Diabetes mellitus is a syndrome of disturbed metabolism of glucose which arises due to hyperglycemia which is responsible for the majority of symptoms. Insulin is actually the key regulator of the metabolism of hydrocarbons, proteins and lipids, and a relative or absolute shortage of insulin effects the complete intermedial metabolism. It is supposed that biochemical disturbances are primarily determined by the amount of the insulin deficiency.
Diabetes of Type I, i.e. IDDM (Insulin Dependent Diabetes Mellitus) is characterized by progressive autoimmune process of the destruction of β cells of Islets of Langerhans by T lymphocytes (Eisenbarth, G S. New Engl. J. Med. 314:1360–1368, 1986). More precisely, IDDM is the result of the destruction of beta cells mediated by CD4+ and CD8+ cells and the function of antigen presenting cells (APC) (Frque F., Had{hacek over (z)}ija M., et al., Proc. Natl. Acad. Sci., USA, 91:3936–3940, 1994).
NOD (non-obese diabetic) mice develop the classical picture of diabetes which is completely identical to IDDM in people (Makino S., et al., Expl. Anim. 29: 1 (1980)). Furthermore, chemical diabetes caused by Alloxan in mice also develops the picture of diabetes with all the accompanying symptoms identical to the human form of diabetes of Type I (Dunn J. S., et al., Lancet II: 384–387, 1943).
The purpose of the therapy of diabetes is the normalization of the following parameters; the concentration of glucose in blood, the concentration of lipids, and the absence of glucose and acetone in urine.
In the therapy of diabetes, two principles of treatment are applied: firstly, the general one, and secondly, special principles of treatment of diabetes.
General Principles of Treating Diabetes
By treating diabetes, it is endeavored to achieve the state of normoglycemia and thus prevent the development of later complications. The general principles of treatment comprise, regardless of the type of diabetes: diet, physical activity, education and self-control. The nourishment must be composed so that it meets the daily needs for nutrient according to the age, sex, activities, height and weight. It is recommended that the diet consists of: 15–20% of proteins, 25–30% of fats, and 55–605 of hydrocarbons. The total daily quantity of substances must be divided into a larger number of smaller meals (5–6 during the day) in order that no major oscillations in the concentration of glucose in the blood would occur, the physical activity is important because it increases the exploitability of glucose in peripheral tissues with a proportionally reduced use of insulin. Education and self-control are important for understanding of the very disturbance, and for the daily self-control of the concentration of glucose in the blood.
Special Principles of Treating Diabetes
Treatment by exogenous insulin is applied in case of Type I Diabetes, where over 805 of the mass of the endocrinous part of the pancreas. The successfulness is high since, before the discovery of insulin, such persons used to die after 1.5 years, and today they live for even 50 years after the appearance of the disease.
Oral antidiabetics are not a replacement for insulin, but a support to the secretion of endogenous insulin or its hypoglycemic effect. Persons with the Type II Diabetes should take oral antidiabetics only in case if the correct nourishment and the corresponding physical activity have not given the desired results. Although the transplantation of pancreas, i.e. Islets of Langerhans, is one of the special principles of treatment of Type I Diabetes, it is still being researched today. Namely, the success of the transplantation depends on the number of transplanted islets, by which the isolation is still a problem, as well as the immunological reaction of rejection due to which various immunosuppressive must be used on a daily basis.
Metabolic Effects of Insulin
Insulin is a protein, with the molecular mass of 5734 Da. It consists of “A” and “B” chain, which are connected by two disulfide bridges, while the third disulfide bridge is within the “A” chain. “A” chain consists of 21 amino acids, and the “B” chain of 30 amino acids.
Insulin is synthesized by β cells of the Islets of Langerhans of the pancreas. The basic physiological function of insulin consists in maintaining of normoglycemia. Thus, insulin effects the metabolism of hydrocarbons so that it firstly suppresses the creation of glucose in the liver, and that happens if the concentration of insulin in the circulation ≈30 mU/L, and secondly so that it stimulates entering of glucose into peripheral tissues (by speeding up the translocation of the glucose transporter GLUT-4 on the surface of cells of skeletal muscles and of adipose tissue, and by activation of intracellular enzymes such as, e.g. glycogen synthetase). That happens if the concentration of insulin in the circulation ≈100 mU/L, which is usually present in the blood after a meal. Insulin is also a very potent inhibitor of lipolysis, and thereby also of the ketogenesis. The antilipolitic effect of insulin is manifested already at the insulin concentration of ≈10 mU/L. Insulin also inhibits the proteolysis, if its concentration in the blood is between 10–30 mU/L.
