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Thematic Division: Chemistry and Technology of Plant Substances. ______________________________________________________ Subdivision: Chemistry of Natural Compounds. Registration Code of Publication: 2pc06 Received June, 24. 2002.
LARCH ARABINOGALACTAN AS A PERSPECTIVE POLYMERIC MATRIX FOR BIOGENIC METALS Svetlana A. Medvedeva,1* Galina P. Aleksandrova, 1 Valentina I. Dubrovina,2 Tatiana D. Tchetverikova,3 Ludmila A. Grishchenko,1 Irina M. Krasnikova,3 Lubov P. Feoktictova,4 and Nonna A. Tukavkina1 1
Irkutsk Institute of Chemistry at Russian Academy of Sciences (Siberian Branch). Favorsky St., 1. Irkutsk 664033. Russia. Tel.: +7 (3952) 511-430. Fax: +7 (3952) 396-046; E-mail:
[email protected] 2 Irkutsk Research and Development Antiplague Institute of Siberia and Far East. Trilissera St., 78. Irkutsk 664047. Russia. Fax: +7 (3952) 220-140. E-mail:
[email protected] 3 Irkutsk State Medical University. Red revolt St., 1. Irkutsk 664003. Russia. E-mail:
[email protected] 4 Irkutsk Institute of Geochemistry at Russian Academy of Sciences (Siberian Branch). Favorsky St., 2. Irkutsk 664033. Russia. Fax: +7 (3952) 464-050. ____________________________________________ *Leader of the thematic course; +Corresponding author Keywords: larch, arabinogalactan, biogenic metals, synthesis, biological activity.
Abstract In the work there have been shown the possibility and perspectives of synthesis of pharmacologically significant compounds containing biogenic metals, with arabinogalactan being used as a biologically active matrix. It has also been shown that arabinogalactan isolated from Siberian larch wood is a biologically active material. It exhibits gastroprotective, membrane-acting properties and has immune modulating activity. Owing to its polymeric base and membrane-acting properties, arabinogalactan can play a role of matrix for directed transport of various medicinal preparations and biologically significant microelements. The designed methods of synthesis have allowed to obtain a series of metal-containing (copper, nickel, cobalt, iron) derivatives of arabinogalactan, the content of metal in the latter varying from 1 to 5%, depending on the conditions of reactions and chemical composition of the initial reagents. As a result of the researches it has been shown that depending on the properties of the metal arabinogalactan is capable of acting as a ligand, or exhibiting the properties of the stabilizer of hydrophobic colloidal systems. So in the case of interaction of arabinogalactan with ions of copper there takes place complexing, at which two vicinal arabinogalactan hydroxyls participate, and depending on рН of the media two types of complexes are formed, which differ in spectral characteristics. In the case of interaction with ferric salts in alkaline media arabinogalactan stabilizes the hydrophobic colloidal system of the generated ferric oxides, transferring them into water-soluble state. The synthesized ferruginous arabinogalactan derivative ferrogal, at the expense of presence of bound iron in its composition, has shown the brightly expressed antianemic activity. Besides, owing to an original method of synthesis, ferrogal has presved not only membranetropic activity of arabinogalactan, but its immune modulating property as well. Moreover, as to the immune modulating effect, ferrogal in some cases even exceeded arabinogalactan. Therefore biologically active arabinogalactan matrix allows and will allow in the long run to obtain a wide series of domestic drugs of a new generation, which will possess in addition to the particular properties of the graft group, membranetropic, immune modulating properties and prolonged operation.
