The biological and cytotoxic activity of fusicoccin: monograph 9786010438347

This monograph contains theoretical and experimental results for the preparation of fusicoccin, as well as the main area

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The biological and cytotoxic activity of fusicoccin: monograph
 9786010438347

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AL-FARABI KAZAKH NATIONAL UNIVERSITY

S. Azat, Z.A. Mansurov, R.L.D. Whitby

THE BIOLOGICAL AND CYTOTOXIC ACTIVITY OF FUSICOCCIN Monograph

Almaty «Kazakh university» 2018

UDC 541.183; 661.183; 631.811.98(477) LBC 35.50 A 99 Recommended for publication by the Academic Council (Protocol №1, 29.09.2018) and the Editorial and Publishing Council of al-Farabi Kazakh National University (Protocol №1, 11.10.2018) Reviewers: Professor N.G. Prikhodko (Almaty University of Power Engineering and Telecommunications) Doctor of biological science I.S. Savitskaya (al-Farabi Kazakh National university) PhD. G.T. Smagulova (al-Farabi Kazakh National university)

Azat S. А 99 The biological and cytotoxic activity of fusicoccin: monograph / S. Azat, Z.A. Mansurov, R.L.D. Whitby. – Almaty: Qazaq University, 2018. – 132 p. ISBN 978-601-04-3834-7 This monograph contains theoretical and experimental results for the preparation of fusicoccin, as well as the main areas of its application and use. The monograph is designed for a wide range of specialists in the field of chemistry, nanotechnology, agrochemistry and technology of production and processing of agricultural products, as well as students, undergraduates and Ph.D. doctoral students of relevant specialties.

UDC 541.183; 661.183; 631.811.98(477) LBC 35.50 ISBN 978-601-04-3834-7

© Azat S., et.al. 2018 © al-Farabi KazNU, 2018

CONTENTS 

  List of abbreviations --------------------------------------------------------- 5 Illustrations -------------------------------------------------------------------- 6 Acknowledgments ------------------------------------------------------------ 10 Introduction ------------------------------------------------------------------- 11 1. Fusicoccin, properties and application -------------------------------- 12 1.1. The properties of fusicoccin and its application as an Anticancer preparation ------------------------------------------------------ 12 1.2. Plants growth stimulators. Physiological activity and mechanism of action --------------------------------------------------------- 17 1.3. Natural small molecules and their use as an anticancer drug ----- 27 1.4 Fusicoccanes, an interesting group of active small molecules ----- 28 References 1 -------------------------------------------------------------------- 30 2. Carbon sorption materials ----------------------------------------------- 35 2.1. Activated carbon based on raw materials ---------------------------- 42 2.2 Characterization of pores in carbon materials ----------------------- 45 References 2 -------------------------------------------------------------------- 49 3. Obtaining of activated carbons based on vegetable raw materials and research methods ------------------------------------------ 54 3.1. Activation process of raw materials ---------------------------------- 54 3.1.1. Physical activation of raw materials ----------------------------- 55 3.1.2. Chemical activation ----------------------------------------------- 55 3.2. Methods of studying the physicochemical properties -------------- 56 3.2.1. Fourier – IR spectroscopy ---------------------------------------- 56 3.2.2. Method of scanning electron microscopy ----------------------- 56 3.2.3. The method of low-temperature nitrogen adsorption ---------- 57 3.2.4. Аutomated mercury porosimeter --------------------------------- 58 3.2.5. Sorption activity of methylene blue on activated carbon ------ 59 3.2.6. Chromatographic separation of fusicoccin ---------------------- 60 3.2.7. High performance liquid chromatography (HPLC) ------------ 62 3.2.8. The mass spectrometric analysis method ------------------------ 63 3.3. Synthesis of carbon sorbents and using for fusicoccin extraction, experimental results and their discussion -------------------- 64

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3.3.1. Computer modeling of activated carbon for fusicoccin ----- 64 3.3.2. Determination of specific surface area of samples of carbon materials Sorbtometre-M ----------------------------------- 65 3.3.3. Methylene blue adsorption -------------------------------------- 68 3.3.4. The investigation of physico-chemical properties of the obtained nanoporous carbon sorption materials. Selection of the optimum conditions for obtaining modified sorption materials---- 70 3.3.5. The study of sorption characteristics of carbon materials --- 72 3.3.6. The results of Fourier – IR spectroscopy of raw materials -- 84 3.3.6.1. IR spectroscopic analysis carbonized rice husk ---------- 86 3.3.6.2. IR spectroscopic analysis carbonized sorbents ----------- 87 3.3.7. Extract of phytohormone of fusicoccin containing components -------------------------------------------------------------- 88 3.3.8. Mass spectrometric analysis. Fragmentation of Fusicoccin using MS/MS -------------------------------------------- 89 3.3.9. Analysis of samples on сhromatogram ------------------------ 93 3.3.10. Determination of the chemical composition of the samples by liquid chromatography ---------------------------------------------- 94 Conclusion ---------------------------------------------------------------------- 98 References 3 -------------------------------------------------------------------- 98 4. Study of biological and cytotoxic activity of fusicoccin ------------ 99 4.1 The examination of the cytotoxic activity of the obtained fractions ----------------------------------------------------------------------- 99 4.2 Antimicrobial activity tests --------------------------------------------- 101 4.3 The study of the analgesic activity of naturally occurring substances and their derivatives -------------------------------------------- 102 4.4 Testing for the phagocytosis-stimulating activity ------------------ 104 Conclusion ---------------------------------------------------------------------- 105 References 4 -------------------------------------------------------------------- 105 5. Using fusicoccin as biostimulant (BS)---------------------------------- 106 5.1 Investigation of the activity of fusicoccin wheat using biotest ---- 106 5.2 Practical application of fusicoccin ------------------------------------ 108 5.3 The use of BS to increase the yield of agricultural crops ----------- 110 5.4 Sowing varieties of tomatoes and cucumbers in the greenhouse with PMF---------------------------------------------------------------------- 113 5.5 Results of laboratory and field experiments to assess the effectiveness of phytomicrofertilizer (PMF) on cereals ----------------- 124 Conclusion ---------------------------------------------------------------------- 130

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LIST OF ABBREVIATIONS  AC WSh RH AS IR PCM CM CMS SEM BET DFT BJH FC ICP HPLC VNM VWM NOM VOC VME MB IUPAC RNA DNA NMR GS-MS

– – – – – – – – – – – – – – – – – – – – – – – – – –

Activated Carbon walnut shells rice husk apricot stones infrared spectroscopy porous carbon material carbon material carbon molecular sieve Scanning Electron Microscope Brunauer, Emmett, Teller Density functional theory Method proposed by Barrett, Joyner, and Halenda Fusicoccin Institute of combustion problems High-performance liquid chromatography Volume of narrow micropores Volume of wider micropores Natural organic matter Volatile organic carbons Volume in mesopores Methylene blue International Union of Pure and Applied Chemistry Ribonucleic acid Deoxyribonucleic acid Nuclear magnetic resonance Gas chromatography–mass spectrometry

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ILLUSTRATIONS  Figures 1 Formula of fusicoсcin – phytotoxin of the phytopatogenic fungus Fusicoccum amygdali Del 2 Natural – zeatin cytokinin formula 3 Chemical structure of Fusicoccin-A, Cotylenin-A and Ophiobolin-A 4 Application of carbon materials 5 Classification of pore size 6 Scheme of chemical transformations during carbonization of cellulose 7 Identification techiques of pores in carbon materials 8 Setting the carbonization of vegetable raw materials 9 Installation for chemical activation of vegetable raw materials 10 Scanning electron microscopy 11 The device is low-temperature nitrogen adsorption autosorb-1 12 Device PoreMaster a mercury porosimeter 13 The apparatus UV detecter connected with column chromatography 14 High performance liquid chromatography 15 Microporous activated carbon and fusicoccin molecular 16 The optimal pore size of carbon for column chromatography 17 The calibration graph of dependence D for solutions of MB 18 The methylene blue value of activated carbon for adsorption ability 19 Electron micrographs of carbon samples from AS, treated with potassium hydroxide in a ratio of 1: 2 (a) and at a ratio of 1: 4 (b) 20 Electron micrographs of carbon samples from RH, treated with potassium hydroxide in a ratio of 1: 2 (a) and at a ratio of 1: 4 (b) 21 Electron micrographs of carbon samples from WSh, treated with potassium hydroxide in a ratio of 1: 2 (a) and at a ratio of 1: 4 (b) 22 Electron micrographs of carbon samples RH:H3PO4 (1:3) (а) and RH:H3PO4 (1:2) (b) 23 The pore size distribution of samples from apricot stones at 500 0C, according to the mercury porosimetry 24 The pore size distribution of samples from apricot stones at 600 0C, according to the mercury porosimetry. 25 The pore size distribution of samples from apricot stones at 700 0C,according to the mercury porosimetry 26 Pore size distribution of samples of rice husk, at 500 0C, according to the mercury porosimetry 27 Pore size distribution of samples of rice husk, at 600 0C, according to the mercury porosimetry 28 The pore size distribution of samples of rice husk, at 700 0C, according to the mercury porosimetry

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29 The pore size distribution of samples of rice husk, at 800 0C, according to the mercury porosimetry 30 The pore size of the carbon material from walnut shells 31 The isotherms of adsorption and desorption of nitrogen samples AS-500 32 The isotherms of adsorption and desorption of nitrogen samples AS-600 33 The isotherms of adsorption and desorption of nitrogen samples CRH-800 34 The isotherms of adsorption and desorption of nitrogen samples 35 The adsorption isotherm of nitrogen and water on the sample WSh 36 Differential pore size distribution of samples AS-500 determined by DFT 37 Differential pore size distribution of samples AS-600 determined by DFT 38 Differential pore size distribution of samples CRH-800 determined by DFT 39 Differential pore size distribution of samples of AS-500, AS-600, CRH-800 determined by the DFT 40 Differential pore size distribution of samples of AS-500, AS-600, CRH-800 determined by BJH method both graphs show the greatest number of pores with a diameter of 4 to 16 nm, which corresponds to small mesopores. 41 IR spectra of apricot stones before carbonization 42 IR spectra of apricot stones (Т=8000С) 43 The IR spectra of carbonized rice husk at 700 °C 44 IR spectra of walnut shells (t=800 0С) 45 Curves of chromatographic separation of fusicoccin on the column WSh-750 (a) and organic gel Octyl Sepharose CL-4B (b) 46 MS conditions for the detection of fusicoccin in full scan 47 Full scan spectra of fusicoccin in methanol ESI (+) 48 Full scan spectra of fusicoccin in methanol ESI (-) 49 Fragmentation (MS/MS) of the ion m/z 703.3 with the amplitude applied in the trap 50 Fragmentation (MS/MS) of the ion m/z 703.3 with the amplitude applied in the trap 51 Fragmentation MS3 of m/z 703 [fusicoccin+Na]+ and analysis of the resultant ions by product ion scan mode 52 Fragmentation MS3 of m/z 703 [fusicoccin+Na]+ (fragmentation route 2) and analysis of the resultant ions by product ion scan mode 53 4A spiked at concentration 0.5 ug/g 54 m/z 703 from the full scan analisys from the solid extract sample 55 Pure Fusicoccin’s peak 56 The fraction extract obtained by elution with 10% ethanol in the separation of 70% ethanol extract of milk-ripe wheat 57 The fraction obtained by elution with a 50% ethanol in the separation of 70% ethanol extract of milk-ripe wheat 58 Grains of beans for biotest 59 Bean seeds put into the cups 60 The plant poured with:a – solution of fusicoccin; b – only with water 61 Influence of a biostimulator on the germination of seeds of wheat variety “Nadezhda” in NaCl solution (2%) 62 Wheat field with a strip treated with a phytohormone solution (yellow ears)

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63 The effect of presowing treatment of sugar beet seeds on the development of the root, I-control, II-exprience 64 Material Safety Data Sheet 65 The variety of tomato with PMF. 1 day on 24 January, 2015 66 The variety of tomato with PMF Study of varieties of cucumbers using innovative technology 67 Varieties of cucumber with PMF on the 3-day, 30 January 2015 68 3 February 2015, cucumber 69 17 February 2015, tomatoes 70 The varieties of cucumbers 71 February 21, 2015 the variety of cucumber started rooting, and put down roots in the fabric 72 February 28, 2015 control with versatile fertilizer 73 Cucumbers and tomatoes 9 March, 2015 74 March 21, 2015 varieties of cucumbers have begun to bear fruit, in varieties of tomatoes (control) there began to appear the first flowers 75 PMF used for cucumbers and tomatoes 10 April 2015 76 PMF and Control biostimulant used for cucumbers and tomatoes 14 February, 2015 77 PMF and Control biostimulant used for cucumbers and tomatoes 28 March 2015

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Tables 1 Advantages and disadvantages are summarized on each characterization technique 2 The data on the specific surface area of the samples of carbon materials 3 The specific surface and sorption capacity of the non-activated carbon materials 4 The specific surface area of the activated carbon samples 5 Characteristics of carbon materials after activation 6 Data on the dependence of optical density on the concentration of MB 7 The methylene blue value of activated carbon for adsorption ability 8 The elemental composition of the samples of activated carbon materials 9 Textural properties of the samples according to data of nitrogen adsorption 10 Result in fractions after preparative separation 11 The cytotoxic activity of the samples A0, А1, А2, А3, А4, А5, А6, А7 pertaining to brine shrimp larvae Artemia salina (Leach) under cultivation conditions in vitro 12 The antimicrobial activity of the samples 13 The analgesic activity of naturally occurring compounds and their derivatives 14 The influence of the samples (0), (1), (2), (3), (4), (5), (6), (7) on the phagocytic activity of blood neutrophils, (М±m) 15 The effect of fusicoccin on yield and weight of 1000 grains of Steklovidnaya-24 winter wheat 16 The effect of BS on increasing the yield of wheat varieties "Steklavidnaya24" 17 Increasing the yield of winter rye varieties "Sholpan" 18 Effect of BS on increasing the yield of sugar beet 19 рН of activation stimulator 20 The results of experiments carried out in homes 21 The temperature and amount of precipitation before planting and during the growing season of crops (according to Kostanai HMS) 22 Results of laboratory evaluation of the physiological activity of PMF on cereals 23 The impact on the PMF spring wheat seed germination and root rot infestation of crops (Karabalyk ACES) 24 Influence of processing of seeds and wheat crops with chemicals and growth regulators on yield and its structure 25 Influence of processing of seeds and crops of wheat chemicals and inducers of resistance against the structure of complex diseases 26 The impact of drugs on the germination of barley seeds and seedling root rot infestation 27 Effect of processing barley seeds and crops on harvest and its structure 28 Structure of the barley harvest in the treatment of seeds and crops during the growing season

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ACKNOWLEDGMENTS   Thanks to: Colleagues in the Chair of chemical physics and materials science of Al-Farabi Kazakh National University; Colleagues in the “Institute of combustion problems”; Colleagues in the Open type National nanotechnological laboratory of AlFarabi Kazakh National University; Colleagues in the Brighton University (UK)

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INTRODUCTION  The problem of creation and use of sorption materials are current interest for the practice of the modern medicine and agriculture. The knowledge of physical and chemical rules of carbonization, activation as well as sorption and desorption processes is of particular importance in the case of application of the nanostructured carbon sorbent agent for high purification of water contaminated with pesticides, as well as for reducing the concentration of cytokines in the blood of sepsis patients. Practical importance is production of a biostimulant using a carbon sorbent for a significant increase in productivity, which is very relevant for the regions of Kazakhstan. As a result of the comprehensive analysis carried out in the work the biostimulant obtained by means of highly-porous carbon sorbent was used in field experiments to stimulate vegetative growth of plants and to form reproductive organs, to increase grain crops yield and their resistance to biotic and abiotic stresses. In addition, biological and cytotoxic activities of obtained fusicoccin open possibilities for their use as anti-cancer drugs. The practical relevance of the work was confirmed by the receipt of the passport safety of chemical production for “PMF” (phyto micro-fertilizer), approved by the Committee of Industry of the Ministry of Industry and New Technologies of the Republic of Kazakhstan from June 16, 2014 (RK-Chemical Production №0000288). The achieved results are aimed at the development of nanotechnology scientific bases in the field of biostimulators produced to improve food crops yield and to determine their biological and cytotoxic activities.

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 FUSICOCCIN, PROPERTIES    AND APPLICATION 

1.1 The properties of fusicoccin and its application as an anticancer preparation Fusicoccin was discovered in 1964 by the Italian scientist Alessandro Ballio as the phytotoxin of the phytopathogenic fungus “Fusicoccum amygdali Del”. This phytotoxin was able to kill young almond trees as follows: this toxin opened stomata of leaves which did not close after. In consequence of the excessive transpiration and because of the weakness of the root system young trees were dying rapidly from drought, i.e. fusicoccin is a natural desiccant. In the laboratory of Professor A. Ballio the structure of this phytotoxin was determined as is shown in Figure1. Considering the fact that fusicoccin refers to natural terpenoids as well as the gibberellin Gronevald et.al. developed a hypothesis about the kinship of gibberellin and fusicoccin. Indeed, they both are diterpenoids, metabolites of phytopathogenic fungi. Over time, the number of known gibberellins increased rapidly from few in 1960 to more than eighty by the end of the 80s. The same for fusicoccins: now more than 15 related compounds of this group are known (denoted by literal characters – A, B, C, etc.) [1]. Preliminary data on the presence of substances of the fusicoccin family in higher plants were published first by Muromtsev and his co-wokers in 1980. Later for the detection of fusicoccin a gas chromatography, mass-spectrometry was used (GC/MS) with a preliminary fractionation of the material by the high performance liquid chromatography (HPLC). Initially, the authors have developed an identification method for fusicoccinal metabolites in the culture fluid of the fungus F. amygdali by the combined method of the gas chromatography and mass spectrometry of trisilil derivatives, when the detection is performed through several chosen characteristic ions. To obtain fusicoccin, the authors used corn cobs and cabbage leaves, 12

which were homogenized in ethanol. The alcohol extracts were concentrated. The concentrate was extracted by chloroform. The chloroform extract was subjected to the liquid chromatography. The obtained peaks were subjected to the gas-liquid chromatography followed by mass spectrometry. The content of the endogenous fusicocin A in plants was 10-11-10-12 M, which is 2-3 orders lower than that of the endogenous gibberellins. Hence it becomes clear that the problem of preparative quantities extraction of natural fusicoccin from higher plants is a very difficult experimental problem.

Figure 1 – Formula of fusicocin – phytotoxin of the phytopatogenic fungus Fusicoccum amygdali Del. [1]

Then, the same authors tried another shot to extract natural fusicoccin from higher plants. At this time, as the object for the fusicoccin extraction the authors used a sterile culture of transformed horseradish roots distinguished by the intensive growth. This objet was chosen for the following reasons. Firstly, it eliminates the possibility of contamination by microorganisms and fungi that may be sources of fusicoccin. Second, the culture of roots cells can be obtained in very large quantities due to their intensive growth. From cultures of roots cells the authors obtained alcohol extracts. The extracts were evaporated in vacuo, the aqueous residues were extracted by chloroform, followed by determination of the presence 13

of fusicoccin in aqueous and chloroform phases by immune methods. Fusicoccin was in chloroform, whereas in the aqueous phase there were found only trace amounts, that eliminates the possibility of effective interaction of such compounds as proteins and sugars with receptors and antibodies. Substances similar to fusicoccin were discovered at all stages of roots cultivation (since 14 days). It was found that the maximum content of fusicoccin in grown sterile roots is up to 150 nM per kilogram of roots. Mass-spectrometric analysis of the obtained fractions showed mainly the presence of fusicoccin type A, also these analyses indicated the possible presence of another unidentified fusicoccin. As a result of undertaken studies Muromtsev and his co-wokers made a conclusion that identification of endogenous fusicoccin is a very difficult task [2]. The first and main of the identified fusicoccins, fusicoccin A, is a glycoside of the carbotricyclic diterpene with a molecular weight of 680 Da gross and the formula С36Н56О12 (Figure 1). The aglycon part of fusicoccin molecules is represented by the tricyclic system including one eight-membered and two five-membered rings. Besides the basic compound (fusicoccin A) the fungus produces some similar compounds (more than 15), which differ in the degree of acetylation, positions of acetyl groups, the degree of oxidation of the aglycone part of the molecule. Thus, ten fusicoccins represent monoacetates, diacetates and triacetates. Another series of fusicoccins has a more simple structure: their C20-atom is not oxidized. The analysis of literature data shows that the fusicoccin molecule is not a unique structure and connections with the similar “skeleton” which are derivatives of the dicyclopentane and cyclooctane (the ring system 5-8-5), are widely distributed in the nature. They were found in fungi and algae, higher plants – liverworts, flowering plants, and even among animals (insects). If and when discovered and identified various representatives of this class of terpenoids the trivial names of organisms-producers are assigned to them. Thus, for example, the fusicoccin extracted from the liverwort Plagiochila acanthophylla is called fusicoplagin C, the fusicoccin extracted from the liverwort Anastrepta orcadensin is anadensin, the fusicoccin extracted from the giant kelp Dictyota dichtoma is epoxydictimen. It was proposed to combine compounds 14

having dicyclopentane and cyclooctane “skeleton” into groups of terpenoids of the fusicoccane range. The basis of the range of such compounds was formed by the hypothetical trans-sin-trans C20 hydrocarbon that was named fusicoccine. 1. The further study of fusicoccin showed that they demonstrate various physiological and biochemical properties. This made it possible to class it among natural regulators of the plant growth. 2. The cells of many organs and tissues of higher plants respond to the treatment by fusicoccin with the increase of the volume, with which such effects can be associated as the opening of the stomata, cells growth and elongation. There are data on the possible role of fusicoccin as an endogenous regulator of seed germination and root formation. 3. Fusicoccin effectively removes seeds from the deep rest. The laboratory of Muromtsev first discovered that fusicoccin actively stimulates the rhizogenesis for number of cultures. Fusicoccin had a very interesting effect on germination of pea seeds. So it stimulated the growth of cotyledon cells and at the same time inhibited the growth of the embryonic axis, which indicates the polarity of the fusicoccin action. It was found that fusicoccin plays an important activating role at early stages of legumes contamination with microorganisms of the genus Rhizobium spp, i.e. fusicoccin activates the nodulation in legumes. 4. Fusicoccin activates absorption of carbon dioxide by columnar cells of the leaf. Also fusicoccin regulates the concentration of ascorbate in the apoplast of coleoptile cells Vigna angularis. 5. One of the most striking hormonal properties of fusicoccin is its anti-stress activity. Muromtsev and his co-wokers showed that fusicoccin can increase the seed germination in conditions unfavorable for germination, for example: at elevated and low temperatures, excessive moisture, salination. The co-wokers of the Institute of Plant Physiology showed that the seeds soaking (0.68 mg/l of fusicoccin) and also spraying of plants of winter wheat, rye, barley (0.34 mg/l of fusicoccin) leads to the increase of the frost hardiness of plants at booting and tillering phases. Moreover, the increase of the frost hardiness correlated well with the degree of development of the photosynthetic apparatus and sugar accumulation, as well as with the development strengthening of the 15

endoplasmic reticulum in cells. It is shown that fusicoccin protects rice plants under salinity, increases the resistance of potato tubers to some diseases. Fusicoccin is undoubtedly one of the most powerful anti-stress compounds for plants. The positive role of fusicoccin in adaptation to osmotic stress is shown in the work [3]. 6. The available data on the study of the biochemical activity of fusicoccin are of particular interest for us. For the first time [4] Ballio and his co-wokers discovered fusicoccinal receptors on the plasma membrane isolated from the roots of maize seedlings. They also created proteoliposomes with the inclusion of isolated fusicoccinal receptors and studied properties of this artificial system. De Boer and his co-wokers developed a method for purification of FC-binding proteins on a special affine sorbent to which the biotinylated fusicoccin is ligated as the active group. A number of authors have also performed some studies on the extraction of fusicoccin-binding proteins [5]. For fusicoccin the alleged receptors are described – fusicoccinbinding proteins (FCBP) of two types. The first type has a high affinity for fusicoccin with a dissociation constant (Kd) about 10-10 М (high-affinity HA), and the second receptors type with a lower affinity, Kd about 10-7 М (low-affinity LA). They were first detected in the fraction containing plasma membranes of maize coleoptiles. It was shown in the publication that the ratio of the quantity of highaffinity receptors sites to low-affinity receptor sites on isolated membranes [HA]/[LA] is approximately one to two. The authors made a conclusion that only the high-affinity binding site is involved in action of fusicoccin. This point of view in the following years became dominant. Thus, the authors suggested that participation of the high-affinity site as a fusicoccin receptor is according to the reconstruction of the system in vitro consisting of fusicoccin-binding protein and Н+-ATPase [А][6-8]. A small quantity or absence of low-affinity sites on most isolated membranes may be due to their inactivation during the isolation process. It is explained by a much greater lability of low-affinity binding sites in comparison with high-affinity ones. Currently, the issue of the functional role of the high- and low-affinity sites is the subject of research.

