake by using the volumetric method The initiation rate and the InH concentration were varied The initial rate

of oxidation, as well as the value of the induction period τ, were determined from the kinetic

curves of oxygen absorption by method described in Ref 15 The interval from the beginning

of the experience to the point of intersection of two straight lines for which tg α1 = 2 tg α2 was

taken as τ in the presence of InH The first line is an extension to a straight of the oxygen

uptake, when the reaction rate is constant after the complete consumption of InH The second line is tangent to the kinetic curve of the oxygen uptake in the point, the reaction rate of which is twice less than that in the absence of inhibitor

The concentration of Q was determined spectrophotometrically at the wavelength λmax =

368 nm The formation of the intermediate product is recorded by the absorption spectrum at the wavelength λmax = 526 nm To prepare the InH solutions, their sample is dissolved in ethanol and then diluted by chlorobenzene The proportion of ethanol in the oxidative cell is not exceeded about 1.3 % (v/v) Methyl oleate was purified by vacuum distillation Dihydroquercetin and Quercetin (Sigma) were used without additional purification

The kinetic data were processed by KINS program given in Ref 16

RESULTS AND DISCUSSION

Methyl oleate is one of the most used model system for the analyzing the antioxidant properties of different individual InH and their mixtures, but the employment of the methyl oleate autooxidation in the thin layer is rare For this reason, in our research it was used the methyl oleate autooxidation in thin layer It is obtained that Q and QH2 were effectively inhibited this process (Fig 1) The dependence of τ on the initial concentration of these antioxidants is closely to linear in the studied range of concentrations The slope of dependences of τ on the [InH]0 concentrations for Q and QH2 is significantly different Besides, there is a need to note that the antioxidant activity of Q is 1.6 times greater than for

QH2 (Fig 1) The similar results were obtained during the oxidation of lard17: it was shown

L I Mazaletskaya, N I Sheludchenko and L N Shishkina 14

that τQ is 1.4 times greater then τQH2 Besides, our results are also consistent of the data presented in Ref 18 about the antioxidant activity of the two flavonoids – fisetin and fustin, the molecular structure of which is also characterized by presence or absence of the C2 – C3 double bond According to Ref.18, the IC50 values for fisetin and fustin during the NADPH-dependent microsomal lipid peroxidation are equal 20.8 and 66 μmol, correspondingly, e i their antioxidant activities differ about 3.2 times

Figure 1 Dependence of the induction period (τ) on the antioxidant concentrations during

autooxidation of methyl oleate 1 – Q; 2 – QH2; 3 – TP

Hence, the obtained results and the literature data point to the fact that the presence of the

C2 – C3 double bond in the C-ring caused the conjugation at all rings in the flavonoid molecule results in the increase of their antioxidant activity

In order to clear the behavior of flavonoids during the oxidation, a detail computer analysis of the kinetics of the methyl oleate autooxidation in the absence and presence of flavonoids was performed It was showed that, in all cases, the peroxide accumulation is well

described by the exponential law: [ROOH] = a×exp(kt), the correlation coefficients of which

are equal 0.99 – 1.0 Earlier the similar relationship was revealed for the methyl oleate autooxidation when the oxygen concentration provides the oxidation in the kinetic range14

The exponential index k, the value of which is proportional to the total oxidation rate14, is not reliable differ for Q and QH2 and is not depend on their concentrations: k = (5.3 ± 0.6)×10-2 h-

1 and k = (4.94 ± 0.04)×10-2 h-1 for Q and QH2, correspondingly Thus, the results of computations indicate the complete consumption of flavonoids within the induction period

The preexponential factor a of the kinetic curves of the peroxide accumulation, the values of

which are due to the rates of radical initiation and the chain propagation [14], is substantially less in the presence of flavonoids than the same during the methyl oleate autooxidation

Influence of the Initiation Rate of Radicals on the Kinetic Characteristics… 15

Furthermore, this parameter decreases with a growth of the flavonoid concentration (Fig 2)

As can be seen from Fig 2, the value a more significantly reduces during the methyl oleate

autooxidation inhibited by Q than that in the QH2 presence These data corroborate the higher antioxidant activity of Q as compared with that for QH2