Introduction In recent years a new approach to obtaining therapeutic and diagnostic drugs has been developed basing on immobilization or grafting medicinal preparations on polymeric carriers [1]. It has allowed to improve pharmacological properties of medicinal preparations - prolong their time of operation, to lower toxicity and side effects, to augment selectivity of effect by an organ - target, as well as to improve stability at storage, that is to provide safety and efficiency of operation. As carriers of medicinal preparations there have been preferably used polysaccharides, products of a vegetative origin,. Many polysaccharides commonly used as a polymeric matrix, such as a dextran, cellulose, starch, cellulose glicollic acid, have no membranetropic properties. Besides, the usage of some of them requires carrying out strictly controlled acid hydrolysis followed by subsequent fractionation for obtaining polysaccharide matrix with indispensable molecular weight. Among natural polysaccharides the larch arabinogalactan is singled out as the most perspective, the latter being water-soluble and having rather low molecular weight 10-14 кD [2]. Besides, the larch wood, in which the content of the substance in question makes up 10-15% [3], is a reliable source for obtaining it. Owing to the polymeric base and membranetropic properties [4], arabinogalactan can act as a matrix for the directed transport of medicinal preparations and biologically active trace elements.
Svetlana A. Medvedeva - professor, doctor of chemical science. Graduated in 1964 from Irkutsk State University. Worked as assistant in department of organic chemistry at Irkutsk State University. Since 1967 worked in A.E. Favorski Irkutsk institute of chemistry at Russian Academy of Sciences (Siberian Branch). She defended theses for Candidate's degree (Ph.D) (1974) on the theme “Study of phenol compounds of needle of some types of fir and larch”. In 1995 was awarded Doctor’s degree on the theme "Conversion of aromatic component of wood in the process of delignification”. At present she is the head of the laboratory of biochemistry of natural polymers at Irkutsk institute of chemistry. Scope of scientific interests: chemistry of natural compounds, chemistry of wood and its components. Has over 160 scientific publications.
Galina P. Aleksandrova - senior researcher, Ph.D. in chemical sciences. In 1977 graduated from the chemical faculty of Irkutsk State University. Worked as researcher in Sibirian Scientific Institute of Cellulose and Cardboard. Since 1985 has worked in the laboratory of biochemistry of natural polymers at Irkutsk Institute of Chemistry. Scope of scientific interests: chemistry of wood and its components. Has over 80 scientific publications.
© Chemistry and Computational Simulation. Butlerov Communications. 2002. Vol.2. No.7.
________ K. Marx St., 68. 420015 Kazan. Tatarstan. Russia. _______ 45
Full Paper __________________________________________________ S.A. Medvedeva, G.P. Aleksandrova, V.I. Dubrovina, T.D. Tchetverikova, L.A. Grishchenko, I.M. Krasnikova, L.P.Feoktictova, and N.A. Tukavkina
In the present work we have revealed the possibilities and perspectives of synthesis of pharmacologically significant compounds, in particular those containing biogenic metals, with the use of arabinogalactan as biologically active matrix.
Results and discussion Recently pharmacological properties of arabinogalactan have become a subject of research. In a number of works it has been noted that Siberian larch arabinogalactan exhibits gastroprotective [5] properties, American larch - membranetropic property [4], and has immune modulating activity [6]. Our research has shown that arabinogalactan isolated from Siberian larch wood stimulates antiinfectious resistance of organism at the expense of increasing function activity of cells of cytophagous system in the case of infecting by Yersinia pseudotuberculosis I-716. It activates all cytophagous processes including chemotaxis, adhesion, absorption, bactericidic ability of peritoneal macrophages. In table 1 there are given the parameters of intensity of formation (oxygenic explosion) of oxygen metabolites in englobing cells corresponding to the number of formasan positive cells and citochemical activity factor. As it is seen high values of HCT-test are observed in 30 mines and in 2 hours they increase even more. The cytophagous activity upon introduction of arabinogalactan rises 1.4 times. Table 1. The effect of Arabinogalactan on phagocytosis of Yersinia pseudotuberculosis I-716, M±m, n=8. Citochemical activity parameter, unit. 30 min 2 hours 81.3±4.5 118.0±0.2 52.6±3.6 70.3±3.8