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Membrane effects of fusicoccin belong to rapid answers: usually stimulation of protons emission begins immediately after adding fusicoccin and has no lag phase. In addition to the rapid membrane effects FC may have a prolonged, generalized action on plants that speaks in favor of its hormonal properties. Conspicuous is the fact that the current fusicoccin doses (10-20 mg / ha) are by two-three orders lower than that of other plant phytohormones and growth regulators (including those for gibberellin). In the publication of Babakov [9] the role of FC in the activation of protein kinase is discussed. Claudio Olivari and his co-wokers [10] first stated that the activation process of Н+-ATPase by fusicoccin requires the presence of another protein. In his work Claudio Olivari found that the phenylarsine oxide was a specific inhibitor of activation process of Н+-ATPase by fusicoccin. Very interesting is the action of fusicoccin on cells. First of all it concerns the acidification of the content of the cell under the influence of fusicoccin. The data that fusicoccin stimulates the protons emission in oat coleoptile cells were first specified in the work. The listed processes can be activated upon condition of the work stimulation of Н+-ATPase or reduction of the ions leakage through ion channels of a plasmalemma. Actually, according to the latest data, fusicoccin affects both the activity of H+-ATPase, and the conductivity of plasmalemma potassium channels [11].

1.2 Plants growth stimulators. Physiological activity and mechanism of action As of today growth regulators include “natural and synthetic chemical substances, applied for plants treatment for the sake of improvement of their quality, changing processes of their vital activity or structure, to increase yields or to simplify winning”. As V.S. Shevelukha points out, this definition is to be supplemented with the most important property of growth regulators – causing respective effects in exceptionally small doses, what is of a great ecological meaning, as well as ability of some of them to protect plants and crops from stress impacts of the environment and pathogens [12]. 17

M. Derfling in his work [13] “Plants Hormones. Systemic Approach” points out main principles of growth regulators action: 1. Regulator imitates endogenous hormone. Thus hormone action increases. In this case growth regulator is identical to the hormone or has similar physiological activity. 2. Regulator disturbs the process of hormone biosynthesis. 3. Physiological activity is inherent not into the introduced regulator itself, but to the product of its decomposition. 4. Regulator has a toxic action. In the first three cases growth regulators change natural hormonal balance in plants, treated therewith. About five thousand of compounds, having regulatory effect, have been found or studied in a varying degree over last decades. About 50 preparations of them have found their practical application. Te overwhelming majority of such physiologically active compounds can be rated among one of the three following categories: 1. Compounds, produced by inferior plants (fungi, mosses) and regulating their development. 2. Compounds, synthesized by microorganisms, which have the ability to influence growth of higher plants. 3. Compounds, formed by higher plants and influencing their growth, but in case of their introducing in intact plants or in tissues segments exogenously. Representatives of two last groups are mainly represented by phytohormones and their synthetic analogs. V.V. Polevoy [14] gives the following definition “phytohormones are signal substances, without which plants organism cannot exist as a comprehensive whole”. Phytohormones cause relatively quick physiological reactions, connected, obviously, with membranes, and more slow changes, depending on synthesis of proteins and nucleic acids, in competent cells. As of today five main groups of phytohormones are known: auxins, gibberellins, cytokinins, abscisins and ethylene. Few more endogenic regulatory substances have been detected lately, they are brassinosteroids, jasmine and cetonitr acids. The question, whether such a compound as fusicoccin can be allocated to phytohormones, is disputable [15]. 18

Auxins. The most important representative is indolil-3-acetic acid (IAA). IAA and its synthetic analogs in essence involve all aspects of vital activity of a plant organism. They affect division, elongation and differentiation of cells, induce root xylem formation. Auxin is necessary for DNA replication initiation; it is capable of maintaining cells trophism and hamper ageing processes [16]. Gibberellins are a large group of compounds of tetracyclic diterpenoids class. Their characteristic effect is acceleration of division of cells of sprouts apical meristems, as well as of cambium. Gibberelins activate seeds germination of many species; they play a significant part in plants transfer to flowering. Principle of action of such wide spread retardants as chlorcholinchloride etc. is based upon ability of these substances to inhibit gibberellins synthesis [17]. Cytokinins. By their chemical nature they are mainly substitutes N-aminopurines. The most important representatives: zeatin, natural cytokinin, as well as kinetin and 6-BAP, are widely used in laboratory studies. Cytokinin activity is also inherent in urea derivatives. Cytokinins stimulate cells division, activate seeds germination, play an important role in the process of cell differentiation and organogenesis of a plant, as well as promote ending dormate state of tubers and buds of woody plants irrespective of a season, are able to delay ageing process [18]. Natural cytokinin was first isolated in 1964 by Letham, who used multistage treatment of corn seed alcohol extract on the stage of milky ripeness for that purpose. As this cytokinin was isolated from maize, it was called zeatin. Natural cytokinin formula is presented in Figure 2. Zeatin was also found in ripening fruits of plump, growing fruit of apple tree, as well as in culture liquid of mycorrhizal fungi. Dihydrozeatin was found in lupine seeds. Cytokinins were also found in ferns, mosses and algae. Another substance, related to zeatin, was isolated from the culture of bacterium, pathogenic for plants, Corynebacterium fascians. Its effect on plants resembles much the effect of cytokinin preparations and is connected with apical dominance disturbance. Thus, zeatin and compounds with cytokinin activity, related to it, are widely spread among the representatives of the plant kingdom.

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Figure 2 – Natural – zeatin cytokinin formula [19]

Of the brightest physiological manifestation of cytokinin action is its ability to delay aging of leaves, that was first demonstrated in the work by Richmond and Lang, in which they succeeded to retain yellowing and protein disintegration in cut off burdock leaves with the help of phytohormone (Xanthium pensylvanicum Wallr.). In these experiments, the cut off burdock leaves, which ended growing, were put into cytokinin solution. Intensive decrease of chlorophyll and protein was in progress in control leaves, so that on the 12th day of testing its loss was 60% of the initial content. Leaves on phytohormone solution remained green and have lost only 15% of protein, that corresponded to its loss for the same period in analogous leaves on a plant. Thus an effective mean for delay of yellowing was found and it seemed rather likely that cytokinins are the product of root metabolism, which are necessary for normal life support of leaves. A more substantial study of the effect of cytokinin on cut off leaves was conducted in works [20-23] by Mothes et al. They showed that biostimulant action in case of its applying to the leaf surface remains strictly localized in the place of its application. That is why, if only one part is sprayed with cytokinin, it remains green and viable for a long period of time, while the other part turns yellow and dies. Such application of phytohormone on one part of a leave was used in studies on cut tobacco leaves, which ended their growth. These leaves were compared to the leaves sprayed with water or cytokinin water solution in whole (10-20 mg/l). For the period of study all the leaves were placed in wet camera under sparse light. In control leaves, which received only water, within 10 days of the test a sharp decrease of protein and chlorophyll content, which was accompanied by outflow of nitrogenous compounds to the footstalk, was registered. Spraying the whole leaf with biostimulator prevented protein content decrease in lamina and outflow of nitrogenous 20

compounds to the footstalk. These results corroborated the data provided by Richmond and Lang [24]. As was mentioned earlier, cut off leaves lose their ability to move metabolites from one part of a leave to another and acquire it only on acceleration. Under the influence of cytokinin moving of metabolites from one part of a leave to another was sped up in cut leaves, and thus, hormone in this respect is a kind of substituted roots action. All this made assumption of the fact, that cytokinins are the factor of root metabolism, with the help of which roots regulate metabolism in leaves, rather probable. This assumption was even more corroborated by stimulating the effect of cytokinin on the growth of young leaves, which didn’t finish growing. Stimulation with cytokinins was expressed both in etiolated leaves in dark, and in green leaves in light. Whereas in certain cases, it was shown that cytokinins can cause secondary growth of leaves, which have already finished their growth. In order to check, how widespread the ability of leaves to react to cytokinin in plant kingdom is, Kulayeva et. al. [25] studied cytokinin effect on cut off leaves of the plants, belonging to various systematic groups, picked up from the point of view of phylogenetic aspect. For that purpose cut off leaves were placed in wet camera under sparse light at room temperature and one half of each leaf was sprayed with water, and the second one – with kinetinin water solution (20 mg/l). The study performed showed that kinetinin slowed down yellowing of the half of a leaf, treated therewith, in representatives of various families of plants from phylogenetic point of view, including the oldest ones (Ranales) and the most advanced (Glumiflorae и Campanulatae) families. Cytokinin affected leaves of both monocotyledonous and dicotyledonous plants. It provided effect on the leaves of plants, belonging to various ecological groups, e.g. on steppe plants – feather grass, and on the representative of the flora of moist places – calla. Archeogoniates proved to be also kinetininsensitive. In particular, leaves of a gymnosperm, Ginkgo bilobа, and a spore-bearing plant, a fern Osmunda claytoniana, reacted to kinetinin. Thus, universal character of cytokinin action on all the types of plants was corroborated. Interesting results were obtained in work [26-29]. The possibility of secondary virescence of cut off 21

leaves under the influence of cytokinin was shown. Already yellowed tobacco leaves were taken for that testing. The leaves were cut from the plants and were placed into moist conditions. One half of each test leave was sprayed with highly active cytokinin – 6benzylaminopurine (BAP) – solution (20 mg/l), and the other one – with water. Probes for biochemical analysis and cytological studies were taken from each half. BAP caused secondary virescence of halves of cut off yellow leaves, treated there with. Halves of all the leaves, which weren’t treated with BAP, continued yellowing until the end of the test (10-11 day) most of them died off. Virescence continued during the whole period of study. It occurred due to both chlorophyll а, and chlorophyll b. The increase in chlorophyll content in initially yellow leaves lasted longer and its content practically reached the content of old, but still yellow leaves on the same plants, from which test leaves were cut. Thus, reversibility of old leaves yellowing occurred the more brimful, the less was the result of the process of yellowing. In a half of a leave, receiving only water, protein and RNA content continued decreasing. As was pointed out earlier, decomposition products were not accumulated in a yellow half of a leaf, but were moved to the part, resuming its vital activity under the influence of cytokinin. The next task was to determine, whether it is roots, which supply leaves with cytokinin-type substances. It was shown in Mothes laboratory that tobacco cut leaves are highly sensitive to kinetinin, whereas leaves on the plant provide no reaction to kinetinin. In order to find out which organs of a plant prevent demonstrating cytokinin action to plant’s leaves, reactions of plant’s leaves to phytohormone in the following variants were compared: 1 – cut leaf; 2 – leaf on a sprout, cut at root collar; 3 – leaf on a stump with deleted remaining top, but preserved root system; 4 – leaf on an entire plant. Third tier tobacco plants pale green leaves were taken for the study. It was found that phytohormone delays yellowing of that part of a leaf, which it was applied to. Difference in pigmentation of the two halves of a leaf, resulting therefrom, is a rather sensitive test of leaf reaction to cytokinin. It was also found that hormone was effective when affecting leaves on remote sprout; at the same time it wasn’t effective when affecting the leaf, connected with stump root, or the 22

leaf of entire plant. Thus it was proven that it is the root which is the producer of cytokinin for leaves. Analogous data as to the importance of the root as the source of cytokininfor plants were obtained for haricot plants in Mothes lab [30-33]. Cytokinins effect was also evident on various cell structures of a leaf. It was clearly seen, e.g., on internal chloroplasts structure. Internal chloroplasts membrane structures are the place of pigments and enzymatic systems localization, providing for water photolysis and photosynthetic phosphorylation. It was shown that under the influence of cytokinins chloroplast structure is preserved. Thus it was found that cytokinins are one of the factor of hormonal regulation of development and maintenance of chloroplasts internal structure, providing for their photosynthetic activity. According to the data provided, cut off wheat leaves also showed that phytohormone delays chloroplasts decomposition in cells. It delayed grains decomposition and chloroplasts filling with osmiophil globules in disks of Brussels sprout leaves in the same way. This effect of cytokinins on chloroplasts structure is to be reflected at their functional state. Membrane apparatus of chloroplasts is the form of spatial organization of pigment and enzymatic photosynthesis system. That is why development of membranes under the influence of cytokinin causes activation of photosynthesis in leaves. Abscisic acid is growth inhibitor and in many cases acts as auxins, gibberellins and cytokinins antagonists. Abscisins extend dormancy of many plants species, accelerate protein and nucleic acids decomposition and improve plants resistance to unfavorable environment factors, are antitranspirants, without affecting photosynthesis intensity. Ethylene is the only gas phytohormone. Ethylene delays mitotic process in root, sprouts and axillary buds meristems, stops auxin polar transport, that causes such processes, as exfoliation, blossom fading and fruits falling, organs ageing. Ethylene accelerates fruits ripening. On the basis of 2-chlorethylphosphonic acid preparations, producing ethylene, viz. ethrel, capozane and others are created, which are successively applied against grain crops lodging. Brassinosteroids. Phytohormones of steroid nature. They were discovered relatively recently: in 1979 chemical composition of 23

brassinosteroid was determined. Brassinosteroids are contained in various plants organs, whereas the highest content of these phytohormones is characteristic of pollen. One of the most important functions of brassinosteroids is their ability to increase plants resistance to unfavorable environmental conditions [34]. Thus, a significant role in response reaction of a plant to various external influences belongs to phytohormones. Diversity of physiological effects of phytohormones is mainly determined by the fact that they regulate realization of cell genetic program, implementation of which needs coordinated action of a variety of enzymatic, structural and regulatory proteins. The primary role of the majority of phytohormones in a plant cell is in genes expression control. Whereas experimental data point out to several levels of phytohormones effect on genes expression, including formation of an active transcription complex, provision of matrix RNA stability as well as regulation at translation level. To implement this function, a hormone is to bind with receptor protein and to get transported to the nucleus in form of hormonereceptor complex. In the nucleus hormone-receptor complex binds with chromatin that causes its activation and transcription induction forming a respective matrix of RNA. Phytohormones directly influence DNA by means of its enzymatic methylation regulation. Auxins, gibberellines and cytokinins inhibit replicative DNA methylation and thus can cause formation of non-methylated sites in DNA, controlled by them, which are necessary for certain genes expression. It was shown that fusicoccin, a regulator of growth of plants of terpenoid nature, is as well able to control the degree of DNA methylation, extracted from wheat seeds embryos. Apart of direct impact on DNA phytohormones actively influence the molecular and genetic processes at transcription and post-transcription levels. Cytokinins and their synthetic analogs increase activity of polymerases I and II, that causes RNA synthesis enhancement, increase the amount of ribosomes as well as activate polysomes formed from ribosomes and mRNA present in the cell. Auxin and its analogs have the ability to activate enzyme RNA polymerase and to stimulate RNA synthesis.

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A more detailed review of phytostimulators action will be given at the example of fusicoccin, a well-known biosynthetic plants growth stimulation of terpenoid nature. Fusicoccin was first discovered in 1964 as product of metabolism of a fungus Fusicoccum amygdali Del., causing cancer in stone and almond trees [35]. According to its chemical nature fusicoccin is a glycoside of carbocyclic diterpenoid with molecular weight 680 and formula C36H56O12. It is stated that fusicoccin shows high and versatile physiological activity in high plants organisms, treated therewith [36-41]. Fusicoccin activates cell growth in various organs of flowering plants: in stems, seed lobes, leaves, coleoptiles and roots, showing greater universality of action than auxin, gibberellin and cytokinin. It is found that fusicoccin induces seeds germination. Seeds treatment with fusicoccin solutions replaces durable cold stratification. Fusicoccin induces root formation in grafts of various plants. Fusicoccin is able to cause leaves stromata opening in darkness and thus to improve transpiration. Fusicoccin increases vital activity of cultivated plants in unfavorable soil and climatic conditions, especially under low temperatures, salinization, overwetting etc. Aggregate of data received as of today: a rather broad spectrum of physiological activity of fusicoccin, its receptors identification in plasmalemma preparations from roots and coleoptiles of cereals and germinated garden radish seeds, as well as from leaves of Vicia Faba mayor L. and, finally, discovery of endogenic fusicoccin in cells of higher plants, belonging to monocotyledons and dicotyledons: maize Zea mays L. Sterling breed and headed cabbage Brassica oleracea L, “Zimovka 1474” breed in amounts of approximately 10-8 and 10-9 g/kg allows making a conclusion that fusicoccin pretends for the role of phytohormone with a highly low content in plant tissues. According to modern views, fusicoccin action shows itself both on membrane level and on the level of protein synthesizing. Fusicoccin specifically activates process of intracellular energy of high-energy phosphate bounds turning into the energy of electrochemical protons gradient on the plasmalemma level. The data, obtained in vivo and in vitro, point to the fact that fusicoccin effect is connected with Mg2+ -dependent, K+ -stimulated ATF-aze, 25

which, in its turn, activates proton pump. Stimulation of H+ ATFazes with fusicoccin is shown on various plant materials [42, 43]. According to the point of view of some researchers, H+ pump activation is not a primary reaction to fusicoccin: fusicoccin is able to acidify cytoplasm in plasmalemma, which leads to ATF-aze activation and protons discharge. Though according to the data of other authors, obtained with application of NMR method, fusicoccin does not cause cytoplasm acidifying in cells of peas internodes. According to numerous data fusicoccin does not provide effect on the synthesis of RNA and protein. An assumption is expressed, according to which fusicoccin effect on functional activity of protein synthesis apparatus occurs through activation of proton pump, as well as by means of influencing proteins, already existing in a cell. Though, fusicoccin prolonged influence on the entire plant, such as protection from stress, root formation stimulation cannot be directly connected to activation of proton pump only or other transport plasmalemma system. It is probable, that various fusicoccin effects are conditioned by participation of various types of receptors, which remain not identified. According to the data of various authors fusicossin influences RNA and protein synthesis in isolated seed lobes of pumpkin and in barley plants. According to the data of D.G. Muromtseva [44], fusicoccin mechanism of action is based upon the increase thereby of activity of RNA-polymerase, bound with chromatin, whereas this activation is of a short-term, transitory character. Stimulation of RNA synthesis with fusicoccin is the result of a regulator effect directly on the ferment, RNA-polymerase, and does not affect chromatin matrix activity. Thus, action of natural and synthetic growth regulators is directly or indirectly caused by their influence on genetic apparatus of a plant cell [45-49].

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1.3. Natural small molecules and their use as an anticancer drug Some small molecules that are used in preclinical as well as clinical studies influence PPIs of the Bcl-2 protein family of antiapoptotic proteins [45]. Several compounds have been identified that target the hydrophobic binding pocket, which forms the BH3 binding site in Bcl-2, and prevent thereby the hetero-dimerization of Bcl-2 with pro-apoptotic members and thereby the induction of apoptosis. Another well known example is the nutlins (cis-imidazoline analogs) that target the p53/MDM2 interaction. These compounds bind selectively to MDM2 and prevent thereby the interaction with p53 and enhance the activity of the p53 pathway. It is interesting, that healthy wild-type p53 cells retained their viability when treated with these nutlins, while wild-type p53 expressing cancer cells are killed. Besides these examples many other small molecules are known that prevent PPIs that are beneficial in the treatment of cancer. However, so far not many small molecules are known that affect cancer cells by stabilizing PPIs. In recent years several reviews have been published about the isolation of (novel) natural compounds and active small molecules with an anticancer activity from various sources. Plants supplied several of these compounds while other anticancer drugs have been isolated from marine organisms and microorganisms, like fungi. Although the working mechanism of many active small molecules is not yet (completely) clarified, most new compounds have been shown to belong to diverse structural classes, including polyketides, terpenoids, terpenes, alkaloids, and steroids and to influence PPIs. Furthermore, many drugs (> 60%) that are currently used in cancer treatment are derived from natural occurring small molecules, either directly or indirectly. Paclitaxel (taxol) is, for instance, isolated from the bark of the Pacific yew tree Taxus brevifolia [46] and the vinca alkaloids vinblastine and vincristine are isolated from the Madagascar periwinkle Catharanthus roseus.