By binding of insulin to the extracellular α-subunit of receptor, the insulin-receptor complex activates the Zn2+ dependent protein tyrosine kinase which makes the transmembrane β-subunit of insulin receptor and, due to that, autophosphorylation of receptor and other proteins with phosphate groups over ATP (Reddy and Kahn, 1988) arises. The activation of phosphatidylinositol-specific phospholipase C leads towards the hydrolysis of membrane phosphoinositides. Thus, the cyclic inositol-phosphate glucosamine arises, the second messenger which activates phosphodiesterases, reduces the content of the intracellular cAMP and produces diacylgycerol which activates protein kinase C (Saltiel, 1986). Protein kinase C regulates numerous enzymes and the very insulin receptor through phosphorylation (Van de Werve, 1985).
During testing of Type I Diabetes by insulin, the slow absorption of insulin from the subcutaneous tissue results in its inadequate pique at the time of the meal and after taking it with hyperglycemia between two meals (due to a low concentration of insulin in the port vein). Insulin deficiency leads to an increased release of glucose by the liver, and that is the reason of hyperglycemia on an empty stomach, i.e. of postprandial hyperglycemia. The consequence of the low concentration of insulin includes the increase of the secretions of glucagon from α-cells of the Islets of Langerhans of the pancreas.
Insulin resistance is often joined with the appearance of obesity, the intolerance of glucose, hypertension, dyslipidemy, disturbances in blood coagulation and the speeded up aterogenesis and it is also referred to as the Syndrome X.
Role of Plant Proteins in Treatment of Diabetes
The results of sequencing of the human genome just published stimulate researches about the role of small target molecules and proteins connected with diseases (Engl. target).
The completed sequencing of the human genome revealed a number of “harmful” genes on all chromosomes. Those results indicate and reveal a number of target places for medicines. In that context, a new strategy of research for obtaining of new medicines is started. That strategy first comprises target places (primarily, those are proteins, but that also includes parts of nucleic acids), and secondly, the ligand, i.e. proteins of small molecules as a substitute to the “ill” target place.
Plants contain four main types of molecules: hydrocarbons, proteins, nucleic acids and lipids. Beside the stated ones, plants contain other types of molecules in smaller quantities, e.g.: alkaloids, terpenoids, phenols, sterols another so-called “secondary metabolites”.
Hydrocarbons in plants include monomers called monosaccharides and polymers called polysaccharides. Polysaccharides which are included in the construction of the plant cells are called structural polysaccharides. The best known structural polysaccharide in plants is cellulose (it makes 40%–60% of the cell wall). Reserve polysaccharides serve as the reserve of food, and the best known in plants are starch and insulin.
After cellulose, proteins make the largest remaining part of the biomass of the living plant cell. Proteins in plant cells are made of 20 various amino acids bound into polypeptides. As well as in case of hydrocarbons, proteins also have an important role in the construction of the cell (structural proteins), and they also serve as a reserve. Unlike hydrocarbons, proteins can also be enzymes. Structural proteins of the cell wall are called extensions and have an important role in its expansion during the very growth of the plant. Extensions are proteins rich in hydroxyproline, serin, threonine and the asparaginic acid. Plant cells contain various kinds of membranes, each has a different protein system. E.g. the inner membrane of mitochondrions and chloroplasts contains about 75% of proteins while the membrane which surrounds the plant cell has about 50% of proteins. The majority of the non-protein part of the membrane which surrounds the plant cell is made by lipids. The reserve proteins in plants are most often present in seeds and they serve as the source of nutrients during germination. The contents of proteins in seeds depends on the plant species. Some reserve proteins of plants are: zein, gliadin, ricin D, abrin, etc. Many plant proteins have the function of enzymes and catalyze biochemical reactions. One example is α-amylase, the enzyme which disintegrates amylose.
Plant Peptides and Amino Acids
In 1981, Khanna et al. published the results of the research about isolation of “polypeptides p” from the fruit and seeds of the plant Moomordica charantia L. (Cucurbitaceae). “Polypetide p” consists of 17 various amino acids and has the molecular mass of 11,000. When the s.c. is applied in rodents, primates and people, its insulinomymetic effect in the dose of 0.5 units/kg (1.8 mg/ml=40 units) is noticed. Sulfoxide amino acids: s-methylcysteine sulfoxide (SMCS) and S-allylcystein sulfoxide (SACS) isolated from plants Allium cepa L. and Allium sativum L. have caused the loss of body mass and the content of glycogen in liver after a month of per oral application in rats with experimentally induced diabetes (Sheela et al., 1995).