Figure 2 Dependence of the preexponential factor a on the quercetin (1) and dihydroquercetin (2)

initial concentrations

Dependences of τ on the initial concentrations of flavonoids [Q]0 and [QH2]0 differ from those for the natural antioxidant TP obtained under the same conditions It can be seen from the comparison of data, which are presented in Fig 1 Unlike Q and QH2, the dependence of τ

on [TP]0 is nonlinear (Fig 1, curve 3) in the range of the studied concentrations TP inhibits

better the methyl oleate autooxidation compared with Q and QH2 at the low concentrations, where the contribution of side-reactions was negligible However, the most active of the investigated flavonoids Q provides the some longer induction period compared with TP already at [InH]0 = 2.5×10-4 mol dm-3 (Fig 1)

The inhibitory action effectiveness is above noted to depend on side-reactions involving the antioxidant One of these reactions is the interaction of InH with the molecular oxidation product – ROOH As known, TP has the antiperoxide activity decomposing the methyl oleate hydroperoxide already at room temperature [19] In this work the reaction of Q and ROOH was studied by the Q consumption under anaerobic conditions at 333 K It is established that

the consumption rate of Q (WQ) is independent on [Q]0 at [ROOH]0 = const in the range of Q concentrations providing the linear chain termination However, WQ increases with the growth in the [ROOH]0 concentration at the fixed initial concentration of Q, as can be seen in Fig 3, The results obtained indicate that Q does not practically interact with ROOH under experimental conditions The observed consumption of Q is due to its interaction with free

L I Mazaletskaya, N I Sheludchenko and L N Shishkina 16

radicals forming during the thermal decay of ROOH From the dependence of WQ on [ROOH]0 presented in Fig 3 the rate constant of the ROOH decay with the free radical

formation (k3΄) was calculated It’s value is equal k3΄ = WQ /[ROOH]0 = 2.8×10-7 s-1

Figure 3 Effect of [hydroperoxide]0 on the initial rate of the quercetin consumption

To compare the antioxidant action effectiveness of studied flavonoids with their

antiradical activity, the stoichiometric inhibition coefficient f and the fk7/k60.5 parameter,

where k7 and k6 are the rate constants of the antioxidant interaction with peroxyl radicals and the square recombination of peroxyl radicals, correspondingly, were measured by the model reaction of the methyl oleate initiated oxidation The kinetic curves of the oxygen uptake during the methyl oleate oxidation in presence of Q and QH2 have the particularly pronounced

induction period It can be seen in Fig.4, curve 1 for Q To calculate f, the τ values were measured from the kinetic curves of the oxygen uptake at the different initiated rates (Wi) and initial concentrations of InH These data are given in Table Then we made plot on the τWi –

f[InH]0 coordinates presented in Fig 5 It is seen that these dependences are linear (Fig 5)

From the slope of these straight lines the f values for flavonoids were calculated which are equal fQ = 2 ± 0.2 and fQH2 = 2,6 ± 0.3 Thus, despite the fact that the molecules of these flavonoids have the same number of OH groups there is a some difference among their

stoichiometric inhibition coefficients The f value for Q obtained from kinetic curves of the oxygen uptake is in good agreement with fQ = Wi/WQ = 2 calculated from the rate of Q

consumption (WQ) (Fig 4, curve 1΄) As observed, there is the formation of coloured

intermediate product with the maximal absorption at the wavelength λ = 526 nm during the initiated oxidation of the methyl oleate inhibited by Q However, the practically complete discolouration of the reaction mixture is observed by the induction period end (Fig 4, curve

1΄΄) This result may indicate that the coloured intermediate product formed in the inhibition

reaction also possesses the antioxidant properties

Influence of the Initiation Rate of Radicals on the Kinetic Characteristics… 17

τ W i × 10 4 , mol dm -3

[InH] 0 × 10 4 , mol dm -3

Figure 5 Plot of τWi versus [InH]0: 1 – Q, 2 – QH2; the methyl oleate initiated oxidation at 333 K

From the kinetic curves of the oxygen uptake at the initial step the rates of the inhibited

oxidation (Wing) were determined To calculate the rate constant of the Q and QH2 interaction

with peroxyl radicals of methyl oleate (k7), the following equation was used:

L I Mazaletskaya, N I Sheludchenko and L N Shishkina 18

ω = W0/Wing – Wing/W0 = fk7[InH]/(Wik6)0.5 = king[InH]/(Wik6)0.5,

where W0 is the oxidation rate of methyl oleate without additivies InH, Wi – the initiation rate

Since Wing is determined at t > 0, the concentration of InH is calculated by equation [InH] =

[InH]0 – tWi/f From the slope of straight lines presented in Fig 6 the values fk7k6-0.5 were calculated which are equal 55 and 25 (dm3 mol-1 s-1)0.5 for Q and QH2,respectively For

comparison, the fk7k6-0,5 parameter for the well-known synthetic antioxidant 4-methylphenol (BHT) obtained under the same conditions was calculated as fk7k6-0,5 = 4,5 (dm3 mol-1 s-1)0,5 which was a good agreement with published data about its smaller reactivity compared with flavonoids [10]

Figure 6 Plot of ωWi versus [InH]0: 1 – Q, 2 – QH2; the methyl oleate initiated oxidation at 333 K

Assuming k6 = 1×107 dm3 mol-1 s-1, king = f k7 were calculated as 1.7×105 and 7.9×104 dm3 mol-1 s-1 for Q and QH2, respectively The obtained value king for Q is satisfactory agreement

with king = 4.3×105 dm3 mol-1 s-1 calculated from the kinetic curves for oxygen uptake during the methyl liloleate oxidation (0,242 M in chlorobenzene), inhibited by quercetin [20] Some

difference in values k ing can be associated with greater reactivity of peroxyl radicals from the

methyl liloleate compared with that from methyl oleate From the values king the rate

constants of flavonoids with peroxyl radicals was calculated, which have values k7 = 8.5×104

and 3.0×104 dm3 mol-1 s-1 for Q and QH2, respectively The ratio of parameters characterizing the antiradical activity of Q and QH2 also shows that the presence of the C2 – C3 double bond

in the C-ring leads to a 2-fold increase in the inhibition constant It is a good agreement with the ratio = 2,6 which are calculated by the concentrations of flavonoids for 50% inhibition of lipid peroxidation from the half-wave potential of the first oxidation wave measured by flow-

Influence of the Initiation Rate of Radicals on the Kinetic Characteristics… 19

through column electrolysis and the octanol/water partition coefficient for fisetin and fustin presented in Ref 18

Thus, the data obtained allow us to conclude that Q has the highest rate constant

interaction with the peroxyl radicals (k7) which is 2,8 and more 10 times greater than that for

QH2 and BHT, respectively The antioxidant activity of flavonoids and also their antiradical properties is above note to depend substantially on the oxidation system [10,18,20] It is associated with the occurrence of the different side-reactions, the lipophilicity of flavonoids

or the hydrogen-bonding ability of solvents [10,11,18,20] Our results showed that the antioxidant activity of Q and QH2 correlates with the parameter fk7k6-0,5, characterizing their antiradical activity under the different conditions of the methyl oleate oxidation From the

data obtained, the magnitudes fk7k6-0,5 for Q and QH2 differ about 2 times while the inhibition action effectiveness for Q is 1.6 times greater than that for QH2 calculated from the dependence of τ on [InH]0 under the autooxidation condition The existence of the direct correlation between the induction period of the methyl oleate autooxidation in the presence of flavonoids and the Q and QH2 concentrations indicates their primary interaction with peroxyl radicals

Table Effect of initial rate of radicals on induction periods ( ) of the methyl oleate

oxidation in presence of antioxidants, temperature 333

[1] U Takahama: Photochem Photobiol 38, 363 (1983)

[2] U Takahama: Plant Physiol 71, 598 (1983)

[3] V.A Kostyuk, A.I Potapovich, S.M Tereshchenko, I.B Afanas΄Yev: Biokhimiya, 53

(8), 1365 (1988) (in Russian)

[4] Yu.O Teselkin, B.A Zhambalova, I.V Babenkova, G.I Klebanov, N.A Tyukavkina:

Biofizika, 41 (3), 620 (1996) (in Russian)