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1.4 Fusicoccanes, an interesting group of active small molecules Fusicoccanes are a large group of active small molecules that refer to the terpenoids. They have a typical dicyclopenta[a,d] cyclooctane skeleton (5-8-5 core ring structure), to which various side chains are linked [50]. They are found throughout nature and are amongst others produced by various fungi, higher plants, liverworts, and even insects. A number of biological effects have been described for fusicoccanes on both plants and other organisms, and several of them are caused by the selective targeting of a particular protein or PPI. Some well-studied fusicoccanes are Fusicoccin-A (FC), Cotylenin-A (Cot-A), and Ophiobolin-A (OPH-A) (Figure 3). Fusicoccin-A was first described in 1964 by Ballio et al., and is produced by the fungus Phomopsis amygdali (formerly known as Fusicoccum amygdali Del.) from which more than 30 fusicoccin derivatives have been isolated, among which at least 16 natural derivatives (Figure 3). The fungus infects peach and almond trees, in which the secreted FC stabilizes the complex formed between the Cterminal tip of the plasma membrane H+-ATPase and a 14-3-3 protein. This stabilization irreversibly activates the H+-ATPase, that drives the opening of stomata resulting in uncontrolled water loss and wilting of the tree. Although the only known FC target in plants is the H+-ATPase, at the cell and organ level FC affects a range of different processes, like opening the stomata, breaking seed dormancy, inducing an apoptotic like cell death in sycamore cells, and reducing hydrogen peroxide and nitric oxide production in guard cells [47-51]. Many of these processes can be (in)directly explained by the activation of the H+-ATPase while others suggest the presence of another FC target. FC is not only active on all higher plants, but on other organisms as well. It randomizes, for instance, the left-right asymmetry early in the Xenopus laevis embryonic development [53] and induces apoptosis in cancer cells derived from different origins, for which the efficacy of FC can be enhanced by combining it with the cytokine interferon (IFNα). Cotylenin-A was first described by Sassa who discovered it in a fungal culture filtrate of Cladosporium sp. And described its 28

growth enhancing effect on plant cotyledons. The structure of Cot-A is rather similar to that of FC, and comparisons made on their effects in plants show that both molecules have similar physiological effects. In some cases FC is more potent (e.g. induction of seed germination and wilting of tomato cuttings), while Cot-A is more effective in others (e.g. stomatal opening and cotyledon elongation). Besides this, Cot-A has been shown to induce differentiation in human myeloid leukemia cells and inhibits the growth of various tumor types both in vitro and in vivo, when combined with IFNα or rapamycin.

Figure 3 – Chemical structure of Fusicoccin-A, Cotylenin-A and Ophiobolin-A [52]

Ophiobolins (OPHs) are roduced by plant pathogenic fungi belonging to the Bipolaris species. Currently 23 analogs have been identified, to which various biological activities are ascribed that are all damaging plants either directly or indirectly. Besides being harmful for plants, OPHs also possess anti-microbial activities as well as toxicity towards animals and even cancer cells. The working mechanism of most OPHs is not completely clear and probably differs per analog. OPH-A, however, has been shown to mainly function through the specific inhibition of calmodulin, an important signalling molecule.

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Anticancer effects have been described for several fusicoccanes, including FC, Cot-A and OPH-A. All show a clear growth reduction at relatively low concentrations (IC50-value mostly in the μM range, depending on the cell line). Although the tumor selectivity differs per molecule, with some being relatively cancer selective while others are indiscriminate and kill healthy cells as well as cancer cells, fusicoccanes form an interesting group of potentially new anticancer compounds. The working mechanism of most fusicoccanes in inhibiting the cancer cell growth is not clear yet. OPH-A most likely acts through the selective inhibition of calmodulin by binding covalently to calmodulin with its C7-aldehyde group [54]. FC and Cot-A both lack this aldehyde group, suggesting that these compounds function through a different cellular target. When combined with IFN, FC and Cot-A both induce apoptosis by the induction of the TRAIL pathway, however, this is most likely the final effect of the combined treatment and the primary target of both compounds is therefore still unknown. Boear and et.al [55] studied properties of fusicoccin-A as anticancer. We have developed a highly efficient method of producing fusicoccin extract from germinated wheat seeds. This method is based on the selective sorption fusicoccin by nanostructured carbon sorbent developed by researchers at Institute of Combustion Problems [56]. Thus, it becomes possible to create a high-performance domestic anticancer drug.

References 1 1 Ballio A., Chain E.B., DeLeo P., Erlanger B.F., Mauri M., Tonolo A. Fusicoccin: a new wilting Toxin produced by Fusicoccum Amygdali Del // Nature. – 1964. – Vol. 203. – № 4642. – P. 297. 2 Tajima N., Nukina M., Kato N. and Sassa T. Novel fusicoccins R and S, and the fusicoccin S aglycon (phomopsiol) from Phomopsis amygdali Niigata 2-A, acid. Biosci. Biotech. Bioch. – 2004. – № 68 (5). – R. 1125-1130. 3 Aducci P., Ballio A., Fogliano V., FulloneM. R., Marra M., Proietti N. Purification and photoaffinity labeling of fusicoccin receptors from maize // Development. – 2003. – Vol. 130. – № 20. – P. 4847-4858. 4 De Boer A.H., Watson B.A. and Cleland R.E. Fusicoccin binding protein from plasma root membrane. – 1989. – Plant Physiol. – №89 (1). – P. 250-259.

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5 Trofimova, M.S., Smolenskaya, I.N., Drabkin, A.V., Galkin, A.V., Babakov, A.V., Plasma membrane, H + ATPase activation // Physiologia Plantarum. – 1997. – Vol. 99. – Issue 2. – p. 221-226. 6 Olivari C., Meanti C., De Michelis M. I., Rasi-Caldogno F. Fusicoccin Binding to the Plasma Membrane H + -ATPase IV. Fusicoccin Induces the Association between the Plasma Membrane H + -ATPase and the Fusicoccin Receptor // Plant Physiol. – 1998. –Vol. 116. – №2. – p. 529–537. 7 Oecking C., Eckerskorn C., Weiler E.W. The fusicoccin receptor of plants is a member of the 14-3-3 superfamily of eukaryotic regulatory proteins // FEBS Lett – 1994. – Vol.352. – P.163–166. 8 Babakov A.V., Abramycheva N.Yu., Bilushi SV., Shevchenko V.P. Study of the interaction of fusicoccin with plasma membranes of higher plants // Biol. membranes. – 1990. – T. 7. M 2. – P. 107-112. 9 Olivari C., Albumi C., Pugliarello M. C., De Michelis M. I. Phenylarsine Oxide Inhibits the Fusicoccin-Induced Activation of Plasma Membrane H + ATPase // Plant Physiol. – 2000. – Vol. 122. – №2. – p. 463–470. 10 Baunsgaard L., Fuglsang A.T., Jahn T., Korthout H.A.A.J., De Boer A.H. and Palmgren M.G. The 14-3-3 proteins associate with the plant plasma membrane H + -ATPase to generate a fusicoccin binding complex and a fusicoccin responsive system. Plant J. – 1998. – 13 (5). – P. 661-671. 11 De Vries-van Leeuwen I.J., Kortekaas-Thijssen C., Nzigou Mandouckou J.A., Kas S., Evidente A. and De Boer A.H. / Fusicoccin-A selectively induces apoptosis in tumor cells after interferon-a priming. Cancer Lett. – 2010. – №293 (2). – P. 198-206. 12 Shevelukha V.S. Plant growth and its regulation in ontogenesis. – M .: Kolos. – 1992. – p. 234. 13 Defling K. Plant hormones: a systematic approach. – M .: World. – 1985. – p. 124-298. 14 Polevoj V.V. Phytohormones. L .: Publishing House of Leningrad State University, 1982. – p. 248. 15 Hamburg K.Z. Phytohormones and cells. – M .: Science. – 1970. – p.103. 16 Sorokin H., Mathur I., Thimann K. The differentiation of the pea on xylem differentiation in the pea epicotyl // Amer. J. Bot. – 1962. – Vol. 49. – P. 444. 17 Leike H. Effect of gibberellic acid and kinetin on dormant buds of various thrashing // Flora A. – 1967. – 158. – P.301. 18 Collier MD, Fotelli MN, Nahm M., Kopriva S., Rennenberg H., Hanke DE, Gebler A. Regulation of nitrogen uptake by Fagus sylvatica on the whole plant level – interactions between cytokinins and soluble N compounds // Plant, Cell and Environment. – 2003. –Vol. 26.- Issue 9. – p. 1549-1560. 19 Formula phytohormone by sitokin // https://en.wikipedia.org/wiki/Cytokinin 20 Lewis D. H., Burge G. K., Schmierer D. M., Jameson P. E. Cytokinins and fruit development (Actinidia deliciosa). Changes during fruit development // Phys. Plantarum. – 1996. – Vol. 98.- Issue 1. – p. 179-186. 21 Brault M., Maldiney R., Miginiae E. Cytokinin binding proteins // Physiologia Plantarum. – 1997. – Vol. 100., Issue 3. – p. 520-527.

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22 Inoue T., Higuchi M., Hashimoto Y., Seki M., Kobayashi M., Kato T., Tabata S., Shinozaki K., Kakimoto T. Identification of CRE1 as a cytokinin receptor from Arabidopsis // Nature. – 2001. – Vol. 409. – P. 1060-1063 .. 23 Sakakibara H., Suzuki M., Takei K., Deji A., Taniguchi M., Sugiyama T. A response-regulator homologue that can be taken in for example, The Plant Journal – 1998. –Vol . 14. – №3. – P. 337-344. 24 Richmond A. A., Lang A. Effect on xanthiun leaves // Science. – 1957. – Vol. 125 No. 3249. – P. 650. 25 Kulaeva ON, Kuznetsov V.V. Recent achievements and prospects in the field of study // Plant Physiology. – 2002. – V. 49. – № 4. – p. 632-638. 26 YamadaH., SuzukiT., TeradaK., Take., Ishikawa., MiwoK., MizunoT. The Arabidopsis HK4histidinekina seisa cytokinin-binding receptor that sign cytokinin across the membrane // Plant Cell Physiol. – 2001. – Vol. 42. – P. 1017-1023. 27 Sakakibara H., Suzuki M., Takei K., Deji A., Taniguchi M., Sugiyama T. A response-regulator homologue that can be taken in for example, The Plant Journal. – 1998. – Vol. 14.- № 3. – P. 337-344. 28 Brandstatter, I.B., Kieber J.J. The response of the regulators are rapidly and specifically induced by cytokinin in Arabidopsis // Plant Cell. – 1998. – Vol. 10. – P. 1009-1019. 29 Kusnetsov V., Herrmann R. G., Kulaeva O. N., Oelmueller R. Gen. Genet. – 1998. – Vol. 259. – P. 21-28. 30 Mothes K., Engelbrecht L. Kinetin und das Problem der Akkumulation 16alicher Stickstoff-Verbindungen // Monatsber. Dtsch. Akad. Wiss Berlin. – 1959. – № 1. – P. 367. 31 Mothes K.Uber das Alterna der Blatter und die Moglichkeit ihrer Wiederverjungung // Naturwissenschaften. – 1960. – № 47. – P. 337-351. 32 Mothes K, Engelbrecht L., Kulajewa 0. Uber die Wirkung des Kinetins auf Stickstoffverteilung und Eiweipsynthese in isolierten Blattern // Flora. – 1959. – No. 147. – P. 445. 33 Grove, Michael D .; Spencer, Gayland F .; Rohwedder, William K.; Mandava, Nagabhushanam; Worley, Joseph F .; Warthen, J. David; Steffens, George L .; Flippen-Anderson, Judith L .; Cook, J. Carter. "Brassinolide, a plant growthpromoting steroid isolated from Brassica napus pollen" // Nature. – 1979. – №281 (5728). – P. 216-217. 34 Ballio A., Chain E.B., DeLeo P., Erlanger B.F., Mauri M., Tonolo A. Fusicoccin: a new wilting Toxin produced by Fusicoccum Amygdali Del // Nature. – 1964. – Vol. 203. – № 4642. – P. 297. 35 Marré E. Fusicoccin: A tool in plant physiology // Annu. Rev. Plant Physiol. – 1979. – Vol. 30. – p. 273-288. 36 Muromtsev G.S., Chkanikov D.I., Kulaeva ON, Hamburg K.Z. Basics of chemical regulation of growth and productivity of plants. – M .: IN Agropromizdat. – 1987. – p. 80-133. 93 Mansurov Z.A., Jandosov J.M., Kerimkulova A.R., Azat S., Zhubanova A.A., Digel I.E., Savitskaya I.S., Akimbekov N.S., Kistaubaeva A.S. Nanostructured carbon materials for biomedical use / Eurasian chemico-tecnological journal. – 2013. – №15 (3). – P. 209-217.

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38 Mansurov Z.A., Azat S., Adekenova A.S., Kerimkulova A.R., Ivasenko S.A., Shulgau Z.T., Gilmanov M.K., Ibragimova S.A. Extraction Fusicoccin From Wheat Seeds Using Nanocarbon Sorbents // Advanced Materials Research. – 2013. – V.647. – P. 67-70. 39 Donaire J.P., Rodríguez-Rosales M.P., Soto M.J., Sanjuan J., Olivares J. Effect of Fusicoccin on Rhizobium spp. // Molecular Plant-Microbe Interactions. – 1999. – Vol. 12. – № 12. – P. 1090-1094. 40 Stout R.G., Cleland R.E. Partial Characterization of Fusicoccin Binding to the Receptor Sites on Root Membranes // Plant Physiol. – 1980. – Vol.66. – № 3. – P.353-359. 41 Baunsgaard L., Fuglsang A.T., Jahn T., Korthout H.A., de Boer A.H., Palmgren M.G. The 14-3-3 proteins associate with the plant plasma H + -ATPase to generate a fusicoccin binding complex and a fusicoccin responsive system // Plant J. – 1998. – Vol. 13. – pp.661-671. 42 Malerba M., Crosti P., Cerana R., Bianchetti R. Fusicoccin affects cytochrome with leakage and cytosolic 14-3-3 accumulation of H + -ATPase activation // Physiologia Plantarum. – 2004. – Vol. 120. – Issue 3. pp. 386-394. 43 Muromtsev G.S. Fusicoccin – a new phytohormone? // Plant Physiology. 1996. – Vol.43. – № 3. pp. 478-492. 44 Korthout HAAJ, de Boer A.H. Brain protein homologs, Plant Cell. – 1994. – Vol. 6. – pp. 1681–1692. 45 Rubinstein B., Cleland R. E. Response of Avena Coleoptiles to Suboptimal Fusicoccin: Kinetics and Comparisons with Indoleacetic Acid // Plant Physiol. – 1981. – Vol. 68. – №3. – pp. 543-547. 46 Johansson F., Sommarin M., Larsson C. Fusicoccin activates the C-terminal inhibitory domain. // Plant Cell. – 1993. – Vol.5. – pp. 321-327. 47 Olivari, C., Albumi, C., Pugliarello, M.C., De Michelis, M.I. – 2000. – Vol. 122. – №2 – pp. 463–470. 48 Oecking C., Eckerskorn C., Weiler E.W. The fusicoccin receptor of plants is a member of the 14-3-3 superfamily of eukaryotic regulatory proteins // FEBS Lett – 1994. – Vol.352. – pp.163–166. 49 De Michelis M.I., Rasi-Caldogno F., Pugliarello, M.C., Olivari, C.Fusicoccin binding the H + -ATPase. // Plant Physiol. – 1996. – Vol. 110. – pp. 957-964. 50 Wurtele M., Jelich-Ottmann C., Wittinghofer A. and Oecking C. 14-3-3 regulatory complex. EMBO J., Chemical structure of Fusicoccin-A, Cotylenin-A and Ophiobolin-A. – 2003. – №22 (5). – pp. 987-994. 51 Wurtele M., Jelich-Ottmann C., Wittinghofer A. and Oecking C. 14-3-3 regulatory complex. EMBO J., Chemical structure of Fusicoccin-A, Cotylenin-A and Ophiobolin-A. – 2003. – №22 (5). – pp. 987-994. 52 Wurtele M., Jelich-Ottmann C., Wittinghofer A. and Oecking C. The 14-3-3 regulatory complex. EMBO J., Chemical structure of Fusicoccin-A, Cotylenin-A and Ophiobolin-A. – 2003. – №22 (5). – pp. 987-994. 53 Wurtele M., Jelich-Ottmann C., Wittinghofer A. and Oecking C. 14-3-3 regulatory complex. EMBO J., Chemical structure of Fusicoccin-A, Cotylenin-A and Ophiobolin-A. – 2003. – №22 (5). – pp. 987-994.

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54 De Boer A.H., Watson B.A. and Cleland R.E. Fusicoccin binding protein from plasma root membrane. – 1989. – Plant Physiol. – №89 (1). – pp. 250-259. 55 De Vries-van Leeuwen I.J., Kortekaas-Thijssen C., Nzigou Mandouckou J.A., Kas S., Evidente A. and De Boer A.H. / Fusicoccin-A selectively induces apoptosis in tumor cells after interferon-a priming. Cancer Lett. – 2010. – №293 (2). – P. 198-206. 56 Pat. 26936 RK. Method for chromatographic separation of biologically active substances / Mansurov Z.A, Gilmanov M.K, Kerimkulova A.R, Azat S., Guckenheimer E.Yu .; publ 15.05.2013, Bull. №5. – MJ RK.

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2

 CARBON SORPTION MATERIALS 

The useful properties of activated carbon have been known since ancient times. This traces back to 1500 BC when Egyptians used charcoal as an adsorbent for medicinal purposes and a purifying agent. Around 420 BC it was observed that Hippocrates dusted wounds with powdered charcoal to remove their odor. Ancient Hindu societies purified their water by filtration through charcoal. In 1773, the Swedish chemist Karl Wilhelm Scheele was the first to observe adsorption of gases on charcoal. A few years later activated carbons began being used in the sugar industry as a decolorizing agent for syrup. In the early 20th century the first plant to produce activated carbon industrially was built for use in sugar refining industry in Germany. Many other plants emerged in the early 1900’s to produce activated carbons primarily for decolorization. During World War I activated carbon was used in gas masks for protection against hazardous gases and vapors. Today, activated carbons are used to remove color from pharmaceutical and food products, as air pollution control devices for industrial and automobile exhaust, for chemical purification, and as electrodes in batteries. 500,000 tons per year of activated carbon are produced globally [1-6]. 80% of this is used for liquid phase applications, and 20% is used for solid phase applications. Activated carbon (AC) is a unique material with its immense capacity for adsorption from gas and liquid phases. It occupies a special place in terms of producing a clean environment involving water purification as well as separations and purification in the chemical and associated industries. In these roles, it exhibits a remarkable efficiency as the international production is a little more than a million tons per year, with perhaps 2 million tones being in continuous use. Broadly AC has been used for three main purposes:

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Figure 4 – Application of carbon materials [7]

1. AC is dominantly used for purposes of adsorption in liquidphase. The wide-ranging scenarios for AC are: ‒ drinking water availability, to improve taste, smell and color including ‒ removal of chlorinated compounds and other volatile organic carbons (VOCs); ‒ purification of ground water purity from contaminants coming from disused sites of heavy industries; ‒ treatment of both industrial and municipal wastewater; ‒ mining operations require feed water treatment, metallic ion adsorption (gold and other metals), adsorption of excess flotation reagents and adsorption of natural organic matter (NOM); ‒ pharmaceutical processes, including purification of process water, use with fermentation broths and purification of many products; ‒ the food, beverage and oil industries for removal of color and unacceptable tastes; ‒ the dry-cleaning industries require purification of solvents; ‒ the electroplating industries require purification of wastewaters containing Pb, Cr, etc; ‒ household water purification, cleaning of aquaria and use in oven-extract hoods;

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‒ the sugar and sweetener industries need decolorization agents for production of white sugar, etc. 2. AC in different forms (granular, extruded, fiber or cloth) is used for production of pure gases in the chemical industry, to reduce pollutant gases to very low concentrations in a single stage, in protection against poisonous gases, in air conditioning, for removal of oil from compressed air, to separate gases in mixtures by sieving, etc. 3. Carbon materials have been used for some time in heterogeneous catalysis, acting as direct catalysts or as a catalyst support. Catalytic particles can be supported within the porosity to promote required catalytic conversions [8-10]. Activated carbon is a microcrystalline form of carbon with very high porosity and surface area. It may be visualized as foam solid that has a large surface within a rigid granule or particle structure of relatively small volume. Its chemical structure allows it to preferentially adsorb organic materials and other nonpolar compounds from gas or liquid streams. Activated carbon has become one of the most technically important and most widely used adsorbents because of its high adsorptive capacity. Present technology demands a very large production of activated carbons with appropriate characteristics for each particular application. In general, activated carbon which is used in any of the most common applications must have adequate adsorptive capacity, chemical purity, mechanical strength, etc. Furthermore, all these specifications should coexist with a low production cost. Activated carbon is obtained from a carefully controlled process of dehydration, carbonization and oxidation of organic substances. It can be prepared for research in the laboratory from a large number of materials. However, the most commonly used ones in commercial practice are peat, coal, lignite, wood and agricultural by-products such as coconut shell, almond shell, rice husk, etc. Pyrolysis of the starting material with exclusion of air and without addition of chemical agent usually results in an inactive material with a specific surface area of the order of several m2/g and low adsorption capacity. One can prepare carbon with a large adsorption capacity by activating the carbonized products with a 37

reactive gas. The majority of activated carbon used throughout the world is produced by steam activation (physical activation). In this process, the carbonized product is reacted with steam over 900 oC. Another procedure used in the production of activated carbon involves the use of chemical activating agents before the carbonization step. The most commonly used activating agents are phosphoric acid, zinc chloride and salts of sodium and magnesium etc. Chemical agents act as dehydration agents and they may restrict the formation of tar during carbonization. Chemical activation is usually carried out at lower temperatures than the simple pyrolysis and the activation process with steam or carbon dioxide. The production at lower temperatures promotes the development of a porous structure because under these conditions elementary crystallites of smaller dimensions are formed [11-12]. Most of the available surface area of activated carbon is nonpolar in nature. However, during production the interaction of surface with oxygen produces specific active sites giving the surface of slightly polar nature. As activated carbon is one of the most commonly used adsorbents in many industrial applications for its adsorptive capacity. In this study, production of activated carbon from apricot stones and the quality of the products have been investigated. Carbon adsorbents tend to be hydrophobic and organophilic [13-14]. According to the IUPAC definition, pores can be distinguished in three groups with respect to their dimensions.  Macropores – Pores with larger than 50 nm;  Mesopores – Pores with diameters between 2 nm and 50 nm;  Micropores – Pores with diameters less than 2 nm. Most activated carbons contain pores of different sizes: micropores, transitional mesopores and macropores. Therefore they are considered as adsorbents with wide variety of applications (Figure 5). Microporous carbons have a unique structure which essentially is a three dimensional network, or labyrinth of carbon atoms some in hexagonal arrangements and some as individual carbon atoms bonded together extremely close but not close packed. This bonding arrangement results in space existing between the atoms to create an interconnecting three dimensional passage way in which every space unit has a connection to all others within the carbon. The dimensions 38

of the passage way (width) are those of molecules, about 0.5 nm, such that any molecule which enters into this space is subjected to intense dispersion forces from the carbon atoms (about eight per space) which make up the “surface” of the space.