In 1998, Sauvaire et al. have isolated, and in 1999, Broca et al. have described the in vivo effect of 4-hydroxyisoleucine as a new stimulator of the insulin secretion from the seeds of Trigonella foenum graccum L, 4-hydroxyisoleucine aids the glucose induced secretion of insulin both on the model of isolated Islets of Langerhans, and in people. Its stimulating effect in a dose of 100 μmol/L to 1 mol/L was dependent exclusively on the stimulation of the secretion of insulin by glucose, i.e. it did not show the activity in case of low concentrations of glucose (3 mmol/L) or the basal concentration of glucose (5 mmol/L).
The fruit of the plant Blighia sapida Koenig (Sapindaceae) contains emetic ingredients: hypoglycine A and its γ-L-glutamyl dipeptide, hypoglycine B, which indicate a hypoglycemic activity, they act in such a way that they inhibit oxidation of long-chain fatty acids. Hypoglycine A is twice stronger hypoglycemic compound than hypoglycine B which is also a teratogen, and thus too toxic for therapeutic use (Tanaka et al., 1972; Oliver-Bever and Zahnd 1979).
Development of Neuropathy in Diabetes
Neuropathy is a common late complication of diabetes which affects somatic and autonomic peripheral nerves. Neuropathy occurs in a certain percentage in Type I and Type II Diabetes (Greene, D A, et al., Diabetes Care. 15:1902–6, 1992). Peripheral nerve abnormalities in people and in the animal model of diabetes are manifested as the decreased conductibility of nerves, axonal reduction, and nerve fiber loss (Behse F F, et al., J. Neurol. Neurosurg. Psych. 40:1072–82, 1977; Brismar. T. Metab. Clin. Exp. 32:112117, 1983; Sima, A A F, et al., Ann. Neurol. 18:21–29, 1985), in connection with metabolic alterations (Green, D A, et al., Diabetes 37:688–693, 1988), including altered calcium signaling (Levy, J, et al., Am. J. Med. 96:260–273, 1994). Existing studies indicate that altered homeostasis of calcium ions is a widespread occurrence in IDDM and NIDDM. Both, in people suffering from diabetes, and in animal models of diabetes, the identical change was observed, which is the increase of the Ca2+ ions in the cytosol (Hall, K E, et al., J. Physiol. 486:313–322, 1995; Nobe, S, et al., Cardiovas. Res. 24:381, 1994.; White, R E, J. Pharmacol. Exp. Ther. 253:1057–1062, 1993.; Kappelle, A C, et al., Br. J. Pharmacol 111:887–893, 1992). The increase of the concentration of calcium ions aids the process of natural atrophy (apoptosis) of nerve cells, but that process has been shown in a number of other experimental models as well (Trump, B F, et al., FASEB J. 9:219–228, 1995; Down, D., Phosphoprotein Res. 30:255–280, 1995; Joseph, R, et al., Mol. Brain. Res. 17:70–76,1993).
The latest researches suggest that “serum factors” have an important role in the pathogensis of diabetic neuropathy in patients with Type I diabetes mellitus. By incubation of β-cells of the Islets of Langerhans in conditions of the general tissue culture, when the serum of patients with Type I or Type II Diabetes was added to the medium (Had{hacek over (z)}ija, M, et al. Period. Bio 1.97:313–317, 1995), apoptosis in β-cells of the Islets of Langerhans is connected with the increase of the concentration of L-type calcium ions (Juntti-Berggren, L, Science 261:86–89, 1993). It was also shown that neuroblastoma cells demonstrated a reduced growth, the increase of entering of Ca2+ ions, i.e. the intensified apoptosis if they were exposed to the serum of patients suffering from Type I Diabetes with neuropathies (Pittinger, G L, et al., Diabetic. Med. 10:925–932, 1993; Pittinger, G L, et al., Diabetic Med. 12:380–386, 1995: Pittinger, G L, et al., J. Neuroimmunol. 76:153–160, 1997; Migdalis, I N, et al., Diabetes. Res. Clin. Pract. 49:113–118, 2000). The complement-independent, Ca2+-dependent inductions of apoptosis of nerve cells aid the appearance of autoimmune immunoglobulins in diabetes on nerve fibres (Srinivasan, S M, J. Clin. Invest. 102:1454–1468, 1998).