[5] M.R Cholbi, M Paya, M.J Alcaraz: Experientia, 47 (2), 195 (1991)

[6] W Bors, W Heller, C Michel, M Saran: Methods Enzymol., 186 343 (1990)

[7] V Roginsky, L.A Eduadro: Food Chem., 92 (2), 235 (2005)

[8] A.I Potapovich, V.A Kostyuk: Biokhimiya, 68 (5), 632 (2003) (in Russian)

L I Mazaletskaya, N I Sheludchenko and L N Shishkina 20

[9] S Ya Sokolov, N.A Tyukavkina, V.K Kolkhir, Yu.A Kolesnik, A.P Arzamastsev, N.G Glazova, V.A Zyuzin, A.I Baginskaya, V.A Babkin, L.A Ostroukhova: Patent

[15] N.M Emanuel, G.P Gladyshev, E.T Denisov, V.F Tsepalov, V.V Kharitonov: The testing procedure of chemical compounds as stabilizers of polymer materials Preprint

Chernogolovka, 1976, 35 p (in Russian)

[16] E.F Brin, S.O Travin: Chem Phys Reports 10 (6), 830 (1990)

[17] S V Antoshina, А.А Selishcheva, G.M Sorokoumova, E.A Utkina, P.S Degtyarev,

V.I Shvets: Prikladnaya biokhimiya i mikrobiologiya, 41 (1), 23 (2005) (in Russian) [18] B Yang, A Konani, K Arai, F Kusu: Analytical Sciences, 17, 599 (2001)

[19] L.I Mazaletskaya, N.I Sheludchenko, L.N Shishkina: Petroleum chemistry, 48, 105

(2008)

[20] P Pedrielli, G.F Pedulli, L.H Skibsted: J Agric Food Chem., 49, 3034, (2001)

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G E Zaikov, R M Kozlowski, pp.21-28 ©2010 Nova Science Publishers, Inc

Chapter 3

1N.M Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia

2D.I Mendeleev Russian Chemical-Technological University,

Moscow, Russia

For the first time, the effect of a synthetic antioxidant – an inhibitor of free-radical processes, phenosan, on the cellulase activity was studied

A considerable increase of product yield of microcrystalline cellulose hydrolysis by

celloviridin (a complex of cellulases from Trichoderma Viride) under the effect of a wide

range of concentrations of phenosan, including ultralow doses, not associated with its antioxidant properties was observed

The concentration dependence of the effect is complex and non-monotonic

Ultralow concentrations of phenosan increase the product yield similarly or greater than “usual” ones

When increasing the process efficiency by phenosan at different concentrations of celloviridin protein, it is shown that phenosan action (including ultralow concentrations) allows a considerable decrease of the amount of an expensive enzymic complex necessary for obtaining the target product

Keywords: antioxidants, hindered phenols, hydrolysis, cellulose, ultralow doses, wide

concentrations

E M Molochkina, Yu A Treschenkova, I A Krylovet al

22

While the population of the Earth increases and food resources based on non-food raw materials and, which is the most important alternative sources of energy are actively searched for, the interest to cellulose-containing substrates (CCS), which renewable resources are almost unlimited, increased Annually, about 1011 tons of plant biomass on the Earth is hydrolyzed by microorganisms’ enzymes, and released energy is equivalent to 640 billion barrels of oil Therefore, it is clear why CCS enzymic hydrolysis using cellulase complexes produced by microorganisms is considered to be the most perspective way for obtaining alternative fuel [1]

Beside searching for and cultivation of new microorganisms – cellulase complex producers, new approaches to increase the yield of end products under the effect of currently used enzymic preparations shall be developed

Celloviridin produced from Trichoderma Viride fungus in two variants – celloviridin

G20x and celloviridin G3x, is one of cellulase complexes used for CCS hydrolysis in Russia The preparations catalyze degradation of cellulose from a plant cell to oligosaccharides, mono- and disaccharides by enzymes of three types: endoglucanase (Е.С 3.2.1.4), cellobiohydrolase (Е.С 3.2.1.91) and beta-glucosidase (Е.С 3.2.1.21) Cellulolytic activity of celloviridin-V G20x equals 2000 ± 200 units/g The content of enzymic system components

in celloviridin G3x is not constant, and its activity equals 100-200, 300-370 or 500-600 units/g [2]