Figure 5 – Classification of pore size [15]

These forces can “trap” the molecule for periods of time much longer than on an open surface and so the phenomenon of physical adsorption of gases is generated. The site, place or space where a molecule can be trapped (adsorbed) is called an adsorption site. These adsorption sites can be modified in terms of their size (widened or narrowed) and in terms of their chemical composition [16]. The surface can be bonded to hydrogen, oxygen, chlorine, nitrogen, etc. to alter the polarity of the surface. Changing this polarity can enhance the adsorption process for polar molecules. The porosity can be widened by gasification with water vapor, oxygen or carbon dioxide. The parent materials may also be impregnated with zinc chloride, potassium hydroxide or phosphoric acid, these treatments improving adsorption characteristics, a process known as activation. The variables, within an adsorption system (dominantly an aqueous solution) controlling extents of adsorption include [17-18]: 1. Volume of narrow micropores, < 0.7 nm (VNM – cm3/g), including the pore-size distributions. 39

2. Volume of wider micropores, 0.7-2.0 nm (VWM – cm3/g), including the pore-size distributions. 3. Volume in mesopores, >2.0 and 7500C. 6. The effects of additions of surface oxygen complexes upon pore volumes and pore size distributions (pore dimensions can be reduced). 7. The characterization of surface oxygen complexes. 8. The pH of the solution. 9. Ionic strength. 10. Temperature. Nowadays in Kazakhstan the necessity of creation of carbon materials with high sorption properties has come. A pioneer in this field was “Laboratory of carbon nanomaterials” at the Institute of Combustion problems and at Al-Farabi KazNU created by Professor R.M. Mansurova. In this laboratory from waste rice grinding production by carbonation the first carbonized carbon biomaterial was obtained which has the ability to adsorb not only molecules, but even whole microbial cells. In the work [19-21] on the carbonized carbon material from rice husk cells of the following microorganisms: rhodotorula, glutinis var, glutinis, pseudomonas aeruginosa, pseudomonas mendocina were sorbed. The choice of these microorganisms was due to the fact that they are widely used for detoxification of sewage. In this work we have obtained very interesting results. It is shown that microorganisms adsorbed on carbon materials remained viable in the presence of highly toxic ions of cadmium, lead and copper [22-24]. If you apply the available microorganisms in the presence of these ions in a day only 20% of the total number of introduced microorganisms remains, whereas the number of living microorganisms adsorbed under the same conditions was 80 %. That is sorption of microorganisms in the 40

carbon sorbent from rice husk four times increased the viability of the microorganisms in the presence of toxic ions, in this case the total sorption capacity of these toxic ions increased several times. All these results suggest that unusual carbon material with sorbed living cells of microorganisms was created and it has the ability to high sorption of highly toxic ions. This new material can be widely used for industrial detoxification of polluted water contaminated with heavy metal ions. In this laboratory headed by Professor R.M. Mansurova, carbon materials have been created by carbonization of the waste from local raw materials. Interesting work has been done on the sorption of toxic ions by obtained carbonized carbon materials derived from rice husk and wheat bran [25-26]. It is stated that activation of the obtained carbon materials with hydrogen peroxide and ammonia significantly increases the sorption capacity for heavy metal ions and radionuclides. Thus, developed on the basis of vegetative raw materials of Kazakhstan carbonized carbon materials have a great potential for detoxification of water contaminated with ions of heavy metals and radionuclides. Moreover, they offer the prospect of using these materials for the enrichment of rare and expensive metals. Owing to excellent sorption quality of such materials it is necessary to expand and deepen the study on the creation of properties of new carbon materials derived from the vegetative raw materials of Kazakhstan [27]. Nanostructured carbonaceous materials are produced and used as sorbents of general and selective purposes [28-38], hydrogenation catalysts and cathode materials for production of lithium batteries. In this work [39-43], a promising new material based on carbonization of apricot stones is obtained. Synthesis of nanomaterials is carried out by the methods of carbonization plant and carbonization of minerals [44-47] raw materials under various temperature conditions, using different catalysts, as well as in flames, at the facilities of various types. This material is characterized as a partially graphitized system with low crystallinity. The initial loss of the battery capacity based on a new electrode material is due to formation of a protective layer on the electrode surface which protects the anode from further destruction. The new anode material showed stable performance during prolonged cycling. Furthermore, this material showed 41

excellent compatibility with the electrolyte based on propylenecarbonate even at relatively high current densities. Further improvement of the performance of the material can be achieved by increasing its dispersion and surface modification of the anode. There also have been studied the processes of carbonization of mineral raw materials (Tonkereys, Chilik, Narynkol, and Saryozek clay) [48-49]. The degree of water purification from ions of lead reaches 85.2 %, copper ion – 94.4 %, and 98.4 % of nickel ions. In the case of carbonized vegetative raw materials the situation is as follows: the concentration of metal ions in water is from 9 to 7 µg/ml, the degree of sorption of lead carbonized by walnut shells is up to 93 %, nickel – 68 %, cadmium – 70 %, and cobalt – 83 % [50-51]. The main trends of development in science and technology is currently focused on the study of nanoscale structural components of matter and functional materials. Progress in this area is of great theoretical and practical importance because such materials can be widely used in ecology, biology and medicine and just because in these areas of science the greatest social and economic effect from the introduction nanomaterials is predicted. It should be noted that prospects for practical use of nanostructured sorbents are obtained by high temperature carbonization of cheap secondary vegetative raw materials. Carbon obtained by the carbonization of plant materials, keeps its original finely organized structure. By varying the conditions of carbonization, it is possible to obtain a complex of compositions of carbon from other materials. Some of these materials can find practical application. Availability and annual renewability of raw materials, low content of mineral impurities, developed porous structure, ecological production allow to obtain cheap, fast regenerable carbon sorbents [52-54].

2.1. Activated carbon based on raw materials A typical carbonization scheme with the following physical activation comprises raw materials preparation stages (separation, drying, cleavage), coking (carbonization without access for air) [55]. The chemistry of pyrolitic processes during carbonization is extremely complex and not fully clear. The main task of the 42

carbonization stage is the removal of easily fugitive components, the maximum increase of carbon density and obtaining of a material with a sufficiently large specific surface and porosity [56, 57]. We would only note that the general thermodynamic analysis of the pyrolysis of carbon-containing materials allows to distinguish three main groups of processes and mechanisms: 1) cracking and dehydrogenation reactions of non-aromatic molecules; 2) cyclization of hydrocarbon chains with n ≥ 6 in the aromatics with the side chains interruption; 3) polycondensation of the aromatics in more stable polynuclear arenas. Together these cleavage (destruction) and synthesis with the recombination (consolidation) reactions lead eventually to accumulation of flat formations of hexagons, structural elements of graphenes. At the same time, these reactions are accompanied by the educing of auxiliary gaseous products entrapping the part of the carbon. More complex transformations happen to the carbohydrate part of vegetable raw materials. The articles [58, 59] show a flow chart of possible chemical reactions behavior of cellulose during the activation. From the flow chart of chemical transformations during the activation presented in Figure 6 it is apparent that ctivation proceeds in four stages. During the first stage (T=25-150 °C) predominantly the process of moisture desorption from the surface takes place. Also, dehydration due to formation of water from hydroxyl and hydrogen groups and the growth of the local ordering of macromolecules mutual arrangement may occur. The processes proceeding at this stage are reversible. The second stage occurs in the temperature interval 150-240 °C and is accompanied by the intramolecular dehydration forming bonds –C=O and –C=C-. The temperature interval 240-400 °C corresponds to the third carbonization stage. In this temperature range macromolecules degradation processes proceed as the result of the destruction of 1,4 glycoside, cyclic –C-O-C and part of –C-C bonds according to the radical mechanism. These processes lead to the decomposition of the initial polymer to separate “rings” with the consequent formation of fragments C4 (-CH =CH-CH=CH-). Simultaneously with dehydration depolymerization competing reactions are possible resulting in the formation of levoglucosan. 43

Figure 6 – Scheme of chemical transformations during carbonization of cellulose [60]

44

It dramatically increases the yield of volatile resinous matters and reduces the final carbon content. During this stage the selection of various products including aromatic proceeds. The main processes of the fourth stage which proceeds in the temperature range 400-700 °C are aromatization with hydrogen evolution and condensation of fragments C4 in the “carbon polymer”, i.e. in turbostratic carbon layers. Condensation of the C4 fragments may occur according to two possible schemes: “longitudinal” and “transverse”. In the case of the “longitudinal” scheme C4 fragments link up forming a polymer chain. Binding of neighboring chains leads to formation of graphite-like layers. In the case of the “transverse” polymerization each fragment links up with its “copy” forming a carbon chain which grows in the transverse direction. Interaction of the neighboring chains creates new layers. All this indicates the complexity of processes occurring during the pyrolysis of even, such a “model” system as cellulose. In spite of nearly identical chemical composition the cellulose samples of different origins may be very distinct in structural and textural characteristics. It is natural to assume that the behavior of carbonization processes for plant samples of various types is much more complex and under the same conditions differs both well in the composition of products educed during pyrolysis and contained in the carbonized sample and as in structural characteristics of the carbonated coal. Nevertheless, the results of the elemental GC-MS analysis, the studies of sample mass changes during the process indicate that the process of carbonization of rice husk and apricot stones generally fits into the above-described scheme and is characterized by chemical transformations presented in Figure 6.

2.2 Characterization of pores in carbon materials Pores in carbon materials have been identified by different techniques depending mostly on their sizes. The techniques for identification of pores in carbon materials are summarized Figure 7. Pores with nano-meter sizes, i.e. micropores and mesopores, are identified by the analyses of gas adsorption isotherms, mostly of N, at 77 K. Other gases, such as CO2, H2, H2O, Ar and CH4 are also 45

used. For the analysis of adsorption/desorption isotherms of gases, different methods have been proposed to extract pore structure parameters, named as BET, DFT, t-plot, BJH, HK,etc. these pores detected by gas adsorption are only open pores. If some pores are too small to accept gas molecules, however, they can not be detected as pores by the adsorbate gas molecules. In other words, these pores are closed pores, which are sometimes called latent pores. By using gases with different molecular sizes, therefore, molecular sieve performance of porous carbons has been evaluated. The fundamental theories, equipments, measurement practices, analysis procedures, and many results obtained so far by gas adsorption have been reviewed in different publications. Small – angle X-ray scattering has an advantage to identify the closed pores, together with open pores. For macropores, mercury porosimetry has been frequently applied, but great care has to be paid to apply this technique to fragile carbon materials as exfoliated graphite. Identification of intrinsic pores between hexagonal carbon layers, interlayer space, in carbon materials, is carried out by X-ray diffraction. The use of internal standard of silicon is emphasized to be essential for accurate determination of the width of these pores. Recently, direct observation of pores on the surface of carbon materials was reported by using microscopy techniques, such as scanning tunneling microscopy (STM) and transmission electron microscopy (TEM) for micropores and mesopores and scanning electron microscopy (SEM) and optical microscopy for macropores. In order to evaluate pores, a large number of observations and statistical treatment are essential, coupled with image processing technique. By these techniques, not only the pore size and its distribution but also the shape and area of pore cross-section and smoothness of the pore wall can be evaluated [62- 64].

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Figure 7 – Identification techiques of pores in carbon materials [61]

Table 1 Advantages and disadvantages are summarized on each characterization technique Characterization technique Adsorption/desorption of N2 gas at 77K BET method BJH method

Advantages and disadvantages Give overall surface area (SS)

DFT method

Differentiate micropores and mesoporous and volume. Give pore size distribution in mesopore range. Give pore size distribution in a wide

HK method

Give pore size distribution

Adsorption/desorption isotherm of various gases (H2, He, CO2, CO. etc.) X-ray small-angle scattering Transmission electron microscopy

Give the information of molecular sieving performance

Detect micropores, both open and closed pores Detect nano-sized pores, even with the size less than 0.4nm. Give localized of information, need statistical analysis of data. Scanning tunneling Detect only pore entrances on the surface. Give microscopy morphological information of the pore entrance. Need statistical analysis with criteria. Scanning electron microscopy Detect only macropores Mercury porosimetry Defect mostly macropores. Difficult to apply for fragile materials.

STM observation gives only information of the entrance of pores on the physical surface of samples. For a quantitative assessment of these pores, criteria for the definition of pores have to be set up in order to distinguish them from depressions on the surface. The following two criteria were used, for example, in the analysis of the pore formation on the surface of glasslike carbon spheres during air oxidation: 1) pore wall must be so steep that its slope must be more than unity, and 2) pore must be so deep that the tip apex of the microscope can not reach its bottom. Defect formation by oxygen plasma treatment and ion bombardment was studied on the basal plane of natural graphite by STM observations. In a TEM image, taken on thin section with sufficiently high magnification, pores can 48

be recognized. Detailed studies were carried out mainly on activated carbon fibers by applying Fourier transformation on TEM images. SEM was successfully applied to characterize rigid intraparticle pores in worm-like particles of exfoliated graphite. For this observation, a novel procedure had to be developed to prepare the fractured surface of worm-like particles along the exfoliated direction. Distributions of cross-sectional area, lengths of major and minor axes, aspect ratio, and fractal dimension were determined as a function of exfoliation conditions. For flexible interparticle macropores in exfoliated graphite, the process consisting of impregnation of paraffin, thin-sectioning, and then transmission optical microscopic observation was successfully applied. Macropores in isotropic high-density graphite were analyzed on their polished cross-sections observed under optical microscope. Relationships of pore structure parameters determined through image processing on optical micrographs to mechanical properties of isotropic high-density graphites were discussed [65, 66].

References 2 1 Dubinin M.M. Adsorbents, their preparation, properties and application. – M., 1978. – p. 4-22. 2 Aygun A., Yenisoy-Karakas S., Duman I. Micropor. Mesopor. Mater. – 2003. – Vol. 66. – P. 189-195. 3 Guo Y., Yang S., Yu K., Zhao J., Wang Z., Xu H. Carbon materials for advanced // Materials Chemistry and Physics. – 2002. – № 74. – p. 320. 4 Daud W.M., Ali W.S. Carbon fiber produced from palm tree and coconut shell // Bioresour. Technol. – 2004. – No. 93. – p. 63-69. 5 Benaddi, H., Bandosz, T.J., Jagiello, J., Schwarz, J.A., Rouzaud, J.N., Legras, D. and Béguin, F.Surface Functionality of Wood and Carbon // Carbon. – 2000. – Vol. 38. – p. 669-674. 6 Asma B.M. Apricot Production. – Malatya Evin Ofset, Turkey, 2000. – 240 p. 7 Smisek M., Cerny S. Active Carbon Manufacture, Properties and Aplications. – New York: Elsevier Pub., Comp., 1970. – 370 p. 8 Khezami L., Chetouan, A., Taou, B., Capar, R. Cellulose, lignin, xylan // Powder Technol. – 2005. No. 157. p.48–56. 9 Hayashi J., Kazehaya A., Muroyama K., Watkinson A.P. Preparation of activated carbons from lignin by chemical activation // Carbon. – 2000. – No. 38. – p.1873-1878.

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10 Zhandosov J.M., Shabanova T.A., Shamalov M.E., Bijsenbaev M.A., Mansurov Z.A. Preparation of carbon material with high specific surface and study of synthesis products. Combustion and Plasma Chemistry. – 2010. – V. 8. – № 3. – p. 257-261. 11 Klijanienko A., Grabowska E.L., Gryglewicz G. Z. Development of the mesoporosity during phosphoric acid in Biosphere // Bioresour. – Technol. – 2008. – No. 99. – p. 7208-7214. 12 Jibril B., Houache O., Maamari R.A. and B.A. Rashidi Effects of H3PO4 and KOH in carbonization of lignocellulosic material // J. Anal. Appl. – Pyro. – № 83. – p. 151-158. 13 Azat S. Synthesis of Carbonized Nano Mesoporous Sorbents Based on Vegetable Raw Materials // Nanoscience and Nanoengineering International journal. – 2013. – № 1 (1). – pp. 41-44. 14 Azat S., Pavlenko V.V., Kerimkulova A.R., Mansurov Z.A. Synthesis and structure determination of carbonized nano mesoporous materials based on vegetable raw materials // Advanced Materials Research Vols. – 2012. – p. 535-537: Online available since 2012 / Jun / 14 at www.scientific.net 15 Fierro V., Fernandez V.T., Montane D. and Celzard A. Phenol. Micropor. Mesopor. Mater. – 2008. – Vol. 111. – pp. 276-284. 16 Cookson J.T. Carbon Adsorption Handbook. Edited by Cheremisinoff P.N., Ellerbusch F. – Ann Arbor Sci., Michigan. – 1980. – pp. 241-279. 17 Keltsev N.V. Basics of adsorption technology. – M .: Chemistry, 1976. – 512 p. 18 Greg S., Singh K. Adsorption, Specific Surface, Porosity. – M .: Mir, 1970. – p. 259. 19 Mansurov Z.A., Zhylybaeva N.K., Ualieva P.S., Mansurova R.M. Plant Raw Material // Chemistry for Sustainable Development. – 2002. – №3 – pp. 321-328. 20 Mansurov Z.A. Nanocarbon materials // Bulletin of the Kazakh National University. Chemical series. – 2003. – № 2 (30). – pp. 29-31. 21 Mansurov Z.A., Shabanova T.A., Mansurova R.M. Morphology of micronano particles of carbonized plant materials // Vestnik KazNU. Chemical series. – 2004. – № 2 (34). – pp. 129-135. 22 Basso M.C., Cerrella E.G. and Cukierman A.L. Ions from Dilute Aqueous Solutions // Activated Carbons from a Rapidly Renewable Biosource for Cadmium (II) and Nickel (II) Removal of Cadmium. Eng. Chem. Res. – 2002. – Vol. 41. – pp. 180-189. 23 Jia Y.F., Thomas K.M. Adsorption of cadmium ions on oxygen surface sites in activated carbon // Langmuir. – 2000. – № 16. – P. 1114. 24 Azat S., Kerimkulova A.R., Mansurov Z.A. Synthesis and determination of the structure of carbonized nanomaterials based on plant materials // VII International Symposium “Physics and Chemistry of Carbon Materials / Nanoengineering”, Almaty. – September 19-21. – 2012. – pp. 124-126. 25 Mansurov Z.A. Synthesis of carbon nanomaterials and their applied aspects. Vestnik. Chemical series. – 2008. – № 2 (50). – pp. 16-31. 26 Azat S., Mansurov Z.A. Wastewater treatment with carbonized nanosorbents // KazNU Bulletin, Chemical Series. – 2011. -№ 1 (61). – pp. 166-169.

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27 Samuranov M.M., Shilina Yu. A., Zhylybaeva N.K., Bijsenbaev M.A., Shabanova T.A., Ryabikin Yu.A., Zashkvara O.V., Mansurova R.M., Mansurov Z.A. A. Nanostructured carbonized multifunctional sorbents. Vestnik NAN RK. Chemical series. – 2006. – №4. – pp. 35 – 41. 28 Bevla F.R., Rico D.P., Gomis A.F. Activated Carbon from Almond Shells // Chemical Activation. Activating Reagent Selection and Variables Influence, Ind. Eng. Chem. Prod. Res. Dev. – 1984. – Vol. 23. – pp. 266-269. 29 Laine J., Calafat A. and Labady M. Preparation and Characterization of Activated Carbons from Coconut Shell Impregnated with Phosphoric Acid // Carbon. – 1989. – Vol. 27, No. 2. – pp. 191-195. 30 Blasco J.M., Cordero T., Gomez Martin J.P. and Rodriguez J.J. A Kinetic on Chemical Activation of Holm Oak Wood // J. of Anal. And Appl. Pyroly. – 1990. – Vol. 18. – pp. 117-126. 31 Balcı S., Doğu T., Yücel H. Characterization of Activated Carbon Produced from Almond Shelland Hazelnut Shell // J. Chem. Tech. Biotechnol. – 1994. – Vol. 60. – pp. 419-426. 32 Toles C.A., Marshall W.E., Johns M.M. Granular Activated Carbons from the Nutshells for the Metals and Organic Compounds // Carbon. – 1997. –Vol.35, No. 9. – pp. 1407-1414. 33 Girgis B.S., Daifullah A.A. Phenols by Activated Carbon Obtained from the Agricultural Waste // Wat. Res. – 1998. – Vol. 32. – pp. 1169-1177. 34 Toles C.A., Marshall W.E., Johns M.M., Wartelle L.H., McAloon A. AcidActivated Carbons from Almond Shells: Physical, Chemical and Adsorptive Properties and Estimated Cost of Production // Bioresource Technology. – 2000. – Vol. 71, Issue 1. – pp. 87-92. 35 Iniesta E., Sanchez F., Garcia A.N., Marcilla A. Yields and CO2 Step Carbonization Process. Effect of Different Chemical Pre-Treatments and Ash Content // J. of Anal. and Applied Pyrolysis. – 2001. – Vol. 58-59. – pp. 983- 994. 36 Özer A., Çam G. The Activated Carbon from the Sugar Beet Pulp Treated with Phosphoric Acid // F. Ü. Müh. Bil. Der. – 2002. – Vol. 14 (1). – pp. 191-197. 37 Yang T., Lua A.C. Characteristics of Activated Carbons Prepared from Pistachio-Nut Shells by Physical Activation // Journal of Colloid and Interface Science. – 2003. – Vol. 267, Issue 2. – pp. 408-417. 38 Onal Y., Akmil-Basar C., Sarici-Ozdemir C. Elucidation of the naproxen sodium carbonic acid, equilibrium and thermodynamic characterization // Journal of Hazardous Materials. –2007. – № 148. – P. 727. 39 Demirbas E., Kobya M., Sulak M.T. Hydrogen source carbon fiber activated carbon // Bioresource Technology. – 2008. – No. 99. – p. 5368. 40 Soleimani M., Kaghazchi T. Activated Hard Shell of Apricot Stones: A Promising Adsorbent in Gold Recovery // Chinese Journal of Chemical Engineering. – 2008. – № 16 (1). – P. 112. 41 Sentorun-Shalaby C., Ucak-Astarhoglu M.G., Artok L., Sarici C. Methods of Activation and Specific Applications of Carbon Materials // Microporous and Mesoporous Materials. – 2006. – № 88. – P. 126. 42 Kobya M., Demirbas E., Senturk E., Ince M. Adsorption of heavy metal ions from aqueous solutions by activated carbon prepared from apricot stone // Bioresource Technology. – 2005. – № 96. – P. 1518.