Preparation Arabinogalactan Control
Formasan positive param., % 30 min 2 hours 76.0±2.9 91.0±0.0 50.3±3.4 65.0±4.7
At simultaneous hypodermic introduction to white mice of the preparation and the alive plague vaccine - Yersinia pestis EV arabinogalactan stimulates immunegenes, promotes 4.8 times lowering of the vaccine immunizing dose and increases life expectancy of the vaccinated animal upon infecting with the highly virulent strain Yersinia pestis 2638 (table 2). The revealed immune modulating of property widen the spectra of independent arabinogalactan application and make very attractive its application as biologically active matrix - carrier of medicinal preparations, in particular ions of metals. It can open major perspectives for creation of the transport forms of biogenic metals. Table 2. Protective action of alive plague vaccine (Yersinia pestis EV) on its introduction to white mice in combination with arabinogalactan. Antigene
Y. pestis EV + Arabinogalactan
Dose of antigene, mln cell 104 105 106 107 108 104 105 106 107
Y. pestis EV
Number of animals In experiment survived 8 1 7 1 8 8 6 6 8 8 7 7 6 6 10
Intact animal
1 2 2 5 0
U50, mln cell ρ (on χ2)
5
1.71·10
8.26.105
>0.2 >0.2 0.2 4.8 and were accompanied with the appearance of maximal values of absorption in UV λ 238 nm and visible 700 nm areas of spectrum, the intensity increasing with the growth of рН up to 9.1. Simultaneously the content of free ions of copper in solution became lower, and in solutions with maximal optical density the latter disappeared altogether. The appearance of maximal absorption and growing intensity of the solution absorption at the expense of the higher content of bound copper ions is the authentic proof of formation of complex compounds in the process of interaction of arabinogalactan with copper ions. It is of interest that at further increase of рН up to 12.5 there have been observed changes of position of maximal absorption bands in UV (250 nm) and visible (625 nm) areas (fig. 1), which is probably caused by the change of ligand media of copper ions. On the basis of the comparative analysis of IR spectra of arabinogalactan and its copper containing complexes (they are identical in the area 1600-1800 cm-1) one could unambiguously state that the formation of new coordination clusters in the area рН 9.1 - 12.5 is caused neither by polysaccharide macromolecule oxidation nor by the appearance of carboxyl groups, which have been revealed in the experiment of adextran complexing with ions of copper [11]. More probable reason for the change of spectral behaviour of complexes at high рН is the change of coordination number of copper and/or intermolecular interaction that will be the subject of our further research. The number of arabinogalactan protons (n), participating in the reaction with ions of copper was calculated by the reaction R(OH)3 + Cu2+ ↔ [CuR(O3H3-n)] n-2 + nH+, it has appeared to equal 2, which testified the involvement of two arabinogalactan hydroxyl groups in the complex formed. Using the method of molar ratios it has been established that each sixth arabinogalactan carbohydrate link enters the reaction (6 carbohydrate links of polymer fall on one ion of metal). The constant of a complexing (Кр) 2.7·10-12 has appeared to be comparable on value with the similar constant calculated for complexes of dextran with Сu(II) ion [11]. This research has shown that arabinogalactan is capable of acting as a ligand: in particular, in reactions with ions of copper complexing takes place. The binding of ions of copper in the complex with arabinogalactan is provided in a wide interval рН 5.0-12.5 and Fig. 1. Absorption spectrums of water solutions containing Cu(II) (c = 4.68 10-3 mol/l) and arabinogalactan (of 1.62 %) at various рН: 4.8 (1, it coincides with a spectrum of arabinogalactan water depending on рН the formation of different solution); 6.5 (2); 7.0 (3); 9.1 (4); 10.8 (5); 12.5 (6). complexes is possible. Quite differently behaves arabinogalactan in reactions with salts of iron(II and III). In this case it is similar to other polysaccharides [12] in exhibiting the properties of stabilizer of hydrophobic colloidal systems, in particular of ferric oxides. To obtain ferruginous derivatives there have been used the reaction of interaction of iron salts with ammonia in the media of arabinogalactan water solution: FeCl2 + 2FeCl3+ 8NH4OH = Fe3O4·n H2O + 8NH4Cl + (4-n)H2O Depending on the actual experimental conditions various variants of hydrolytic transformations could be realized, one of which is the formation of colloidal solution of polynuclear forms of hydrated ferric oxide by the common formula Fe3O4·nH2O (colloid particles) [13]. Arabinogalactan thus adsorbed on the surface of colloid particles prevented their aggregations. Ironarabinogalactans [8], as well as all metal derivatives of arabinogalactan were obtained with as high yield as 60-90%. It is possible to enter up to 5% of iron into arabinogalactan macromolecule, and the content of metal varies from 0.32 up to 5.10% depending on chemical composition of salts of the reaction mixture and their ratios (table 4). Table 4. Content of iron in Ironarabinogalactans depending on chemical composition and amount of iron Fe(II) and Fe(III) salts entering the reaction . Amount of iron salts, mmol on 1 g arabinogalactan 0.36 0.46 0.56 0.75 0.96 1.16 1.61 2.08
Fe, % 1.71 2.61 2.67 4.52 4.33 4.21 5.02 4.93
FeCI3 yield, % 74 79 80 65 68 72 57 45
FeCI3+FeSO4 Fe, % Yield, % 3.20 86 3.41 82 4.20 89 5.10 92 5.02 67 3.40 81 1.81 60
Fe, % 0 0.40 1.51 0.32 0.80
FeSO4 yield, % 66 42 71 56 61
The product with maximal iron content and maximal yield was synthesized using the mixture of iron salts (II, III). At that, with the increase of iron salts in the reactionary environment with the interval of values 0.36-0.96 mmol there has been observed the increase of iron content in the products of reaction from 3.20 to 5.10%. The yields of products of reaction reached 92%. The further increase of the amount of salts entering the reaction resulted in lowering up to 60% of both the yield of products of the reaction, and the content of iron in them from 5.00 of 1.8%. Extreme dependence of iron content in a Ironarabinogalactan macromolecule on the amount of iron in the reactionary media (fig. 2) has obviously proved the existence of critical concentration of iron salts (II, III), and as a consequence, critical concentration of hydrated ferric oxide, outside of which there took place violation of stability of colloid particles of the product. In the case of arabinogalactan reaction with FeCl3, in this interval of concentrations no such dependence has been observed. © Chemistry and Computational Simulation. Butlerov Communications. 2002. Vol.2. No.7.
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Full Paper __________________________________________________ S.A. Medvedeva, G.P. Aleksandrova, V.I. Dubrovina, T.D. Tchetverikova, L.A. Grishchenko, I.M. Krasnikova, L.P.Feoktictova, and N.A. Tukavkina
The carried out experiment visually testified that the chemical composition of salts taking part in the reaction, or to be more exact, the rate of oxidation of iron, probably, through the structures of the formed oxides and, probably, the size of particles, influence the quantitative composition of the reaction products. Actually the radiographic method has demonstrated that ironarabinogalactans have amorphous - crystalline structure. The crystalline part is represented by microspheroids of ferric oxides, which have different sizes and are formed differently. Their formation depends on the conditions of reaction, first of all on chemical composition of the reaction mixture. If the salts Fe(III) participate in synthesis, diamagnetic samples of ironarabinogalactans are obtained, in which iron is bound in the form of ferrihydrite (δ-FeООН and γ-FeООН). If the mixture of salts Fe(III) and Fe(II) is introduced into the reaction, then ferromagnetic samples are produced, EPR – spectra of which represent two intensive singlets with parameters: g1=2.034, ∆H=450e and g2=1.97, ∆H=130e. Fig. 2. Influence of change of amount of iron salts in the The metal cell of ironarabinogalactans is shaped