Celloviridin production from microbiological material is rather labor consuming and expensive It is of importance to find possibilities of increasing the action efficiency of cellulases of celloviridin complex and, thus, to reduce the amount of required celloviridin Celloviridin represents a complex set of components containing a biomass, in particular, having lipids subjected to peroxidation (LPO), which products may affect the enzyme activity

To regulate LPO intensity in biological and chemical systems, antioxidants (AO), including free-radical reaction inhibitors from the group of hindered phenols, are used Recently, it has been found that AO of this type relate to substances, which ultralow doses (ULD) affect biological systems of different complexity [3]

In view of possible increase of process efficiency, we consider perspective to study the effect of a wide range of AO concentrations from the class of hindered phenols – phenosan K (potassium salt of beta-(3’,5’-ditert.butyl-4’-hydroxyphenyl)propane acid), on cellulose hydrolysis by celloviridin complex

Phenosan in the form of industrially produced acid is used in cattle breeding by inducing the growth-stimulating action in the forage composition for the farm livestock (chicken broilers, veals) It is assumed that phenosan acts as a bioantioxidant, preventing oxidation of fat-soluble vitamins A and E in the combined fodder composition [4 - 6]

Being the inhibitor of free-radical oxidation, phenosan may stabilize celloviridin preparation, because the latter may contain biomass components, lipids, in particular, capable

of easy oxidation, giving products capable of damaging enzymic proteins

However, for us the main precondition of phenosan use is the fact that this substance is one of the agents in ULD affecting various biological systems [3] In particular, it is known as

a superactivator of enzymic activity, this effect being not associated with antioxidant

An Antioxidant from Hindered Phenols Group Activates Cellulose Hydrolysis… 23

properties This fact is described in ref [7], which shows that injection of this preparation to smooth muscle cells of mouse aorta in the cellular culture in 10-18 M concentration induces protein kinase C activation by 400%

The aim of this work was to determine the presence and to estimate the level of phenosan

K effect on cellulose hydrolysis by celloviridin complex, using a wide range of AO concentrations, including ultralow doses

The tasks included:

1 Determination of microcrystalline cellulose (MCC) hydrolysis products yield - total sugars and glucose - in different periods after initiation of the process, carrying out the reaction in the presence of various phenosan concentrations, including ultralow doses

2 Investigation of phenosan effect on the target product yield at different concentrations of celloviridin

In the work microcrystalline cellulose (MCC) by Merck Company (Microcrystalline Cellulose, Charge Nr.: 5010160202) was used; enzymic preparation celloviridin G3x by OAO (BK) “Vostok” was presented by Biotechnology Department, D.I Mendeleev RCTU The studies were performed on two celloviridin G3x samples taken from different batches of preparation (CV-1 and CV-2 samples), which demonstrated different cellulolytic activity estimated by the rate of total sugars accumulation with the help of phenol-sulfuric method [8, 9] Activity of CV-1 sample estimated by this method was by 65% higher than activity of CV-2 sample

To purify the “grout” of the initial celloviridin powder in the acetate buffer (pH) from components insoluble in the aqueous medium (filler, biomass residues) and to obtain samples containing cellulases soluble in the aqueous medium, the “grout” was centrifuged, and supernatant fluid was used in the work Protein concentration in supernatant fluid was determined by the Lowry method [10]

Synthetic AO phenosan K was synthesized in N.M Emanuel Institute of Biochemical Physics, Russian Academy of Sciences

The rate of cellulose enzymic hydrolysis was estimated by accumulation of total sugars, which quantity was determined by the phenol-sulfuric method [8, 9], and glucose, which concentration was determined by glucose oxidase test [11] using the assay kit “Novogluk-K, M”