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43 Mansurov Z.A. Some Applications of Nanocarbon Materials for Novel Devices // Gross R. et al (eds.). Nonoscale Devices – Fundamentals.- Springer. – 2006. – P. 355-368. 44 Guo Y., Yu K., Wang Z., Xu H. Effects of activation conditions on preparation of porous carbon from rice husk // Carbon. – 2003. – № 41. – P.1 645. 45 Yalcin N., Sevinc A. Studies of the surface area and porosity of carbons prepared from rice husks // Carbon. – 2000. – № 38. – P. 1943. 46 Faust S.D., Aly O.M. Chemistry of Water Treatment. – Woburn: Butter Wort Pub., 1983. – Р. 113. 47 Hameed B.H., Rahman A.A. Removal of phenol from aqueous solutions by adsorption onto activated carbon prepared from biomass material // J. Hazard. Mater. – 2008. – Vol. 12. – Р. 34-40. 48 Tazhkenova G.K., Mansurov Z.A., Mansurova R.M., Erkasov R.Sh., Sydykov M.Kh. // Sorption of metal ions of Carbon based on Tonkerys clay. International Symposium dedicated to the 100th anniversary of academician A. B. Bekturov. – 2001. – Almaty. – P.195. 49 Azat S., Seitzhanova M.A., Kerimkulova M.R., Kerimkulova A.R., Mansurov Z.A. Development and study of the physico-chemical characteristics of sorbents based on carbon, clay and silver compounds / Proceedings of the VIII International Symposium "Physics and Chemistry of Carbon Materials / Nanoengineering, – Almaty. – September 17-19. – 2014. – P. 199-203. 50 Tazhkenova G.K., Urmashev B.A., Urazalin A.K., Biisenbaev M.A., Mansurov Z.A., Mansurova R.M., Erkasov R.Sh. Study of the process of absorption of heavy metal ions by carbon-mineral sorbents // II Int. simp "Combustion and plasma chemistry". – Almaty. – 2003. – pp. 214-219. 51 Tazhkenova G.K., Mansurova R.M., Erkasov R.Sh., Ryabikin Yu.A., Sergaziev A.D. / IR and EPR spectroscopic study of carbon-containing Tonkerys clay, I International Symposium “Combustion and Plasma Chemistry. – Almaty. – 2001. – pp. 220-223. 52 Aygun A., Yenisoy-Karakas S., Duman I. Production of granular activated carbon from fruit stones and nutshells and evaluation of their physical, chemical and adsorption properties // Micropor. Mesopor. Mater. – 2003. – № 66. – pp. 189–195. 53 Razvigorova M., Budinova T., Petrov N., Minkova V. Purification of water by activated carbons from apricot stones, lignites and anthracite // Water Res. – 1998. – № 32. – P. 2135–2139. 54 Azat S., Busquets R., Pavlenko V.V., Kerimkulova A.R., Raymond L.D., Whitby, Mansurov Z.A. Аpplications of activated carbon sorbents based on greek walnut // Applied Mechanics and Materials. – 2014. – Vol.467. – Р. 49-51. 55 Azat S., Meldebekova G.S, Kerimkulova M.R, Seytzhanova M.A, Kerimkulova A.R, Mansurov Z.A., Study of properties of carbonated sorbents based on rice husk. Bulletin of KazNU, biology series. – 2014. – № 1/2 (60). – pp. 3-6. 56 Tseng R.L., Tseng S.K., Wu F.C., Hu C.C., Wang C.C. / Effects of micropore development on the physicochemical properties of KOH-activated carbons / J. chen. Inst. Chem. Eng. – V.39. – P. 37-47. 57 Kennedy L.J., Vijaya J.J., Sekaran G. Effect of two stage process on the preparation and characterization of porous carbon composite from rice husk by

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phosphoric acid activation // Industrial & Engineering Chemistry Research. – 2004. – № 43. – P.1832. 58 Kolyshkin DA, Mikhailova KK Active Words: Properties and Methods of Testing: Advocacy. – L. Chemistry, 1972. – P.37. 59 Mahorin K.E, Pishchai I.Ya. Physico-chemical properties of carbonaceous adsorbents // Demineralization water. – 1996. – №2. – pp. 74-83. 60 Orfao J.J., Antunes F.J., Figueiredo J.L. Pyrolysis kinetics of lignocellulosic materials-three independent reactions model // Fuel. – 1999. – № 78. – P. 349-358. 61 Michio Inagaki. Pores in carbon materials – Importance of their control / New carbon materials. – 2009. – V.24., No.3. – P. 194-222. 62 Donnet J.B., Papirer E., Wang W., et al. The observation of activated carbons by scanning tunneling microscopy. Carbon. – 1994. – V.32. – P. 183-184. 63 Oshida K., Kogiso K., Matsubayashi K., et.al. Analysis of pore structure of activated carbon fibers using high resolution transmission electron microscopy and image processing. О Mater Res. – 1995. – V. 10. – P. 2507-2517. 64 Inagaki M., Suwa Е. Pore structure analysis of exfoliated graphite using image processing of scanning electron micrographs. – Carbon. – 2001. – V. 39. – P. 915-920. 65 Oshida K, Ekinaga N, Endo M. et.al. Pore analysis of isotropic graphite using image processing of optical micrographs. TANSO. – 1996. – № (173). – P. 142-147. 66 Vignal V., Morawski A.W., Konno H., et.al. Quantitative assessment of pores in oxidized carbon spheres using scanning tunneling microscopy. JMater Sci. – 1999. – № 14. – P. 1102-1112.

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3

  OBTAINING OF ACTIVATED CARBONS     BASED ON VEGETABLE RAW            MATERIALS AND RESEARCH            METHODS   3.1. Activation process of raw materials In this work, a series of experiments on the physical and chemical activation patterns of raw materials was performed. Physical activation of raw materials samples was performed in a rotating stainless steel reactor of 0.5 dm3 at 19 rpm. Carbon dioxide gas, was chosen as an activating agent which is supplied from the bubbler into the reaction zone at a rate of 50 cm3/min. Carbonization was carried out in a horizontal pyrolysis installation with adjustable electric heating (Figure 8) in the temperature range from 400 to 900 °C, duration 1 hour after carbonization yield at a predetermined temperature. For the experiment, crushed walnut shells, highlighting the sieving of the product of crushing the working fraction with a diameter of 2-4 mm [1].

Figure 8 – Setting the carbonization of vegetable raw materials

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Activated carbon (AC) does not just happen; it has to be synthesized. The porosities of carbon, as initially prepared by carbonization, are not sufficiently developed for most applications and some amelioration is a prerequisite step. This is done in several ways involving creation of further porosity, widening of existing porosity, modifications to the surfaces of porosities and also modifying the carbonization process itself. There are two industrial processes used to maximize the adsorption potential of carbonaceous material.

3.1.1 Physical activation of raw materials Thermal or physical activation is a process of selective gasification (removal) of individual carbon atoms. Physical activation is carried out using either carbon dioxide or steam or mixtures of these two gases. Carbon atoms can be removed from within porous carbons by gasification using carbon dioxide or water vapor, usually at 800-900 oC according to the following stoichiometric equations, equation I and II. Activation by carbon dioxide and steam produces carbons with different porosities. 2C + 2H2O  2CO + 2H2; C + H2O  CO + H2

H  +159 kJmol

1

H  +117 kJmol

2

3.1.2 Chemical activation Chemical activation is a process of co-carbonization by additions of such material as phosphoric acid (H3PO4), or zinc chloride (ZnCl2) or potassium hydroxide (KOH) and potassium carbonate (K2CO3). Mechanism for these activations is all different, with zinc chloride promoting the extraction of water molecules from the lignocellulosic structures of parent materials, and phosphoric acid combining chemically within the lignocellulosic structures. There is no selective removal of carbon atoms as during physical activation and carbonization yields are improved. 55

As some industrial adsorption processes require “fine-tuning” of the porosity of a carbon, it is possible to combine thermal and chemical activation processes to obtain a desired activated carbon [2].

Figure 9 – Installation for chemical activation of vegetable raw materials

3.2 Methods of studying the physicochemical properties 3.2.1 Fourier – IR spectroscopy FTIR spectra were recorded using specially pressed tablets consisting of KBr, and the test substance previously crushed to obtain a fraction of ~ 50 microns. Measurements were conducted in the wave number range 4000-450 cm-1 at room temperature on the device «Spectrum 65” of the firm PerkinElmer.

3.2.2 Method of scanning electron microscopy Research of surface morphology in the obtained samples were carried out on a microscope QUANTA 3D 200i (FEI, USA) with an accelerating voltage of 30 kV, Figure 10. 56

The samples were fixed on a copper holder with a conductive glue or tape. Previously on the sample surface in a special vacuum system Zeiss Sigma, using emission gun SEM (Zeiss NTS) with an accelerating potential of 5 kV was applied to the conductive thin platinum layer thickness of 4 nm to eliminate charging effects.

Figure 10 – Scanning electron microscopy

3.2.3 The method of low-temperature nitrogen adsorption Information on the micro-mesoporous texture (area of 17 to 3,000 Ǻ) samples of carbon materials was obtained by lowtemperature nitrogen adsorption-on device Autosorb 1 (Quantachrome, USA) after preliminary treating samples and 150200 0C performed at a residual pressure of less than 0.001mer.colmn, Figure 11. Further, measurements of adsorption isotherms of nitrogen were made at liquid nitrogen temperature, 77 K, in the range of relative pressures between 0.005 and 0.991 and their standard processing using the theory of BET (Brunauer-Emmett-Teller) for calculating the total specific surface area SBET; on the model of Barret-Joyner-Halenda (BJH), the adoption of a conditional model of cylindrical pores to calculate the volume of mesopores (Vmeso) and average pore diameter (dav); by the method of Dubinin-Radushkevich (DR) to calculate the volume of micropores (Vand also on the method of DFT (Density Functional Theory – DFT) with the 57

adoption of models of slot / cylindrical pores (QSDFT) for calculating the specific surface area (SDFT) and pore volume (VDFT). Accuracy of the method is 10%.

Figure 11 – The device is low-temperature nitrogen adsorption autosorb-1

3.2.4 Аutomated mercury porosimeter The pore structure of activated carbon materials has been investigated using a mercury porosimeter PoreMaster (Quantachrome Instruments, USA), Figure 12. The operation of all mercury porosimeters is based upon the physical principle that a nonreactive, non-wetting liquid will not penetrate into fine pores until sufficient pressure is applied to force its entry. The relationship between the applied pressure and the pore diameter into which mercury will intrude is given by the Washburn equation: D = (-4γ cos θ)/P, where P is the applied pressure, D is the pore diameter, γ is the surface tension of mercury (480 dyne cm-1) and θ is the contact angle between mercury and the pore wall, usually taken as 140º. Monitoring mercury volume intruded as a function of pressure permits the generation of pore size/volume distributions from the Washburn equation.

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Figure 12 – Device PoreMaster a mercury porosimeter

3.2.5 Sorption activity of methylene blue on activated carbon Before carrying out the analysis standardized test solutions were prepared for the construction of the calibration curve. Construction of the calibration curve. Blank solutions are prepared for the construction of calibration curve. For this purpose in 10 volumetric flasks of 50 ml each, 0.5; 1.0; 1.5; 2.0; 3.0; 4.0; 5.0; 6.0; 7.0; 8.0 ml of methylene blue (MB) solution (1500 mg/l) are infused, then the volume is made up to the mark with water at a temperature of 20 ± 2 °C. The obtained solutions contain 1l, respectively, 15; 30; 45; 60; 90; 120; 150; 180; 210; 240 mg/l. Optical density of the obtained blank solutions is measured on photoelectric colorimeter with a blue colour filter with a wavelength of (λ) of 400 nm in cuvettes with the distance between the active faces of 10 mm. Distilled water is used as a test solution. According to the obtained data the calibration curve of optical density dependence on the concentration of the blank solution is constructed. The data on the dependence of optical density on the concentration of the calibration solutions of MB. Carrying out an analysis. Approximately 0.1 g of carbon, predried and crushed in an agate mortar, is weighed with an accuracy of less than 0.001 g. A weighed sample of carbon is placed in a conical 59

flask of 50 ml with addition of 25 ml methylene blue solution (1500 mg/l), stoppered and stirred for 20 minutes. After shaking, the carbon slurry is transferred to a centrifuge tube and centrifuged for 15 minutes, 5 ml of clarified solution gently pipetted, and its absorbance is determined on the same photoelectric colorimeter. If the optical density of clarified solution is greater than 0.8 absorbance units, then 5 ml of this solution is transferred into a volumetric flask of 25 ml or 50 ml, depending on its optical density. The solution in the flask is diluted with distilled water up to the mark. The optical density of the solution after dilution should be from 0.1 to 0.8 absorbance units. The dilution factor in this case is equal to 5 or 10. Upon optaining of the value of optical density, using the calibration curve the residual concentration in the methylene bleached solution is determined. Activated carbon adsorption of methylene blue (X) in milligrams of methylene blue per 1 g of activated carbon is calculated by the formula: Х = 0.025* (С1 – С2*К) /m

(3)

wherein: С1 – concentration of original dye solution (1500 mg/l); С2 – concentration of the solution after contacting with carbon, mg/l; K – dilution factor taken for analysis, after contacting with carbon (if necessary, the solution is diluted 10 times); m – mass of activated carbon weighed portion, g (≈0.1 g); 0.025 – amount of methylene blue solution, taken for lightening, l. For the analysis result, the arithmetic mean of two parallel determinations is taken, permissible differences between them should not exceed 10 mg/g.

3.2.6 Chromatographic separation of fusicoccin For the experiment, 1 kg of seeds of spring wheat cultivar “Steklovidnaya-24” was taken. The seeds which were soaked for one day in a sterile cool tap water supplemented with 1 mM 6-BAP (6benzyl aminopurine). 6-BAP was added as an inducer of the BS. Then germinated seeds were homogenized in 3 liters of 70% ethanol on the blade type homogenizer MPW-302 (Poland). The homogenate 60

was centrifuged for 10 minutes at 10,000 x g. The resultant alcoholic extract was purified by column nanostructured carbon sorbent of size ø 3 cm by 20 cm which was previously equilibrated with distilled water. In the column was applied to 100 ml of alcohol extract. Then to completely remove unbound materials the column was washed with 200 ml of 10% ethanol, desorption of BS was conducted with 50% ethanol, then for complete removal of all substances that final column was washed with 96% ethanol. Sorption and desorption were carried out on a glass column of their own manufacture, control of the elution was carried out on type UV monitor Uvicord SII manufactured by LKB (Sweden). In the presented work was used a column manufactured as follows (Figure 13).

Figure 13 – The apparatus UV detecter connected with column chromatography

For a glass tube a rubber tube so of the desired size was selected that it enters the tube only half or less. Then, with the narrow end of the plug blade we chose gentle concave surface, deepening the center plug, and then polishing the surface with sandpaper and the exact center of the plug was inserted into the plastic nozzle or syringe with a diameter of 1.5-2.5 mm. The nozzle must be inserted so that one end accounted for exactly the bottom surface of the funnel, and the other free exit from the wider end of the plug. If possible, it is desirable to have a plastic plug screw thread, which can be tightly 61

screwed to various connecting plastic tubing or plug. After making the bottom plug with a hose, the narrow end of its snug nylon fabric (preferably from the children’s bow) was inserted into the cork with a cloth in the lower end of the column. After the plug firmly, but not much comes to a column sticking out of the column pieces of nylon fabric carefully cut with a razor cap more tightly planted on the column and the column was checked for leaks. It was very convenient to use the plug with the union in the upper part of the column. In this case, the cut surface of the funnel is not necessary, and the nozzle or needle is located on both sides of the plug. The top tube provides complete sealing of the column and continuity in the elution solution from a supply vessel with a minimum hydrostatic pressure [3].

3.2.7 High performance liquid chromatography (HPLC) HPLC is basically a highly improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres, Figure 14. That makes it much faster.

Figure 14 – High performance liquid chromatography

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It also allows to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the stationary phase and the molecules flowing past it. This allows a much better separation of the components of the mixture.

3.2.8 The mass spectrometric analysis method Mass spectrometric analysis of chromatographically extracted compounds was performed on an apparatus Agilent 1100 mass spectrometer trap Esquire 3000 plus. Type ionization – electrospray, flow rate 0.4 ml / min., Temperature – 35 0C. The total time of the experiment was 25 minutes. HPLC conditions: 2 minutes – 10% AcN, 100 minutes – 100% AcN, 15 minutes – 90% AcN, 18 minutes – 80% AcN, 21 minutes – 50% AcN. This method is based on chromatographic separation of peptides (fragments in solution) on column converts an RP-18 phase. Pre-dried sample was diluted by a buffer containing 10% aqueous solution of formic acid. The measurement error is 3%. A separation method using LC-MS with stationary phase using fused core particles has been developed. The column was an Ascentis®Express C18 (2.1mmx100cm, 2.7 μm). The advantages of that type of stationary phase is that it provides high peak efficiency, equivalent to a stationary phaste if 1.7 μm particles, but with the need of less inlet pressure. The fragmentation of fusicoccin with multistage mass spectra (MS2 and 2 independent fragmentation pathways in MS3) with an ion trap have been studied to provide a reliable identification even in presence of possible interferences from the matrix. We have attempted to identify fusicoccin in ethanol extracts obtained from wheat (solid extract) and in these extracts spiked with standard.

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3.3 Synthesis of carbon sorbents and using for fusicoccin extraction, experimental results and their discussion 3.3.1 Computer modeling of activated carbon for fusicoccin We have retrieved an X-ray structure of fusicoccin from the Brookhaven Protein Database (entry 3IQV) and optimised its geometry using molecular mechanics (MMFF) followed by semiempirical calculations (PM3). The size of the molecule (to its van der Waals limit) is about 1.9 x 1.5 x 1.1 nm. As it can rotate freely in solution it is unlikely to be bound within pores that are smaller than the compound’s largest dimension. Overlaying the molecule on a model of a graphene sheet and removing carbon atoms until fusicoccin just fits leaves a pore just over 2.1 nm in diameter. To allow for any solvent and rapid molecular rotation, we would suggest a pore size of 2.5 nm as the minimum that could be expected to bind the molecule.

Figure 15 – Microporous activated carbon and fusicoccin molecular

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According to the results of computer modeling, cleaning composite components of fusicoccin using microporous carbon adsorbents not suitable as the size of the molecule of fusicoccin more than micropores. And in the following figure (Figure 16) the optimum pore size for purification of constituents of fusicoccin was determined by computer simulation. When cleaning the composite components of fusicoccin using microporous carbon adsorbents, computer modeling was first used.

Figure 16 – The optimal pore size of carbon for column chromatography

As the results of computer simulation in this work study show that the most effective recognized mesoporous activated carbons.

3.3.2 Determination of specific surface area of samples of carbon materials Sorbtometre-M Summary thermal desorption method is that a mixture of argon with carrier gаs of helium uptake of argon the sample is cooled to liquid nitrogen temperature. Cooling reduces the concentration of argon in the mixture flowing through the meаsuring cell detector that registered the adsorption peak by potentiometer and noted it on a chart. After determining the adsorption equilibrium the sample is 65

heated to room temperature. Concentration of argon аs a result of desorption increаses, and this change in the concentration is recorded on the diagram аs a peak of desorption, directed in the opposite side with respect to the peak of adsorption. The square of obtained peaks are proportional, respectively, the adsorption and desorption amount of the argon. The calculation of the specific surface area of the sample is made by area of desorption peak, like less blurred, using the calibration graph constructed in the coordinates: the peak area related to the hitch, the specific surface of adsorption m2/g. Investigated the specific surface of samples obtained vapor-gаs and chemical (phosphoric acid) activation WSh-600, RH-900, АS600, АS-500, WSh-600. Table 2 shows the data obtained by device Sorbtometer-M. Table 2 The data on the specific surface area of the samples of carbon materials Sample WSh-600 RH-900 АS-600 АS-500

Sample weight (gram) 0.0534 0.0556 0.0552 0.0589

Specific surface area (m2/g) 216.623 165.303 207.025 178.510

Pore volume (cm3/g) 0.028 0.246 0.089 0.065

The characteristics of non-activated materials are presented in Table 3. The nonactivated carbon materials based on RH, and AS and based on WSh differ little from each other in specific surface and little porous substances. Table 3 The specific surface and sorption capacity of the non-activated carbon materials Sample WSh АS RH

Specific surface, m2/g 0.32 0.34 0.32

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The average pore size, m2/g 1.714 1.710 1.710

Sorption capacity, cm3/g 0.07 0.10 0.09

Terms passage of the activation process of carbon materials have been selected in such a way that after activation the carbon material had a high surface area and save a high mechanical strength. Previously, it was found that treatment of carbon materials with inorganic activating reagents was followed by the increase in their specific surface area to 1000 m2/g. Changing the ratio of the activating reagent and the activation temperature rise to 950 °C leads to the fact that the reaction of carbonization proceeds simultaneously with oxidation of the material. The combined influence of two processes leads further to the growth of the specific surface to 12002100 m2/g (Table 4), however, it reduces the mechanical strength of the samples. Table 4 The specific surface area of the activated carbon samples T ºC 650 650 650 650 650 750 750 750 650 650 650 650 650 650 650 650 650 950

Sample WSh/КОН 1/3 AS/КОН ½ AS/КОН ½ AS/КОН ground ½ WShКОН ½ WSh/ Н3PO4 ½ RH/Н3PO4 ½ RH/ Н3PO41/3 WSh/КОН 1/3 RH/КОН ¼ WSh/КОН ¼ RH/КОН ¼ АS/КОН ¼ WSh/NaOH ¼ АS/NaOH ¼ AS/KOH ¼ AS/КОН ¼ RH/K2CO3 ½

Specific surface,m2/g 676.929 373.530 313. 258 384.791 290.677 793.748 739.680 634.900 1276.301 1233.500 1652.500 754.900 1942.900 2099.300 1468.800 592.900 785.420 475.200

Characteristics of carbon materials after activation given in Table 5.

67

Table 5 Characteristics of carbon materials after activation Sample

Sample weight (gram)

WSh:NaOH (4:1) AS:КОН (1:4) WSh:КOH (1:3) WSh:КOH (4:1) RH:K2CO3 (1:2)

2099 1943 1200 1652 475

Specific surface area (m2/g) 311 360 237 345 268

3.3.3 Methylene blue adsorption As a result, activation of raw materials significantly changed the value of specific surface area and sorption capacity (Table 6). If before activation the samples have sorption capacity by the methylene blue 0.07-0.10 cm3/g, after activation the samples showed sorption capacity of 268.75 – 400 mg/g. Some activated carbon had a mesopore (2 to 5 nm) structure which adsorbs medium size molecules, such as methylene blue. Thus, the quantity of methylene blue which is adsorbed onto absorbent also indicated the number of mesopore. Table 6 Data on the dependence of optical density on the concentration of MB Сonsentration, mg/l Optical density

15

30

45

60

90

120 150 180 210 240

0.025 0.05 0.09 0.13 0.2 0.29 0.37 0.45 0.52 0.59

The calibration curve of linear dependence of the optical density (D) of the concentration I solution of MB (the law of BouguerLambert) is shown in Figure 17.