as a spinel, but with reaction mixture on the iron content in ironarabinogalactans: different ratios of oxides Fe(III) and Fe(II): iron enters the preparation as an 1 - product of arabinogalactan interaction with the mixture analog of minerals of the series magnetite- maghemite. These conclusions FeCl3 and FeSO4; 2 - product of arabinogalactan interaction with FeCl3. follow from the values of parameters of element cells of the preparation (table 5). Thus ironarabinogalactan (3.5% of iron) has the composition close to that of magnetite. The content of γ-Fe2O3 in its structure does not exceed 20-30%. Ironarabinogalactans (5.1% of iron) by its composition comes nearer to maghemite, the part of γ-Fe2O3 compounds being not less than 80%. Table 5. The ironarabinogalactans radiographic data. Parameter Iron content, % α - Parameter of cell, А L - Size of fragment, А Part of γ-Fe2O3, %
Ironarabinogalactans Fe(III) + Fe(II) Fe(III) + Fe(II) 3.53 5.01 8.390 8.360 160-170 250 20 80
Fe(III) 4.50 10
magnetite Fe3O4
maghemite γ-Fe2O3
8.396
8.350
Basing on the obtained results of radiographic research, we can accept that similar to dextraneferrites [14] ironarabinogalactans synthesized by us represent microspheroids, which have cores composed either from ferric oxides with crystalline structure of magnetite / maghemite, or from amorphous ferrihydrites coated with a layer of arabinogalactan molecules. It is quite obvious that in this case we deal with ultra dispersible polymeric fragments of hydrated ferric oxide, which by virtue of the dimension should have a high surface energy. The natural tendency of the system to spontaneous decrease of free energy can be accompanied with adsorption on the surface of molecules or atoms from a dispersion media. Thus, it is quite possible that interaction between arabinogalactan and nanoparticles of hydrated ferric oxide takes place owing to the activity of surface of the latter and results in the formation of the colloidal system. So the research carried out by us has shown that the chemical arabinogalactan properties in reactions with salts of metals are polyhedral: arabinogalactan, on the one hand, is capable of participating in the processes of complexing, exhibiting the properties of a ligand, and, on the other hand, can fulfil the functions of stabilizer of colloidal systems. The research of physicochemical characteristics of ironarabinogalactans has shown that the formed colloidal systems are agregately stable. They failed only in the conditions of acidic media (0.01 M НСl). Iron from preparations passed into the solution at different rates (3-12% per hour) depending on the composition of preparation. The most stable system turned out to be ironarabinogalactans containing 3.5% of iron; the latter was obtained with the use of the mixture of iron salts Fe(III) and Fe(II): its decomposition took 48 hours. Therefore, this preparation can provide the most prolonged isolation of iron in the cell. This ironarabinogalactan (ferrogal), the conditions of obtaining of which were standardized, had average weight molecular mass somewhat less than the initial arabinogalactan (14 S.D), it varied in an interval 9-10 S.D. The given IR-spectroscopy proved the absence of structural transformations in the macromolecule of arabinogalactan itself: the spectra of arabinogalactan and ferrogal have appeared to be identical. This allowed to suppose that the chemical effect of the reaction did not result in deep chemical change of a arabinogalactan acromolecule, and consequently, in the change of its biological activity. In this connection, ferrogal was investigated as an antianemic drug and is researched its immune modulating ability was studied Iron deficiency anemia is one of the most wide-spread diseases of blood circulatory system. The existing low molecular weight medicinal forms of the ferruginous drugs applied for treatment of the given pathology do not always render the desired therapeutic effect and perform low assimilation. In this connection the search of new preparations possessing improved properties for treatment of this nosological form is still actual. Table 6. Hhaemoglobin and erythrocyte content in the blood of animals with iron deficiency anemia. Haemoglobin, g/l Experimental group 145.17±4.31 95.75±6.67 р