The reaction was performed in round-bottomed flasks in the acetate buffer solution with

pH 4.5, containing 50 mg/ml of cellulose CV “solution” was added to the reaction vessels so that the final concentration of protein containing the enzymic complex equaled 0.15-0.62 mg/ml Flasks were suspended on a rubber hose strained over the thermostat bath so that their bottoms were dipped into the water The incubation temperature was 40°C Rather intensive and uniform mixing of the reaction mixture was provided by flasks swinging due to water motion in the thermostat To each flask solution of the substance under test in corresponding concentration or acetate buffer solution (in the control) was added The stock phenosan K

E M Molochkina, Yu A Treschenkova, I A Krylovet al

24

solution (10-3 М) was prepared by dissolving a weighed portion of the preparation in the acetate buffer solution with pH 4.5 The effect of phenosan was studied at its concentration in the reaction mixture ranged within 5⋅10-17 − 10-5 M, every time adding solutions of corresponding concentrations obtained by subsequent dilutions of the primary solutions by

100 times into reaction vessels

From celloviridin lipids were extracted and concentrations of total lipids and phospholipids were determined, using the guidelines [12]

The results were treated and graphically designed using the software Sigma Plot for Windows v 8 (demo)

RESULTS AND DISCUSSION

Celloviridin analysis performed demonstrated that the concentration of lipids in the solid preparation is 0.001±0.0001 g/g, and concentration of phospholipids (the main LPO substrates) is 0.1±0.01 mg/g (of about 1.2·10-4 М) For possible effect of their oxidation products on enzymic proteins in the solid celloviridin, this is rather high concentration, and addition of an antioxidant may stabilize potential cellulase activity of the powder

However, the aim of this work was to estimate the possibility of direct phenosan

participation in the enzymic process Therefore, it was added to the reaction mixture together with the homogeneous solution containing enzymic proteins, obtained from celloviridin powder (see Experimental)

Figure 1 illustrates the phenosan effect on the yield of products of MCC hydrolysis by celloviridin It shows the dependence of the amount of sugars formed on the antioxidant concentration in the incubation medium 30, 60 and 120 min after the process beginning Every column represents the average value obtained from three-four parallel measurements ±

As shown in the figure, this effect of phenosan in ULD is either comparable with its action in “usual” concentrations (10-7 - 10-5 М), or is much greater expressed (For “usual”

AO concentrations the concentrations are taken, in which AO from the group of hindered phenols are able to inhibit free-radical oxidation processes)

The glucose percentage in total sugars in the presence of phenosan either increased insignificantly compared with the control (except of a considerable increase from 13 to 23%

in the case of 30-minute incubation with 10-4 М) or even demonstrated a tendency to decrease This indicates that, apparently, the effect of phenosan mostly stimulates the stages associated with the pool of enzymes catalyzing formation of oligo- and disaccharides

An Antioxidant from Hindered Phenols Group Activates Cellulose Hydrolysis… 25

Figure 1 The yield of total sugars and glucose in different times from the incubation beginning in the presence of different phenosan concentrations CV-1 protein content in the incubation medium is 0.62 mg/ml Abscissa axis - common logarithm of phenosan concentration; ordinate axis – concentrations of total sugars (at the top) and glucose (at the bottom) in mg · ml-1

E M Molochkina, Yu A Treschenkova, I A Krylovet al

26

Figure 2.Phenosan effect on the initial accumulation rate and yield of glucose 2 h after the process beginning at different celloviridin (CV-2) protein concentrations in the reaction mixture At the right – phenosan concentrations used Arbitrary units – mg · ml-1· min-1· 104

Thus the expressed increase of the product yield of MCC hydrolysis by celloviridin complex under the effect of a wide spectrum of phenosan concentrations, including ultralow doses was observed Ultralow concentrations of phenosan increase the product yield similarly

or greater than “usual” ones

An Antioxidant from Hindered Phenols Group Activates Cellulose Hydrolysis… 27

Apparently, the observed action of the preparation, even in “usual” concentrations, is associated not with its antioxidant (antiradical) properties, but with direct effect on the enzymic system components

The results obtained allow a suggestion that the phenosan effect on productivity and weight gain of livestock (associated with its antioxidant activity) used in the agriculture may

be contributed by its activating effect on cellulases Apparently, ultralow dose may have more expressed effect than those currently used