68

Figure 17 – The calibration graph of dependence D for solutions of MB Table 7 The methylene blue value of activated carbon for adsorption ability Sample АS 8000C АS 7000C АS 6000C WSh Rice husk WSh:NaOH (4:1) AS:КОН (1:4) WSh:КOH (1:3) WSh:КOH (4:1) RH:K2CO3 (1:2)

Adsorption capacity mg/g 332 325 272 310 305 400 360 237 345 268

Next figure shows that the highest value of methylene blue was WSh:NaOH (4:1), compared to other types. It was 400 mg/g. The activated carbon had a higher surface for adsorption of methylene blue because of large numbers of pores.

69

Figure 18 – The methylene blue value of activated carbon for adsorption ability

3.3.4 The investigation of physico-chemical properties of the obtained nanoporous carbon sorption materials. Selection of the optimum conditions for obtaining modified sorption materials To reveal the morphological and structural features of carbon materials, the method of electron microscopy was used. Investigations were carried out on the microscope Quanta 3D 200iDualSystem, FEIso with the built system of energy-dispersive microanalysis. As seen in Figure 19-22, all the samples of the carbon materials have non smooth and rough surface with a high porous structure. As seen from the photomicrograph the increase ratio of activating reagent destroys the porous structure and reduces the mechanical strength of the samples. With the help of modern energy-dispersive spectrometer of the type Jidda-2300 EDS company «JEOL», Japan, which is an additional device of scanning electron microscope the elemental composition of the carbon samples was determined before and after activation. The results of these studies are presented in Table 8.

70

a)

b)

Figure 19 – Electron micrographs of carbon samples from AS, treated with potassium hydroxide in a ratio of 1: 2 (a) and at a ratio of 1: 4 (b)

a)

b)

Figure 20 – Electron micrographs of carbon samples from RH, treated with potassium hydroxide in a ratio of 1: 2 (a) and at a ratio of 1: 4 (b)

a)

b)

Figure 21 – Electron micrographs of carbon samples from WSh, treated with potassium hydroxide in a ratio of 1: 2 (a) and at a ratio of 1: 4 (b)

71

a)

b)

Figure 22 – Electron micrographs of carbon samples RH:H3PO4 (1:3) (а) and RH:H3PO4 (1:2) (b) Table 8 The elemental composition of the samples of activated carbon materials Elements At%

АS

WSh

RH

C O Na S K Р Fe

62.16 34.67 1.54 0.92 0.71

62.87 34.71 0.73 0.17 0.66 0.58

58.90 36.47 1.59 1.97 1.07 -

АS Activ. 91.54 8.46 -

WSh Activ. 92.07 6.85 0.33 0.48 0.28

RH Activ. 84.43 12.65 0.89 0.85 1.17 -

Table 8 shows, cetonitrile resulted in a substantial reduction of the amount of oxygen and sulfur, which are volatilized during carbonization, this beneficially affecting the quality of the carbonized material. If before carbonization the content of carbon is 62%, after carbonization it is 91%.

3.3.5 The study of sorption characteristics of carbon materials One of the tasks was to control the texture-sorption characteristics of the materials produced – namely, the mesopore volume according to mercury porosimetry, the nature of nitrogen 72

adsorption isotherms and pore size distribution, surface area, volume and average pore diameter. During the work samples of activated carbon from rice husk, walnut shells and apricot stones were synthesized. Samples AS-500, AS-600 and AS-700 were obtained by carbonization in current of argon for 1 hour at temperatures of 500, 600, 700 °C, respectively. Activated carbons (CRH – 600, 700, 800, 900) have been obtained by carbonation in the current of argon for 1 hour at a temperature corresponding to the indices of samples, followed disilication 1M NaOH. In Figure 23 shown a graph of pore size distribution of samples from apricot stones at 500 0C according to the mercury porosimetry. In Figure 24 shown a graph of pore size distribution of samples from apricot stones at 600 0C according to the mercury porosimetry.

Figure 23 – The pore size distribution of samples from apricot stones at 500 0C, according to the mercury porosimetry

According to the mercury porosimetry, in all samples of carbon materials derived from apricot stones, peaks are observed in the range of pore distribution of from 7 to 15 nm, indicating that the mesoporous materials, although substantial amounts of pores attributable to a range of 70 to 100 nm for sample AS-500 and 100 to 300 nm for the other samples correspond to small macropores. 73

Figure 24 – The pore size distribution of samples from apricot stones at 600 0C, according to the mercury porosimetry in Figure 25 shown a graph of pore size distribution of samples from apricot stones at 700 0C according to the mercury porosimetry.

Figure 25 – The pore size distribution of samples from apricot stones at 700 0C, according to the mercury porosimetry

74

In Figures 26-29 shows the pore size distribution of samples of rice husk obtained under various temperature settings in a stream of argon.

Figure 26 – Pore size distribution of samples of rice husk, at 500 0C, according to the mercury porosimetry

Figure 27 – Pore size distribution of samples of rice husk, at 600 0C, according to the mercury porosimetry

75

Figure 28 – The pore size distribution of samples of rice husk, at 700 0C, according to the mercury porosimetry

Figure 29 – The pore size distribution of samples of rice husk, at 800 0C, according to the mercury porosimetry

From the presented curves of pore size distribution (Figures 31 and 32) it is seen that the samples obtained from carbonized rice husk by leaching silica are more meso and macropores, as compared 76

with samples of apricot stones, and have pores in the range of 7 to 80 nm. In Figure 30 shows the pore size distribution of samples from walnut shells, obtained at 700 0C degrees under argon. From the presented curves of pore size distribution (Figure 30) follows shows that the sample obtained from carbonized walnut shells by physical activation, have more meso pore compared with samples of apricot stones and rice husk, and have pores in the range from 1 to 7 nm. Further study of porous samples were performed by lowtemperature nitrogen adsorption for the best carbonized samples from rice husk and apricot stones.

Figure 30 – The pore size of the carbon material from walnut shells

Table 9 shows the values of textural properties of the samples according to the low-temperature nitrogen adsorption. Standard processing was performed by the BET method using a conventional cylindrical pore model (BJH) with the calculation of the total specific surface area, total pore volume; dav median pore diameter (based on micro- and mesopores), and density functional theory (DFT) with calculation with a total specific surface area, total pore volume.

77

Table 9 Textural properties of the samples according to data of nitrogen adsorption Sample АS-500 АS-600 CRH-800

SBET, m2/gr 230 393 641

VBJH, cm3/gr 0,246 0,347 0,540

SDFT, m2/gr 215 320 626

VDFT, cm3/gr 0.295 0.426 0.684

VMIP, cm3/gr 0.0498 0.0638 0.1681

Diameter of pore, nm 3.3 3.1 3.0

The table also shows the data on the volume of pore according to the mercury porosimetry.

Figure 31 – The isotherms of adsorption and desorption of nitrogen samples AS-500

From the above table it follows that the sample of AS-600 has a higher BET specific surface area (393 cm2/g) and pore volume than the sample of AS-500, due to the higher temperature carbonization. The sample CRH-800 has a surface area of 641 cm2/g BET, and a large pore volume according to mercury porosimetry, which corresponds to the graph of the distribution of pore sizes (Figure 29). Figures 32-34 shows that the adsorption / desorption isotherms of nitrogen, which are classified by the International Union of Pure and Applied Chemistry (IUPAC), belong to the first type. 78

In Figure 32 presents the isotherms of adsorption and desorption of nitrogen samples AS-500. In Figure 33 presents the isotherms of adsorption and desorption of nitrogen samples AS-600. In Figure 33 presents the isotherms of adsorption and desorption of nitrogen samples CRH-800. According to the method of nitrogen adsorption the method accounts for the largest number of pores per mesopores, evidenced by the results of measurements of the surface of the pore volume and shape of the hysteresis loop of the formed isotherms. For samples AS-500, AS-600 and CRH-800 were built nitrogen adsorption isotherms as shown in Figure 34.

Figure 32 – The isotherms of adsorption and desorption of nitrogen samples AS-600

79

Figure 33 – The isotherms of adsorption and desorption of nitrogen samples CRH-800

Figure 34 – The isotherms of adsorption and desorption of nitrogen samples

The adsorption isotherm of nitrogen and water of the sample WSh are shown in Figure 35. 80

1 adsorption-desorption of liquid nitrogen, 2 adsorption-desorption of water Figure 35 – The adsorption isotherm of nitrogen and water on the sample WSh

When considering the isotherms of adsorption-desorption of nitrogen, it turns out that there occurs a capillary condensation, due to the presence of mesopores (2-50 nm) in material of walnut. This is evidenced by the shape of the hysteresis loop formed by the adsorption and desorption branches of the isotherms that classification BDDT refer to the 4th type, characteristic of the micromesoporous materials. – Water contains information about the surface chemistry of the sample (but nitrogen can not). - The kinetic diameter of water molecules is less than that of nitrogen molecules, so water can pass more deeply into the very small micropores. On the other hand, water can fill only small mesopores (while nitrogen may completely fill mesopores (2-50 nm)). Figures 36-38 shows the differential pore size distribution in the samples of the AS-500, AS-600 and 800-CRH. The figure shows that all samples have maximum distribution of mesopores at a pore with the diameter of 3 nm and 4, however, the samples contain a sufficiently large number of pores with sizes ranging from 4 to 16 nm. 81

Figure 36 – Differential pore size distribution of samples AS-500 determined by DFT

Figure 37 – Differential pore size distribution of samples AS-600 determined by DFT

82

Figure 38 – Differential pore size distribution of samples CRH-800 determined by DFT

Figure 39 – Differential pore size distribution of samples of AS-500, AS-600, CRH-800 determined by the DFT

83

Figure 40 – Differential pore size distribution of samples of AS-500, AS-600, CRH-800 determined by BJH method both graphs show the greatest number of pores with a diameter of 4 to 16 nm, which corresponds to small mesopores

Figures 39, 40 shows comparative graphs of differential pore size distribution in the samples of the AS-500, AS-600 and 800-CRH calculated using DFT and BJH.

3.3.6 The results of Fourier – IR spectroscopy of raw materials Very important and interesting information about the presence of various functional groups and radicals in the samples gives the IR spectrometry. Was studied the IR spectra of the carbon material, carbonized at different temperatures. The results are shown in Figures 41-44. For IR spectrum of the feedstock (crushed apricot stones) are characteristic absorption bands NH2 (3431,92 cm-1), ОН (3009,97 cm-1) group, С=О (1643,25 cm-1), С–О (1241,55 cm-1), С–ОН (1055,64-1157,28 cm-1), С=С, С=N (1662,55 cm-1) stretching vibrations of СН2 (1378,87 cm-1) (Figure 42). For the IR spectrum of the carbon material, carbonized at a temperature 800 0С, is an increase in the intensity of the characteristic absorption bands NH2, 84

СОН, С=О, ОН and absorption bands СН2 of the aromatic ring (Figure 42). The emergence of fullerenes indicates a serious restructuring in the structure of the carbonized material. These results were confirmed by direct electron-microscopic study of this carbonized material.

Figure 41 – IR spectra of apricot stones before carbonization

Figure 42 – IR spectra of apricot stones (Т=8000С)

85

3.3.6.1 IR spectroscopic analysis carbonized rice husk On Figure 43 shows the IR – spectrum carbonized rice husk samples which obtained at a temperature of 700 °C, respectively. The bands of silica as well as carbon materials pyrolyzed in rice husk dominate in the IR spectrum. 700 °C temperature carbonization peaks disappear, indicating that the carbon burnup surface forms containing fragments of C-H (Figure 43). In this form of oxidized carbon with absorption at 1599, 1453 and 1383 cm-1 are present in larger quantities. The absorbance at 1599 cm-1, attributed to the C = O vibrations of a ketone and aldehyde groups. The peaks with low intensity at 1453 and 1383 cm-1 correspond to the vibrations of C-O in the carboxyl group. With increasing temperature carbonization to 700 °C, the relative intensity of the peaks at 1599, 1453 and 1383 cm-1 remains unchanged. It can be assumed that the formation of oxidized forms of surface carbon by carbonizing rice husk in these conditions occurs: by thermal decomposition of the starting fragments of cellulose, hemicellulose and lignin to form a surface of ketone, aldehyde and carboxyl groups.

Figure 43 – The IR spectra of carbonized rice husk at 700 °C

86

Figure 44 – IR spectra of walnut shells (t=800 0С)

By IR – spectroscopy method found the presence of carbonyl, carboxyl, hydroxyl and siloxane groups on the surface of rice husk.

3.3.6.2 IR spectroscopic analysis carbonized sorbents On the IR spectrum observed characteristic absorption bands 883 and 1050 cm-1 corresponding to C = C, deformation oscillations of C = C aromatic ring and band relating to the stretching vibrations of aromatic rings 1600, 1578 and 1510, and C-H group 3053, 3030 cm1 . By IR spectroscopy revealed the presence of structures of polyaromatic hydrocarbons. It is shown that increase in carbonization temperature increases the intensity of the bands of aromatic condensed systems, . The carbonized sorbents at temperatures 800 С, identified the following polycyclic aromatic hydrocarbons: pyrene by absorption bands (cm-1) 720, 850; crown – 547, 1320; fluoranthene – 600, 740, 820. On the IR spectra of intense absorption bands at wave numbers 1284, 1183, 577, 528 cm-1 revealed the presence of the C60 in Raman 87

spectra. Thus, the study of carbonized sorbents by IR spectroscopy revealed the presence of carbonyl, carboxyl, amine, phenolic groups, and polycyclic aromatic hydrocarbons, and structures of the fullerene. Comparing three different sorbents based on raw materials, it was stated, that walnut shells shows good results and more developing meso pore structure. So, in future work we will use walnut shells for column chromatography.

3.3.7 Extract of phytohormone of fusicoccin containing components Fusicoccin was obtained by a technique developed in the ICP, as part of the bouquet of organic compounds. In this connection, there arose the task of separation of biologically active substances. To solve this task, the technique has been used with liquid chromatography sorbent made of WSh. A distinguishing property of the selected sorbent is that it contains carbon and silica in its structure, this leading to the presence of both hydrophobic and hydrophilic properties. Supernatant – supernatant liquid resulting from the above method was placed on a column of a separating material (in this case cetonitri WSh). To control the chromatographic separation, a UV monitor type Uvicord S II manufactured by LKB (Sweden) was used. To release columns from unadsorbed substances, the column was washed with 10% ethanol to completely remove them, and then bounded with sorbent phytohormone was eluted with 50% ethanol. The spectroscopic study of purified WSh was conducted on spectrophotometer Ultrospec +1100 pro company of Amersham Biosciences (UK) in the ultraviolet and visible regions of the spectrum. Fot the test, СWSh samples obtained at temperature of 800 °C were chosen. To compare the specific characteristics, nanomaterial organic gel Octyl Sepharose CL-4B (Sweden) used in the world today was taken. The liquid containing FC was passed through a separation column with the experimental material. The results of the chromatographic separation are shown in Figure 45.

88

а

b

Figure 45 – Curves of chromatographic separation of fusicoccin on the column SGA-750 (a) and organic gel Octyl Sepharose CL-4B (b)

The first peak (Figure 45) contained substances that do not bind with sorbent. For release the column from unadsorbed substances, the column was washed with 10% ethanol prior to complete removal, and then the phitohormone bound with the sorbent was eluted with 50% ethanol (2nd peak). Analysis of the figures of separation suggests that the synthesized sorbent has separation characteristics which are not inferior to the world analogues. Also from Figure 45 it follows that when using a nanostructured composite material, the process of separation takes a shorter amount of time. However, the greatest advantage of this material is that it has a high resistance with respect to the microbiological media and the lack of parasitic sorption, unlike organic gel Octyl Sepharose CL-4B (b), having agarose in its structure.

3.3.8 Mass spectrometric analysis. Fragmentation of Fusicoccin using MS/MS Fusicoccin was infused with methanol at a concentration of 11.7 µg/g and a flow rate of 180 ul/h in methanol. The MS conditions are shown in Figure 46. 89

Figures 46 and 47 show the spectra obtained when the polarity of the voltage applied in the electrospray (ESI) (source coupling the LC and MS) was positive and negative, respectively.

Figure 46 – MS conditions for the detection of fusicoccin in full scan

The full scan spectra in Figure 48 shows the preferential formation of a sodium adduct of fusicoccin [fusicoccin+Na]+. Other species without such adduct were not detected. Multiple low intense peaks appeared, when infusing fusicoccin with ESI (-).

Figure 47 – Full scan spectra of fusicoccin in methanol ESI (+)

ESI (+) was chosen as the optimal conditions to monitor fusicoccin given that ESI (-) caused low intense peaks that we could not relate to fusicoccin. The full scan spectra in Figure 48 show the preferential formation of a sodium adduct of fusicoccin [fusicoccin+Na]+. Other species without such adduct were not detected. The most abundant 90

product ions are 633 and 431. Fragmentation (MS/MS) of the ion m/z 703.3 with the amplitude applied in the trap shown in Figure 49.

Figure 48 – Full scan spectra of fusicoccin in methanol ESI (-)

Figure 49 – Fragmentation (MS/MS) of the ion m/z 703.3 with the amplitude applied in the trap

The optimal fragmentation amplitude (0.7V) was chosen as the one causing the highest intensity of product ions keeping at least 5% of precursor ion.

91

1600000

1400000

1200000

Intensity (a.u.)

703.3 643

1000000

634.2 633.2

800000

573 431

600000

432.3 413

400000

200000

0 0

0.2

0.4

0.6

0.8

1

1.2

Amplitude (V)

Figure 50 – Fragmentation (MS/MS) of the ion m/z 703.3 with the amplitude applied in the trap

Fragmentation MS3 of m/z 703 [fusicoccin+Na]+ and analysis of the resultant ions by product ion scan mode shown in Figure 51. MS3 1600000 1400000

Intensity

1200000

633 573 545 431 413 353

1000000 800000 600000 400000 200000 0 0

0.2

0.4

0.6

0.8

1

1.2

Amplitude (V)

Figure 51 – Fragmentation MS3 of m/z 703 [fusicoccin+Na]+ and analysis of the resultant ions by product ion scan mode

92

MS3 200000 180000 160000 140000 634 Intensity

120000

573 545

100000

431 413

80000

353 60000 40000 20000 0 0

0.2

0.4

0.6

0.8

1

1.2

Amplitude

Figure 52 – Fragmentation MS3 of m/z 703 [fusicoccin+Na]+ (fragmentation route 2) and analysis of the resultant ions by product ion scan mode

Respectively, in Figure 52 shown fragmentation MS3 of m/z 703 [fusicoccin+Na]+ (fragmentation route 2) and analysis of the resultant ions by product ion scan mode.

3.3.9 Analysis of samples on сhromatogram Ethanol extracts obtained from the elution of the retained fraction of wheat samples from a carbon column. In addition, there was a solid extract that was extracted at Brighton University by mixing it with 5ml methanol and sonication (30 min), Figure 53. In Figure 54 shown the full scan analisys from the solid extract sample. Two intense peaks can be detected, the first one with m/z 701 as most abundant ion (see spectra below), which also contains m/z 703 as isotopic ion, and in the second one, m/z 703 (rt 30-35 min) can also be detected (fusicoccin m/z).

93

Intens. x107

2.5

2.0

1.5

1.0

0.5

0.0 0

5

10

15

20

25

30

35

Time[min]

Figure 53 – 4A spiked at concentration 0.5 ug/g

3.3.10 Determination of the chemical composition of the samples by liquid chromatography Objects of the study: extract obtained by elution with 50% ethanol in the separation of 70% ethanol extract of milk-ripe wheat and extract obtained by elution with 10% ethanol in the separation of 70% ethanol extract of milk-ripe wheat, Figures 55-57. The fraction extract obtained by elution with 10% ethanol in the separation of 70% ethanol extract of milk-ripe wheat shown in Figure 56.

94

Figure 54 – m/z 703 from the full scan analisys from the solid extract sample

95

Figure 55 – Pure Fusicoccin’s peak

1000

2.583 Ar ea :1 45 12 .4

VWD1 A, Wavelength=205 nm (051012E3.D) mAU

800

0

10

20

30

A45.725 re a: 64 7. 28

6

Fusicoccin A33.055 re a: 35 .4 35 5

0

A13.745 re a: 5. 20 A16.637 re 24 a: 9 18 .9 71 9

200

A3.635 re a A5.159 re : 92 a: 6 19 .87 2. 9 31 4

400

A2.756 A2.965 reA3.403 ar:e rea 1a3: : 6 8421 24 .102 1.3 1.9 2

600

40

Figure 56 – The fraction extract obtained by elution with 10% ethanol in the separation of 70% ethanol extract of milk-ripe wheat

96

m

The fraction obtained by elution with a 50% ethanol in the separation of 70% ethanol extract of milk-ripe wheat shown in Figure 57. VWD1 A, Wavelength=205 nm (051012F1.D)

Ar 32.356 ea :6 75 38 .1

mAU

800

600

200

0 0

10

20

A29.856 re a: 12 82 0.

8.168 AA8.707 rreea a:: 8 A10.954 11.713 re 128.9 AA12.187 rreeaa: 3 0.118 a:: 12 553 A14.477 re 464..236 AA15.390 15.895 rrea: 7.78353 A16.846 reeaa:: 448. 188 a 25 1 A18.624 re : 131.466296 a: 2 .63 52 .7748 A21.332 .8 7 rea A22.508 re 58 :7 a: 2 30 12 6. 72 48 .7 8

4

400

30

40

50

60

mi

Figure 57 – The fraction obtained by elution with a 50% ethanol in the separation of 70% ethanol extract of milk-ripe wheat

The results of the quantitative content of fusicoccin in fractions after preparative separation are shown in Table 10. Table 10 Result in fractions after preparative separation The samples consists of fusicoccin or no?

Samples The fraction obtained by elution with a 50% ethanol in the separation of 70% ethanol extract of milk-ripe wheat The fraction extract obtained by elution with 10% ethanol in the separation of 70% ethanol extract of milk-ripe wheat

97

Yes No

Conclusion 1. For the first time a method of determining the optimum pore size of sorbents for the selection of fusicoccin was developed using the program Spartan Graphical interface. 2. It was found that as optimal sorbent with mesoporous structure for cleaning fusicoccin was used walnut shell. 3. Physical and chemical activation rules for rice husk (RH), apricot stones (AS) and walnut shells (WSh) was determined. 4. A sensitive and selective method is developed in samples of milky ripe wheat by analysis of fusicoccin by liquid chromatography and mass spectrometry.

References 3 1 Pavlenko V.V, Anurov S.А., Mansurov Z.A, Biysenbaev M.A, Konkova T.V, Azat S., Tanirbergenova S.K, Zhilibaeva N.K., Acquisition of microporous active carbons on the bases of carbonized wheat acorns / / Bulletin of KazNU, chemical. – №3 (75) – 2014 – pp. 103-113. 2 Pat. 26936 RК. Biologically active assets of chromatographic separation / Mansurov Z.A, Gilmanov M.K, Kerimkulova A.R, Azat S., Gukenyimmer E.J; on, 15 May 2013. # 5. – the Ministry of Education and Science. 3 Azat S., Adekenova A.S, Ivasenko S.A, Seydahmetova R.B, Kerimkulova A.R, Mansurov Z.A., Development of technology of drug-free phusicoccin on nanohydrocarbon sorbent and study of biological activity // The pharmaceutical journal of scientific-practical journal. – 2012. – №2-3 (164). – pp. 57-60.

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4

  STUDY OF BIOLOGICAL AND     CYTOTOXIC ACTIVITY OF FUSICOCCIN  

  4.1 The examination of the cytotoxic activity of the obtained fractions Objects of the study were 8 sample substances for the presence of cytotoxic activity pertaining to brine shrimp larvae Artemia salina (Leach) under cultivation conditions in vitro. Samples designation (name): 1. A0 – crude extract; 2. А1 – fraction after the preparative separation; 3. А2 – fraction after the preparative separation; 4. А3 – fraction after the preparative separation; 5. А4 – fraction after the preparative separation; 6. А5 – fraction after the preparative separation; 7. А6 – fraction after the preparative separation; 8. А7 – fraction after the preparative separation. Cytotoxicity was assessed with the help of the survival test of brine shrimp larvae of Artemia salina (Leach). Experiments were carried out in 2-day-old larvae in cultivation conditions in vitro. The larvae were grown up by dipping of brine shrimp eggs Artemia salina (Leach) into the artificial sea water and by the 48 hours incubation at a temperature of 37 0C. A weighed portion of the test sample was dissolved in 2 ml of methanol, and then it was taken from that solution by 500 μl (3 parallels), 50 μl (3 parallels), 5 μl (3 parallels). After evaporation of methanol 5 ml of artificial seawater were added to each vial. Thus, if the initial sample weight was 2 mg, the final sample concentrations were 100 µg/ml, 10 µg/ml and 1 µg/ml, respectively of each concentration in 3 replications. 10 2-dayold brine shrimp larvae Artemia salina were put into each vial containing the sample using the Pasteur pipette. Thereafter, all the vials were left at room temperature exposed to light for 24 hours. After 24 hours survived and dead larvae were counted. Then, using the obtained data of the upper and lower toxic limits a half-toxic sample dose was calculated. 99

Statistical results reporting was conducted using FNI computer program. Findings of investigations. The results of the testing of the cytotoxic activity of the samples A0, А1, А2, А3, А4, А5, А6, А7 pertaining to brine shrimp larvae Artemia salina (Leach) under cultivation conditions in vitro are listed in the Table 11. Table 11 The cytotoxic activity of the samples A0, А1, А2, А3, А4, А5, А6, А7 pertaining to brine shrimp larvae Artemia salina (Leach) under cultivation conditions in vitro Substance name 1 A0 Crude extract A1 Fraction after the preparative separation A2 Fraction after the preparative separation A3 Fraction after the preparative separation A4 Fraction after the preparative separation A5 Fraction after the preparative separation A6 Fraction after the preparative separation A7 Fraction after the preparative separation

Quantity of survived larvae 95% Concent LD 50, confist nd rd ration 1 2 3 µg/ml dence µg/ml parallel parallel parallel interval 2 3 4 5 6 7 1 10 10 10 60.0410 10 7 9 140.39 932.24 100 7 6 4 1 10 10 10 20.6310 6 6 8 39.22 96.74 100 4 3 4 1 10 10 10 10 10 9 9 100 9 7 8 1 10 10 10 10 8 9 8 100 8 6 8 1 9 10 9 10 7 7 6 15.92100 4 3 3 32.62 91.15 10 7 10 7 100 6 3 5 1 10 10 10 40.1710 8 7 7 95.55 559.30 100 7 5 7 1 10 10 10 56.0810 7 8 10 135.57 984.11 100 5 6 6 1

10

10

100

10

76,11

37.34263.85

Activity 8 has

has does not have does not have

has

has

has has

As the table shows the crude extract A0 and the following fractions after the preparative separation (A1, A4, A5, A6, A7) exhibit the cytotoxic activity pertaining to brine shrimp larvae Artemia salina (Leach).

4.2 Antimicrobial activity tests Materials and methods: Objects of the study were 8 samples for the presence of antimicrobial activity. Samples designation (name): The study of antimicrobial activity of the samples mentioned above was performed pertaining to gram-positive bacteria strains Staphylococcus aureus, Bacillus subtilis, gram-negative strains Escherishia coli and Candida albicans yeast fungus by the method of diffusion into the agar (wells). Comparator agents were gentamicin for bacteria and nystatin for C. albicans yeast fungus. Cultures were grown in a fluid medium with pH 7.3 ± 0.2 at a temperature of 30 to 350C during 18-20 hours. The cultures were diluted 1:1000 in a sterile 0.9% sodium chloride isotonic solution; then they were put into cups with 1 ml of solution with relevant elective nutrient media for the studied test-strains and inoculated according to the method of “continuous lawn”. After drying on the surface of the agar wells of size 6.0 mm were formed, which were filled with the solution of the test sample, gentamicin and nystatin. Ethanol in equivoluminar quantities was used ss control. Thus, the sample was tested in quantity of 1 µg and the comparator agent in quantity of 1 mg. The inoculations were incubated at 37 0C, the accounting of growing cultures was performed after 24 hours. The antimicrobial activity of the samples was estimated by the diameter of test-strains inhibition zones (mm). The diameter of inhibition zones lower than 10 mm and the continuous increase in the cup were estimated as the absence of the antibacterial activity, 10-15 mm – as a weak activity, 15-20 mm – as a moderate activity, over 20 mm – as an expressed one. Each sample was tested in three parallel experiments. Statistical reporting was performed by methods of the parametric statistics with the calculation of arithmetic mean and standard error.

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Findings of investigations. The results of the testing of the samples’ antimicrobial activity are listed in Table 12. Table 12 The antimicrobial activity of the samples Substance name (0 ) Crude extract (1) Fraction after the preparative separation (2) Fraction after the preparative separation (3) Fraction after the preparative separation (4) Fraction after the preparative separation (5) Fraction after the preparative separation (6) Fraction after the preparative separation (7) Fraction after the preparative separation Gentamicin, Nystatin

S. aureus 505

Bac. Subtilis

E. coli M-17

C. аlbicans

14.0±0.3

16.0±0.1

14.0±0.3

13.0±0.1

16.0±0.1

16.0±0.2

15.3±0.3

13.0±0.2

14.0±0.3

15.0±0.3

12.0±0.2

15.0±0.2

16.0 ±0.2

14.0±0.1

14.0±0.3

13.0±0.3

15.0 ±0.2

16.0±0.4

14.0±0.2

14.0±0.1

14.0±0.2

14.0±0.2

15.0±0.3

13.0±0.3

15.0 ±0.2

16.0±0.3

14.0±0.1

12.0±0.2

14.0 ±0.2

14.0±0.4

15.0±0.2

13.0±0.3

26± 1 -

24 ± 1 -

23 ± 2 -

22

It was stated that all presented samples exhibit an antibacterial activity pertaining to the gram-positive microorganisms Staphylococcus aureus and Bacillus subtilis, and the gram-negative test-strain Escherichia coli.

4.3 The study of the analgesic activity of naturally occurring substances and their derivatives The experimental objects were studied in the dose range from 25 to 100 mg/kg when administered intragastrically. The comparator agent diclofenac sodium was tested in the dose range from 25 to 100 mg/kg. The experimental objects and comparator agent were administered 30 minutes before administration of the 0,75% acetic acid solution. 102

The screening results of the analgesic activity of the crude extract and its fractions after preparative separation are listed in the table. The analgesic effect of the submitted samples was determined by the ability to reduce the number of “writhes” (in %) calculated for 10, 15, 20 and 30 minutes and compared to the corresponding figures in animals in the control group, Table 13. Table 13 The analgesic activity of naturally occurring compounds and their derivatives Substance name, dose

Crude extract (0)

Fraction after the preparative separation (5)

Fraction after the preparative separation (6)

Fraction after the preparative separation (7)

10 minutes

15 minutes

20 minutes

30 minutes

Control Diclofenac 25 mg/kg 50 mg/kg 100 mg/kg Control

28±1.4

42±3.2

55±3.5

71±4.5

Diclofenac 25 mg/kg 50 mg/kg 100 mg/kg Control Diclofenac 25 mg/kg 50 mg/kg 100 mg/kg Control Diclofenac 25 mg/kg 50 mg/kg 100 mg/kg

10±2.4 18±3.6 16±2.4 17±2.1 32±3.7 16±2 28±5 18±2.8 22±4.2 20±1.5 7±1.3 21±2 12±0.6 11±2,4

17±2.6 35±5.5 28±3.2 33±4.5 42±4.3 23±1.6 41±4.7 29±4.3 33±5.3 35±3.5 12±1.9 31±2.1 26±1.5 19±2.3

23±2.8 47±5.9 36±4.5 43±5.8 51±4.1 30±2.2 53±4.8 37±5 39±6.3 48±3 19±1.5 38±2.1 36±1,6 25±2.5

34±1.7 66±6.5 44±5.3 50±4.9 70±3.3 34±1.8 66±7 46±6.6 50±8.3 69±2,5 25±2.3 45±1.5 45±2.4 35±4.2

The study revealed that the fraction after preparative separation (7) has an expressed analgesic effect at a dose of 100 mg/kg, the remaining samples showed a weak analgesic activity.

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4.4 Testing for the phagocytosis-stimulating activity The comparator agent “Immunorm” (juice of Echinacea purpurea in alcohol, “Merkle”, Germany) which was tested at a dilution of 0.9% sodium chloride solution in the final concentration of 8%. Blood sampling was taken from a healthy donor in the fasting state from the ulnar vein into heparinized test tubes. As an object of the phagocytosis the daily culture Staphylococcus aureus (strain 209) was used. At the microscopic examination (the magnification 15x90, oil immersion) the number of phagocytic cells (the phagocytic index, PI) out of 100 neutrophils (quantitative indicator) and the number of staphylococci, absorbed by one neutrophil – the phagocytic number (PN, qualitative indicator of the phagocytosis) were counted after 1 hour of study, Table 14. Table 14 The influence of the samples (0), (1), (2), (3), (4), (5), (6), (7) on the phagocytic activity of blood neutrophils, (М±m) Neutrophils Substance name

Control with dilution medium Immunorm (0 ) Crude extract (1) Fraction after the preparative separation (2) Fraction after the preparative separation (3) Fraction after the preparative separation (4) Fraction after the preparative separation (5) Fraction after the preparative separation (6) Fraction after the preparative separation (7) Fraction after the preparative separation

PI, %

PN, unit

27.0±1.5 51.3±4.8 24.1±3.6 25.3±1.3 29.7±2.4 22.7±1.8 17.1±1.5 24.3±5.5 23.1±2.3 32.7±2.9

4.1±0.4 8.2±1.2 3.3±0.23 3.1±0.37 3.4±0.2 4.1±0.1 4.5±1.3 3.9±0.4 3.8±1.1 3.4±0.4

75 mg of the dry sample with account of its solubility were diluted in 0.2 ml of 96% alcohol until fully dissolved and made up to 2 ml of 0,9% sodium chloride solution. The substances were tested at a concentration of 1 mg/ml in three parallel experiments. The smears

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of the blood incubated with the dilution medium (96% alcohol and saline 1:9) were served as controls. Statistical results reporting was performed by methods of the non-parametric statistics with the calculation of the arithmetic mean (M) and its standard error (m). The results of the screening study of the blood cells phagocytic activity are listed in the table. Based on data from the table the comparator agent “Immunorm” has an expressed phagocytosis-stimulating action pertaining to both quantitative and qualitative indicators of the blood neutrophils phagocytosis[1-4].

Conclusion 1. As a result of the investigation of the cytotoxic activity of the extracts fusicoccin, it was found that the extract fractions after preparative separation exhibit cytotoxic activity against larvae of marine crustacean Artemia salina (Leach) and studied biological activity.

References 4 1 Pat. 26936 RК. Biologically active compounds of chromatographic separation / Mansurov Z.A, Gilmanov M.K, Kerimkulova A.R, Azat S., Gukenyimmer E.J; on 15 May 2013. # 5. – the Ministry of Education and Science. 2 Azat S., Adekenova A.S., Ivasenko S.A., Seydahmetova R.B., Kerimkulova A.R., Mansurov Z.A. Development technology of the pharmaceutical fusicoccin on nano carbon sorbents and studying biological activity // The pharmaceutical journal of scientific-practical journal. – 2012. – №2-3 (164). – pp. 57-60. 3 Azat S., Kerimkulova A.R., Gilmanov M.K., Mansurov Z.A., Techniques for obtaining fusicoccin compounds by carbon sorbents // Volume II of the International Conference "High Technologies – Dialogue of Steady Development". – Almaty, May 23-24. – 2013. – pp. 171-174. 4 Mansurov Z.A., Azat S., Adekenova А.S., Kerimkulova A.R, Ivasenko S.A, Shulgau Z.T, Gilmanov М.К., Ibragimova S.А. Extraction Fusicoccin From Wheat Seeds Using Nanocarbon Sorbents // Advanced Materials Research. – 2013. – V.647. – P. 67-70.

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5

 USING FUSICOCCIN    AS BIOSTIMULANT (BS) 

5.1 Investigation of the activity of fusicoccin using biotest Of course, any bioregulator is necessary to be examined first using biotest. Using this biotest, it is possible to determine what type of hormones among regulators relates to bioregulator. For this reason, the impact of fusicoccin on plants were investigated with multiple biotest. The exciting thing in bioassay is to destroy the influence of the apical dominance. Among all phytohormones only because of the effect of the cytokinin performed biotest. Apical dominance is characterized by the presence of the terminal bud on the stem. In case when growing indicated bud, other additional leaves can not grow on this stem. If remove this apical bud, among phito hormones may be additional leaves and shoots only because of the impacts of cytokinin. Beans were placed by 12 pieces in 3 Petri dishes, poured water was poured in a ratio of 1/3, and left in a well-lighted place, Figure 58. About 2 weeks the beans were watered, and changed the paper under the laid seeds was changed. When the beans sprouted and began to grow, were opened a petri dish, and continued to care for the plants were continued to be cared. After 2 weeks, sprouted bean seeds were placed into cups. Thus, daily pouring water on the basis, take care of the plants for 1-2 weeks, Figure 59. Then, the “apical dominance” (availability of the terminal bud on the stem), was destroyed, i.e. removed the terminal buds were removed. In case where the apical bud grows, no other additional leaves can grow on this stem. If we remove this apical bud, among plants of phytohormones only because of the impact of cytokinin there may be additional leaves and shoots.

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Figure 58 – Grains of beans for biotest

To carry out this bioassay, wetted foam 50 ng/ml with solution of wheat fusicoccin, and with a piece of that foam rub the stem stepson removing the terminal bud, within 1 week once a day.

Figure 59 – Bean seeds put into the cups

To determine the efficacy of a given dose of 50 ng/ml, one specific bioassay was performed. To do this, cut the stem of a conventional flower, placed them in three different glasses, and observe the index of rooting. In the first glass there is water, in the second – fusicoccin with water in a ratio of 1: 1000, in the third – fusicoccin with water in a ratio of 1: 500.

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We can make the following conclusion based on this comparison study: rooting in wheat of fusicoccin proceeds faster than in water, and optimal concentration of wheat fusicoccin is 50 ng/ml. As a result of rubbing the stem stepson within 1 week once a day with a piece of foam soaked in 50 ng/ml solution of wheat fusicoccin, removing terminal buds, we obtained the best results. This plant was growing 2-3 times faster than the beans growing on the water only, i.e. without fusicoccin.

a

b Figure 60 – The plant poured with: a – solution of fusicoccin; b – only with water

In conclusion, we can say that using physiological bioassay our fusicoccin can be attributed to cytokinin.

5.2 Practical application of fusicoccin The unusual properties of the biostimulator make it very promising for the rapid vegetative reproduction of woody plants with any type of root system. The use of the obtained sorbent allowed us to obtain the amount of biostimulant, sufficient for field trials. Given the harsh environmental and climatic conditions of Kazakhstan, it was very interesting to study the effect of the biostimulant on increasing the resistance to stress of the most important cereal crops of Kazakhstan. Thus, the effect of purified 108

fusicoccin on the germination of wheat seeds of the Nadezhda variety in 2% NaCl solution, i.e. under conditions simulating strong chloride salinization. It was shown that under these conditions, fusicoccin increases the germination of wheat grains by 19% by weight. The results of the experiments are presented in Figure 61.

2% NaCl

2% NaCl + Fusicoccin

Figure 61 - Influence of a biostimulator on the germination of seeds of wheat variety “Nadezhda” in NaCl solution (2%)

As can be seen from Figure 61, seedlings on fusicoccin have a better developed root system. The use of this biostimulator for increasing winter-hardiness of winter wheat has a great prospect. The tests were carried out in the Research and Production Center of Agriculture and Plant Industry of the Republic of Kazakhstan. Table The effect of fusicoccin on yield and weight of 1000 grains of Steklovidnaya-24 winter wheat Productivity with Version 1000 grain weight 0.1 weave kg % г % control 2,950 100 43,73 100 biostimulator 3,250 110 50,41 115

109

15

As shown by the experiments, the use of fusicoccin increases a mass of 1000 grains by 15% and productivity by 10% (Table 15). In the course of further research, field tests of fusicoccin were carried out (Fig. 62). It was noted that wheat treated with a biostimulant ripened 15 days earlier than wheat without treatment (green shoots).

Figure 62 - Wheat field with a strip treated with a phytohormone solution (yellow ears)

Plants (Fig. 62) were grown on the fields of the Republican State Enterprise "Scientific Production Center of Agriculture and Plant Growing" of the Ministry of Agriculture of the Republic of Kazakhstan (Almalybak village, Karasai district, Almaty region).

5.3 The use of BS to increase the yield of agricultural crops Given the extremely high stimulating activity of BS, it was impossible not to study the effect of BS on the yield of the most important crops. For the experiment, an aqueous solution of BS was taken at a concentration of 50 ng/ml. Seeds before sowing were soaked in this solution for 3-6 hours. After that, the seeds are dried and sown with standard tractor seeders. Field experiments were 110

carried out on sites ranging in size from 2 to 4 hectares. The experiments were conducted on the fields of the RSE "Research and Production Center for Agriculture and Plant Growing". Village Almalybak Karasay district of Almaty region. The results are presented in table 16. Table 16

Plots 1 2 3 4 the average

The effect of BS on increasing the yield of wheat varieties "Steklavidnaya-24" Yield centners / Version ha Control 11,40 Experience 15,80 Control 12,60 Experience 16,20 Control 12,00 Experience 15,00 Control 12,00 Experience 16,00 Control 12,00±0,6 Experience 16,00± 0,2

Yield (% ) 38,60 28,60 25,00 33,30 33,30±6,8

The following experiment to increase the yield of winter rye was carried out on the fields of the Bishkul poultry farm on field plots, each of which had an area of 8 hectares. The results of the experiments are presented in table 17. Table 17 Increasing the yield of winter rye varieties "Sholpan" Yield increase centners / ha Version swath the average Control 19,9 20,0 20,1 20,0±0,1 Experience 27,9 28,0 28,1 28,0±0,1 Yield % 40,2 40 39,8 40±0,2

As can be seen from the table and the official act signed by the administration of the factory, pre-sowing treatment of seeds of winter rye of the Sholpan variety gave an increase of 40% to the yield.

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Such a high increase in yield of winter wheat and winter rye is explained by the following reasons. The pre-sowing treatment of seeds with BS preparation significantly accelerates the development of plants and, before winter, plants of winter wheat and winter rye form the root system better and therefore overwinter better. In spring, plants with a well-developed root system make better use of soil moisture, and these plants grow better than control plants. And all this provides a significant increase in yield. Field trials of the biostimulator obtained by us were carried out to increase the yield of sugar beet. Kazakhstan provides only 10-15% of its needs with its own sugar, while the rest of the sugar is imported. And this led to more than two-fold increase in the price of sugar. The agricultural workers are tasked with a sharp increase in sugar beet production in the Republic. The use of biostimulants to increase the yield of sugar beet will play a major role in this task. Therefore, our task was to study the effect of the BS preparation on the yield of sugar beet. Field tests were carried out in the fields of the Production Agricultural Cooperative "Zher Ana" of Almaty region on an area of 2 hectares With an aqueous solution of BS preparation at a concentration of 50 ng/ml, the seeds of sugar beet were soaked for 4-6 hours; after the seeds were dried, they were planted. The results of the experiment showed that the use of purified biostimulant concentration of 50ng/ml for presowing treatment of sugar beet seeds gave a yield increase of 20% as can be seen from table 18. Table 18 Effect of BS on increasing the yield of sugar beet

Version Control Experience Yield %

1 swath centners / ha 227 288 26,8

2 swath centners / ha 265 302 14

3 swath centners / ha 273 321 23

4 swath centners / ha 235 289

the average 250±23 300±17 20,3±6,5

Figure 63 shows the effect of presowing treatment of sugar beet seeds with a purified biostimulant on the development of a root crop. So, for example, 1 gram of BS is enough for pre-sowing treatment of seeds of grain crops sown on an area of more than 500 hectares. 112

Figure 63 – The effect of presowing treatment of sugar beet seeds on the development of the root, I-control, II-exprience

Pre-sowing treatment of grain seeds gives an increase in yield: winter rye by 40.0%, winter wheat by 33.3% and sugar beet by 20%. Presowing seed treatment accelerates ripening for half a month, winter crops, which allows you to get away from the summer drought.

5.4 Sowing varieties of tomatoes and cucumbers in the greenhouse with PMF Using as a bio-stimulator compound components fusicoccin and enriching part, a new microfertilizer phyto microfertilizer (PMF) was developed and tested in the greenhouse, in the sowing fields. Thanks to the obtained positive results, issued a Material Safety Data Sheet by Ministry of Industry and New Technologies of the Republic of Kazakhstan dated June 16, 2014 (SC-HP number 0000288) (Figure 61) was issued.

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Figure 64 – Material Safety Data Sheet

The test of biostimulator is conducted in a greenhouse of Kazakh National Agricultural University in Almaty. The objects of investigation were varieties of tomatoes and cucumbers. As a reference, water, and the Russian biodyne “wagon” were taken. When sowing tomato varieties, the experiment was carried out in three versions (Figures 65-77): 1) sowing seeds with plain water (control); 2) soaking of seeds with PMF; 3) pouring the soil with PMF. First, tomato seeds were put to soak with PMF. The next day, taking soaked seeds, as well as other methods according to the above embodiments, PMF and water poured on the tableware, which were placed tomato seeds. Each dish was planted the 7 seed 8 types of Russian varieties of tomatoes: “The giant raspberry”, “Consul”, “Scarlet mustang”, “Little Man”, “King of the Giants”, “Darya”, “Pride of Siberia”, “Persimmon”; two types of Dutch varieties of 114

tomatoes: “Pink Lady”, “Bayan”. After sowing, 2 ml of water was poured.