At the next stage we studied the phenosan effect on the target product (glucose) formation in cellulose hydrolysis with different celloviridin concentrations In this part of the work, CV-2 was used Figure 2 shows results on the phenosan influence on glucose formation

at different CV-2 concentrations

As CV-2 protein concentration in the reaction mixture increased from 0.15 to 0.62 mg/ml, the initial reaction rate and glucose yield 2 h after beginning also increased All studied phenosan concentrations caused similar stimulating effect on these parameters From the Figure 2 one may calculate that in the presence of phenosan reaching of the initial rate of glucose formation equal to control requires almost twice less celloviridin; obtaining of the yield of glucose 2 h after the reaction beginning equal to control requires 1.5 times lower amount of the enzyme preparation Thus, the possibility to economize the expensive enzyme complex applied in the industry for CCS hydrolysis using phenosan to stimulate the process, including in ULD, is obvious

At this stage of investigation, we may not indicate what namely components of the studied enzymic system and stages are touched by the phenosan action, and what the mechanisms of its influence are Nevertheless, the fact of stimulating effect of this antioxidant

on the action of the complex cellulase system may be considered established As a prospect, this may be used for production of fuel and foodstuff from CCS Since cellulase complexes obtained from different microorganisms act by similar mechanisms, apparently, phenosan will also activate other cellulase complexes produced from microbiological materials more

effective than Trichoderma Viride

The authors are thankful to T Onishchenko (Murashova) and M Zakharov for great help

in the experimental work

[1] Rabinovich M.L.// Prikl Biokhim Mikrobiol 2006 V.42(1).P.5-32

[2] http://www.sibbio.ru/products/bird/celo_bird.php

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[8] Klesov A A., Chernoglazov V M., Rabinovich M L., Glazov M V., Adamenkova M

D // Biokhimia 1983 V.48(9) P.1411-1420 (in Russian)

[9] Pustovalova L M Laboratory Manual on Biochemistry Rostov-on-Don Phoenix

American Elsevier Pub Co Inc New York N.Y 10017 1973

[13] Molochkina E M., Ozerova I B // Radiats Biol Radioecol 2003 V 43(3) P 294-300

(in Russian)

[14] Treschenkova Yu A., Burlakova E B., Goloschapov A N // Radiats Biol Radioecol

2003 V 43(3) P.320-323 (in Russian)

In: Chemical Reactions in Gas, Liquid and Solid Phases… ISBN: 978-1-61668-671-0 Editors: G E Zaikov, R M Kozlowski, pp.29-43 ©2010 Nova Science Publishers, Inc

Chapter 4

N.M Emanuel Institute of Biochemical Physics RAS, Moscow, Russia

α-Tocopherol (α-TP) is an effective natural antioxidant and important component of biological membranes localized in the lipid bilayer and capable of changing their structural dynamic state For this reason, it was of interest to study α-TP effect in a wide range of concentrations (10-25 М - 10-4 М) on viscosity parameters and thermally-induced

structural transition of the lipid bilayer of plasmatic membranes of liver cells of mice in

vitro The changes of structural parameters, namely, the order parameter of surface and

micro-viscosity of hydrophobic regions of the lipid bilayer in membranes, were measured

on Bruker EMX ESR-spectrometer (Germany) by the spin probe method by use two stable nitroxyl radicals - 5- and 16-doxylstearic acids localized at the different depths in the membrane ~8 A0 and ~20 A0 correspondingly The “dose - effect” dependencies were nonlinear and polymodal, with statistically reliable increases of viscous parameters of the membrane in three ranges of α-TP concentrations: (1) in the range of traditional

“physiological” concentrations of 10-9 – 10-4 M; (2) in the range of ultra-low doses of 10

-17 – 10-9 М, and even (3) in the range of apparent concentrations or dilutions of 10-25 – 10

-17 М The mechanisms of the effect of α-TP in each of these ranges localized in the lipid bilayer are discussed By study the temperature dependencies of micro-viscosity value a new thermally induced structural transition has been found at “physiological” temperatures of 309 – 3130K for α-TP concentrations, including ultra-low doses, to which maxima on dose dependencies

* Corresponding Address to: N.P Palmina (Ph.D., D.Sci.), IBCP RAS, Kosygin str.4, 119334 Moscow, Russia