Figure 65 – The variety of tomato with PMF. 1 day on 24 January, 2015

On January 26, 2015 all the tomatoes were poured with 20 ml of water, and the seeds of cucumbers were sown in two versions, i.e. only one series each hydroponics was poured with 60 ml of water with PMF, and the remaining five rows of each hydroponics was poured with 60 ml of water with addition of the universal stimulator. Temperature of water with biostimulant was 23 °C. Biostimulator was prepared with addition of 1-2 ml of water 1-1.5l PMF.

a – 3-day, January, 26, 2015

b – 5-day, January 28, 2015

Figure 66 – The variety of tomato with PMF Study of varieties of cucumbers using innovative technology.

115

Figure 67 – Varieties of cucumber with PMF on the 3-day, 30 January 2015

Figure 68 – 3 February 2015, cucumber

Only tomatoes were poured. On 5 February, 2015 – cucumbers were poured with only 100 ml of water, and tomatoes filled with water completely. The temperature of hydroponics was 19-20 °C. February 17, 2015 – gave kristalon in roots and bushes of cucumber and tomato, tissue temperature was 21 °C. In February 19, 2015 varieties of tomatoes were poured with water. February 20, 116

2015 held a work on moisturizing. Leaves of cucumbers varieties were sprayed with water, the roots were poured with 10-20 ml of water.

Figure 69 – 17 February 2015, tomatoes

Figure 70 – The varieties of cucumbers

117

Figure 71 – February 21, 2015 the variety of cucumber started rooting, and put down roots in the fabric

On 26 February 2015 the varieties of cucumber were poured with 200 ml of water, tomato humidity was sufficient and the average value was 7. On 27 February 2015 the first three rows of cucumbers were not watered, and the last three rows were poured with 150 ml of water, tissue temperature was 37 °C.

Figure 72 – February 28, 2015 control with versatile fertilizer

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February 25, 2015 there were tendrils of cucumber, the varieties of cucumber poured with 11.5 liters of water by dilution with 6 caps of universal fertilizer. The temperature of tissue was 38.5 °C. March 4, 2015 the temperature of tissue was 29 °C. March 5-6, 2015 tomatoes were transfered to the ground, the distance between planted tomatoes was 40 cm. Varieties of cucumber were sprayed with calcium nitrate, poured the roots of approximately 10-20 g. Tissue temperature was 35 °C. Cucumbers bloom. At this time, cut the lower leaves and stems of cucumber that not waste power on the leaves and stems. Then it increased possibility to obtain large and high-yielding crops.

Figure 73 – Cucumbers and tomatoes 9 March, 2015

On 9 March, 2015 the varieties of carried over cucumbers and tomatoes were poured. March 10, 2015 Humidity of tissue 4-5, the temperature was 35 °C. The varieties of cucumbers were irrigated through a drip for 2 minutes, tomato varieties of the first, second and third row were irrigated with drip for 20 minutes and the fourth row was manually poured with about 500-700 ml of water. The temperature of soil remaining in the bowl of tomatoes was 23 °C and humidity – 2-3. March 11, 2015 the emperature of tissue was 40 °C, since the varieties of cucumbers have taken root more deeply, ie in fabric, the moisture content in cubes began to decrease. For this reason, 6 caps 119

of universal fertilizer were diluted with 11.5 liters of water, to water tomatoes 250 g. The temperature of the tomatoes in a cutlery is 13 °C, humidity was 4. March 12, 2015 pH of first-line of varieties cucumber was 7.5, and in the fifth row pH = 7.7. Humidity of the tissue is in the range 4-5. Humidity of tomatoes was 5-7.5, ie humidity was sufficient. March 23, 2015 the varieties of cucumber were sprayed with a calcium fertilizer. March 24, 2015 tomatoes watered with water, varieties of cucumber were sprayed with a universal fertilizer. 25 March, 2015 tomato varieties were drip irrigated with 500 ml of water and cucumber varieties for 3, 4, 5, 10 minutes with 150-200 ml water. March 27, 2015 varieties of cucumbers were drip irrigated from the third, fourth row for 5 minutes, and the fifth, sixth, seventh, eighth series for 8 minutes. Cucumbers from the eighth row were sprayed with fertilizer of potassium.

Figure 74 – March 21, 2015 varieties of cucumbers have begun to bear fruit, in varieties of tomatoes (control) there began to appear the first flowers

March 26, 2015 – tomato varieties were poured with water, and strapped them. March 27, 2015 varieties of cucumber drip irrigated for 5-8 minutes. 30 March 2015 cucumbers from the third, fourth and fifth rows were superfused for 4 minutes, and the seventh and eighth row – 6 120

minutes. The room temperature was 11 °C, according to the weather forecast, it snowed. Visible yellowing of cucumber fruits because of frost was observed. 10 April, 2015. Room temperature 21 °C, the first row of cucumbers were irrigated by drip water for 5 minutes, the last two rows – 7, 8 minutes.

Figure 75 – PMF used for cucumbers and tomatoes 10 April 2015 Table 19 рН of activation stimulator Sample Fusicoccin Fusicoccin

diluent

Universal fertilizer Universal fertilizer diluent

рН 7.58 6.85 8.24 7.5

Compare with the control variant, the samples treated with PMF and sieved water. In the course of work during the period of flowering, the lower leaves and stems of cucumbers and tomatoes were cut off for the plant not to spend the extra power, and direct them to the development of the fruit during the tuber. It is found that compared with biostimulant “universal” and water, the application of biostimulant based on PMF accelerates the growth period of cucumbers and tomatoes from 1 week to 10 days. 121

Table 20 The results of experiments carried out in homes Month, Day

t°аir

t°cucumber

t°tomatoes

рН

1 27 January 28 January 30 January 31 January 2 February 3 February 4 February 5 February 6 February 9 February 10 February 11 February 12 February 13 February 14 February 16 February 17 February 18 February 19 February 20 February 21 February 23 February 24 February 25 February 26 February 27 February 28 February 2 March 3 March 4 March 5 March 6 March 10 March

2 10 12 10 14 15 14 16-17 11

3 20 14 14-17 27-25

4 14-18 14 15-17 28 19-20 22 22 19-20

5

28 19 31 18 18 14

24 29-32 28-30 24-24.5 21-24 18

17

16-17 23 21-22 20-24 19 21 19 18 23-24 20-21 19-20 26 23-25 22-26 19-23 19-22 26-28.3

15 19 15 20 20 19 21 18 18 26 21 21 23 22 27

16-18 19-20

27 25-33 25-30 24.5-29 23-27 13.5-16 25 16 21 18-22.5 17-18 17-19 20 21 18.5 18-19 17-18 16-17 24 20 15-16 16-17 15-16

122

4-7

7.5

7.6

Humidity cucumbers 6

Humidity tomatoes 7

5

4.5

3-4 6-7.1 3 3-4 4-6 5 4 4-4.5 3-5 2-6 4-6 2-5 4-7 5-7 4-6 4-6.5 3-6.5 4-7.5 4-7 5-7.5 5-7 4-7 4-8 4-8 4-6.5 2-6

3-4 2-3 2.5 2-5 3-5 6-9 5-7.5 4-6 3-4 5-6 2-6 4-8 2.5-7 3-4 2-6 5-9 4-9 3-9 3-6 4-9 3-9 3-8 3-5

Continuation of table 20 1 11 March 12 March 13 March 14 March 16 March 26 March 27 March

2 17 15 27 22 13 22 27

3 22-25 21-23 23-28 21-25 18-22

4 14-15 14-16 13-15 14.5-18 13.5-15

5 7.7

6 2-7 4-5 1.5-6 1-5 2.5-7 2-5.5

7.8-8

Figure 76 – PMF and Control biostimulant used for cucumbers and tomatoes 14 February, 2015

Figure 77 – PMF and Control biostimulant used for cucumbers and tomatoes 28 March 2015

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7 5-7 5-7.5 4.5-6 3-5 5-6

5.5 Results of laboratory and field experiments to assess the effectiveness of phytomicrofertilizer (PMF) on cereals In laboratory experiments there were determined the growth stimulating preparation and fungicidal activity at dilutions of 1:10, 1:25, 1:50, 1:100. As a reference, we take registered in Kazakhstan as plant growth regulators Russian drug biosil established on the basis of the triterpenoid acid. Seeds of wheat and barley were treated with different dilution of the original product on the basis of PMF. They germinate in Petri dishes on moist sterile sand by 100 seeds in 4 times repetition. At the 4th day it was determined vigor, on the 7th day germination and musty. In parallel experiments on laid paper rolls filled with a mixture of sand and soil in the ratio 1: 1, where the plants were grown to the phase of 2 leaves. Field experiments were laid in the Karabalyk ACES. As standards there were chemicals recommended for use on cereals in Kazakhstan. Seeds of spring wheat and barley were treated at the rate of PMF 0.2 and 0.4 l/m. They correspond to the drug dilutions 1:25 and 1:50, with the water flow rate of 10l/t for seed treatment of cereals, which is accepted in production environments. Scheme of experience: PMF – 0.4l per 1 ton of seeds PMF – 0.2l per 1 ton of seeds Vitavaks 200FF – 2.5 kg/t (etalon-1) Kinto duo – 1,5 l / t (etalon-2) Control – untreated seeds Sowing the seeds treated with medication with humidifying water (10-15 l/t) was carried out at the beginning of the third decade of May batch drill. Predecessor – fallow. Sort of wheat is Karabalyk 92, barley – early Karabalyk. Seeding rate of 3.5 – 4 mln pcs/ha, depth – 4-5 cm. The size of the plots – 10 m2, repetition – quadruple. Basic surveys and observations made in the experiments: – The energy of germination, seed germination and musty (analysis of 100 seeds in four repetitions); – Phenological observations during the growing season; – Defeat seedling root rot in the 2-3 leaf stage (50 plants in 4 fold repetition);

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– Affecter ears of corn smut in the flowering stage (analysis of 200-250 ears for each repetition); – Consideration of the harvest, the analysis of its structure and biometric indicators of plants (50-100 pcs. for each option). There were drier with significant insufficient precipitation, but it was not reflected to receive good sprouts. The summer months were generally favorable to the growth and development of crops and manifestations of disease with a sheet-hypophyseal infection. In June or during sprouting – tillering plants dropped more than 120 mm of rain or 2.5 – 3 times the long-term norm. The average daily air temperature was closer to the long-term. July was characterized by significant shortfalls in precipitation and higher average temperature. However, soil moisture was sufficient, in the second decade of the month 10-11 am on crops remained plentiful dew. The first decade of August was also damp, 39.5 mm of rain or more fell that 2 times as much long-term average, the average temperature was lower by 1.5 °C, which delayed the ripening of grain crops (Table 17). Laboratory screening. To determine the growth-stimulating activity of the PMF at different dilutions of the parent drug, a series of laboratory experiments were carried out. It was stated that it exhibits a specific physiological activity. Low temperatures (12-17 °C) significantly accelerates the formation of the second leaf spring wheat. Table 21 The temperature and amount of precipitation before planting and during the growing season of crops (according to Kostanai HMS) Month May June July August

Mean air temperature, °С by decades ave- multi-

Precipitation, mm by decades multiyea amount r 1 2 3

1

2

3

rage

year

15.1 19.1 22.6 15.2

13.9 17.6 19.2 20.8

16.8 19.5 23.8 13.8

15.3 18.7 21.9 16.5

13.4 4.6 15.4 18.9 35.2 13.1 65.4 20.4 18.0 13.2 3.4 18.0 39.5 - 21.5

20.0 113.7 34.6 61.0

31.4 46.3 66.9 36.8

On the accumulation of leaves-hypophyseal mass exceed prototype version control from 11.5 to 47.2%, and the weight of the root system – from 8.2 to 27.9%, and barley – from 7.4 to 16 20,5 125

and from 4 to 39.7% respectively. The most growth -stimulating activity of the drug was shown at dilutions of 1:10 to 1:50, and at 1: 100 it declined. Prevalence of barley root rot in the experimental variants was lower 1.7-1.8 times. This indicates that the drug may be inducers of plant resistance to disease (Table 18). Table 22 Results of laboratory evaluation of the physiological activity of PMF on cereals Spring wheat CultiGermi wet weight, g Medici- vation with 2 nati-on ne or norm leaves, above ml / t ability % ground fibrous % PMF 1:10 97.0 89.0 2.57 2.68 PMF 1:25 100.0 94.0 2.68 2.69 PMF 1:50 99.0 77.0 2.40 2.70 PMF 1:100 99.0 49.0 2.03 2.28 Biosil 1:100 100.0 90.0 2.18 2.72 Control 100.0 50.0 1.82 2.11

germin ation ability, % 98.0 94.0 95.0 93.0 87.0 95.0

barley wet weight, g with 2 leaves, above % ground fibrous 94.0 89.0 94.0 92.0 87.0 89.0

4.34 3.93 4.41 4.12 4.15 3.66

2.99 2.50 2.69 2.49 2.40 2.14

On the basis of laboratory research the optimum concentration or dilution of PMF has been verified, which stimulates the growth and development of seedlings. Taking into account that in seed dressing chemicals crops for their hydration it is recommended to take water at the rate of 10 liters per 1 ton of seeds, field trials were established at a rate of preparation 0.4 and 0.2 liters per 1 ton of seeds that correspond to its PMF dilutions 1:25 and 1:50. The results of surveys and measurements are shown in Tables 3-5. Accounting for laboratory and field germination of spring wheat showed that in the control variant it is very high (97.3%) and under the influence of PMF there is no noticeable change in its figure. Germination of seeds was increased by treatment with chemicals which significantly reduced prevalence of plant root rot (Table 19). In phase two heading replicate plots planted with seeds treated with PMF, this drug was sprayed at the rate of 0.250 liters per 1 ha. Determination of wet weight of ears during breast ripeness showed that its index is much higher in the plots where seeds and crops during the heading stage were treated with a new drug. If selective harvesting combine found that a significant increase obtained by pre126

sowing treatment of phytomicrofertilizer at a rate of 0.2 l/t – 3.4 and 0.4 l/t – 7.3c/ha. Further spraying their crops during the heading level of allowances does not exceed 4.8 kg/ha. Analysis of the structure of the crop and biometric indicators revealed that it was obtained by the stimulating effect of the drug on the growth of plants, the formation of the reproductive organs, resulting in increased grains of ear and weight of 1000 grains. In many respects the structure of the crop option of treating seeds and crops during the growing season clearly excelled over the control (Table 20 and 21). It is advisable to continue research to clarify the effectiveness of phytomicrofertilizer on cereals depending on weather conditions, terms and methods of application. To improve competitiveness and reduce the cost of seed treatment is necessary to determine the physiological activity of the drug in a dilution of 1: 100, 1: 250 and 1: 500 which meet the standards of its rate of 100, 25 and 12 of drug per 1 ton of seeds. Table 23 The impact on the PMF spring wheat seed germination and root rot infestation of crops (Karabalyk ACES)

Variants

Phytomicroferti lizer Phytomicroferti lizer Vitavaks 200FF-etalon 1 Kinto-Duoetalon 2 Control

Laboratory findings of Conseeds, % Density of sumpsprouting, tion rate, germinat pcs/m2 l/t energy ion molding ability

Root rot in sprouting stage, % P

R

0.4

97.0

97.0

5.0

344

30.2

8.7

0.2

96.0

97.3

3.0

350

29.7

8.2

2.0

95.3

97.3

1.0

372

16.5

4.5

1.5

96.6

97.3

1.0

356

19.0

5.3

-

97.3

97.3

3.0

346

34.0

9.8

The experience laid on barley showed that phytomicrofertilizer slightly reduces the germination of seeds, although its laboratory component was high – 98.6%. Chemical drug kinto-duo effectively suppressed the development of molds and reduced the prevalence of 127

plant root rot 1.6 times. Number of affected loose smut ears in the control variant does not exceed 1.3%, the disease is not detected in the processing of seeds with kinto-duo, PMF has reduced prevalence of the disease in the plants 2 times. When barley seed were treated with kinto-duo, an additional yield of 3.3 c/ha was produced in case of phytomicrofertilizer – 5.7 c/ha. However, the processing of the crops was not gain, although this method has a positive influence on the formation of grain yield and weight. Table 24 Influence of processing of seeds and wheat crops with chemicals and growth regulators on yield and its structure Variant Vitavaks 200FF Кinto-duo Control PMF PMF PMF + PMF

Crop, c/ha Consumpti Patchy Mass of Grain Mass of on rate, l/t; affected 20 ears in content, 1000 by l/ha leaves, % KML*, g pcs grains, g average control 2.0

21.6

24.0

23.6±1.1

35.2

46.1

5.9

1.5 0.4 0.2

22.2 21.8 25.1 30.1

25.7 28.2 29.2

25.7±0.9 22.4±0.9 24.8±0.7 23.3±0.8

32.4 34.5 36.6 34.7

45.4 40.2 43.6 47.5

5.2 3.4 7.3

0.4+0.2

18.0

27.1

25.4±1.2

35.5

45.0

4.8

*- kernel milk line

НСР0,5=2.5g

НСР0,5=4.1c/ha

Table 25 Influence of processing of seeds and crops of wheat chemicals and inducers of resistance against the structure of complex diseases Consumpti Tilling Stalk on rate, l/t; capacity, height, cm l/ha pcs Control 1.8±0.1 114±1.1 PMF 0.2 2.1±0.1 114±0.9 PMF 0.4 1.8±0.1 111±1.5 PMF+ PMF 0.2+0.2 2.3±0.1 117.2±1.1 Variant

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Length, cm interstitial of ear site 37.3±1.0 6.2±0.1 38.1±0.7 6.2±0.1 36.6±0.8 6.3±0.1 39.3±0.7 6.8±0.2

Grain content, pcs 22.4±0.9 24.8±0.7 23.3±0.8 25.4±1.2

Table 26 The impact of drugs on the germination of barley seeds and seedling root rot infestation

Variant

Кinto-Duoetalon Phytomicrof ertilizer Control

Laboratory findings of seeds, % energy

germination ability

molding

Density of sproutin g, pcs/m2

1.5

93.3

98.0

0.0

1.50

99.3

99.6

-

98.6

98.6

Consum ption rate, l/t

Root rot in sprouting stage, %

Smutt y, %

P

R

0.0

344

30.5

9.6

0.0

1.0

335

47.0

13.2

0.7

1.0

351

50.2

14.4

1.3

Table 27 Effect of processing barley seeds and crops on harvest and its structure Variant

Crop, c/ha Mass of Grain Mass of Consumpti Affected 25 ears in content, 1000 By on rate, l/t leaves, % KML pcs grains average control 0.2 29.9 24.0 42.4 46.9 5.7

PMF PMF+PM 0.2+0.2 F Кinto-duo 1.5 38.2 Control 30.9 НСР 0.5=1.9g

-

18.5

-

29.6 24.8

18.1 18.7

41.6 41.2

45.1

3.9

44.5 3.3 41.2 НСР 0.5=4.8 c/hа

Structure of the barley harvest in the treatment of seeds and crops during the growing season given in Table 28. Table 28 Structure of the barley harvest in the treatment of seeds and crops during the growing season Variant Кinto-duo PMF1:50 Control

Tilling Stalk height, capacity, pcs cm 2.89±0.22 3.02±0.16 2.92±0.15

79.9±1.24 77.1±1.18 76.5±1.11

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Length, сm upper interstitial of ear site 27.2±0.91 7.0±0.21 27.0±0.89 7.0±0.13 25.3±0.66 6.8±0.16

Grain content, pcs 18.1±0.5 18.5±0.41 18.7±0.42

Phytomicrofertilizer significantly stimulated the initial growth of spring wheat and barley. In the treatment of wheat seeds of the drug dilutions from 1:10 to 1: 100, prototype options exceeded the retention sheet for hypophyseal mass from 11.5 to 47.2% of the root system – from 8.2 to 27.9%, and on barley – from 7.4 to 20.5 and from 16.4 to 39.7%, respectively. Higher stimulating effect of the drug was observed at dilutions 1:25 and 1:50. At low temperatures (12-17 °C) phytomicrofertilizer significantly accelerates the growth of spring wheat, which indicates the increase in the resistance of plants to abiotic stress. In field experiments the stimulating effect of phytomicrofertilizer was confirmed on the growth and development of wheat and barley. As a result, stimulation of vegetative growth of plants and the formation of the reproductive organs, increasing grains of ear and weight of 1000 grains in the processing of their seeds the obtained yield of spring wheat increased from 3.4 to 7.7 c/ha with the average yield in the control of 40.2 c/ ha, and on barley – from 3.9 to 5.7 c/ha. It reduced the prevalence of ears of barley loose smut 2 times. On the basis of laboratory and field experiments there was determined the best optimal consumption rate or concentration of the drug to stimulate the growth and development of crops. The obtained data allow applying for an innovative patent to use the drug to increase grain yields and its resistance to biotic and abiotic stresses.

Conclusion 1. The Material Safety Data Sheet approved by the Committee of Industry, Ministry of Industry and New Technologies of the Republic of Kazakhstan dated 16 June 2014 RK-Chemical production number №0000288 was obtained. 2. It is stated that fusicoccin significantly stimulates initial growth of spring wheat and barley. On the accumulation of leaves, shoots hypophyseal mass culture experienced the first variants exceeded the control from 11.5 to 47.2%, the root system – from 8.2 to 27.9%, and barley – from 7.4 to 20.5 and from 16.4 to 39.7%, respectively. Higher stimulating effect of the drug was observed at dilutions 1:25 and 1:50. At low temperatures (12-17 °C) fusicoccin accelerates the growth of spring wheat, which indicates the increase 130

in the resistance of plants to abiotic stress. In field experiments, as a result of stimulation of the vegetative growth of plants and the formation of the reproductive organs, increasing crop spike and 1000 grains weight of seed treatment of spring wheat, the yield increase was from 3.4 to 7.7 t / ha with the average yield in the control of 40.2 t / ha and on barley – from 3.9 to 5.7 t/ ha.

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Scientific issue

Azat Seitkhan Mansurov Zulkhair Aimukhametovich Whitby Raymond THE BIOLOGICAL AND CYTOTOXIC ACTIVITY OF FUSICOCCIN

Monograph Computer page makeup: A. Aldasheva Cover designer: A. Kaliyeva IS No. 12430 Signed for publishing 19.11.2018. Format 60x84 1/16. Offset paper. Digital printing. Volume 8.25 printer’s sheet. Edition 500. Order No. 7926. Publishing house «Kazakh University» Al-Farabi Kazakh National University, 71 Al-Farabi, 050040, Almaty Printed in the printing office of the «Kazakh University» publishing house