the characteristics of zeolites, such as the possibility of ionic exchange, existence of in intercrystalline pores which let through molecules of various dimensions, the existence of strong acidic places and places active for the reactions catalyzed by metals, etc. make them very interesting for a wide industrial implementation, as well as for fundamental researches (Flanigen, E. M. in: Proc. Fifth. Int. Conf. Zeolites (Ed. L. V. C. Rees), Heydon, London-Philadelphia-Rheine, 1980, p. 760; Vaughan D. E. W., Chem. Eng. Prog., 25, 1988; Cornier, J., Popa, J. M., Gubelman, M, L'actualite, 405, 1992; Subotić, B., Bronić, J., {hacek over (C)}i{hacek over (z)}mek, A., Antonić, T., Kosanović, C., Kem. Ind. 43:475, 1994). The interest for using zeolites as catalysts, absorbents and means for softening of water in detergents has largely increased in the last three decades (Cornier, J., Popa, J. M., Gubelman, M., L'actualite, 405). There are annually millions of tons of zeolites used in the production of washing means (Cornier, J., Popa, J. M., Gubelman, M., L'actualite, 405), hundreds of thousands of tons in oil processing and in the petrochemical industry (Naber, J. E., de Jong, K. P., Stork, W. H. J., Kuipers, H. P. C. E, Post, M. F. M., Stud. Surf. Sci. Catal., 84C:2197, 1994), and the use in other fields is increasing too (Flanigen, E. N. in: Proc. Fifth. Int. Conf. Zeolites (Ed. L. V. C. Rees), Heyden, London-Philadelphia-Rheine, 1980, p. 760; Waughan, D. E. W., Chem. Eng. Prog., 25, 1988; Cornier, J., Popa, J. M., Gubelman, M., L'actualite, 405, 1992; Naber, J. E., de Jong, K. P., Stork, W. H. J., Kuipers, H. P. C. E, Post, M. F. M., Stud. Surf. Sci. Catal., 84C:2197, 1994.; Breck, D. W. in: The properties and Application of Zeolites, Special Publication (Ed. R. P. Towsand), 33:391, 1980.; Vaughan, D. E. W. in The properties and Application of Zeolites, Special Publication (Ed. R. P. Towsand), 33:294, 1980.; Flanigen, E. M., Pure Appl. Chem., 52:2191, 1980.), including the use of zeolites in medicine, agriculture and cattle breeding (Ramos, A. J., Hernandez, E., Animal Feed Sci. Technol., 65:197, 1997; Eriksson, H., Biotechnology Techniques 12:329, 1998.; Mumpton, F. A., J. Nat. Acad. Sci., 96:3463, 1999.).
Zeolites or molecular sieves are hydrated natural and synthetic aluminosilicate compounds with a unique spatial-network structure consisting of SiO4 and AlO4 tetrahedrons linked through common oxygen atoms (Breck, D. W., J. Chem. Educ. 41:678, 1964), as it is schematically presented in FIG. 1.
The negative charge of the aluminosilicate structure is caused by an isomorphic replacement of the silicium with the valence of four by aluminum with the valence of three is neutralized by hydrated cation (Na+, K+, Ca2+, Mg2+ etc.). In reality, SiO4 and AlO4 do not create single-dimensional chain structures in the structure of zeolites, as it is simplified in FIG. 1, but they create two-dimensional and three-dimensional basic structural units, the combination of which gives rise to three-dimensional spatial-network structures characteristic for zeolites (FIGS. 2–5). The specific structure of zeolite, unique, both in the relation with other aluminosilicates, and in relation to other crystal materials, is reflected in existence of structural cavities mutually linked by channels of a certain form and size (FIGS. 2–5). However, unlike other porous materials characterized by a specified arrangement of pores which are statistically distributed in various directions, the form and size of cavities and channels, as well as their mutual relationships are constant and exactly defined as structural parameters of a specific type of zeolites (Barrer, R. M., Zeolites and Clay Minerals as Sorbents and Molecular Sieves, Academic Press, London, 1978, p. 23), as can be seen in the stated examples of the structure of a unit lattice of the zeolite A (FIG. 2), faujasites (X and Y zeolites, FIG. 3), mordenite (FIG. 4) and the ZSM-5 zeolite and silicalite (FIG. 5).
The chemical composition of zeolites is usually presented by a general formula in the oxide form:M2/n.Al2O3*ySiO2-zH2OWhere n is the charge number of cations M, while y≧2 and z depend on the type of the zeolite. The “zeolite” water results from hydration membranes of the compensation M cations. Therefore, the value z depends on the type of the M compensation cations, the number of M cations in a unit zeolite lattice and the level of M cations hydration in the zeolite lattice. By heating of the zeolite up to approx. 600° C., the “zeolite” water can be removed from the zeolite without the change of the structure. By cooling to the room temperature, the same quantity of water is bound to the zeolite, i.e. the processes of desorption and adsorption of zeolite water are strictly reversible.
In touch with electrolyte solutions, cations from a zeolite (Breck, D. W., J. Chem. Educ., 41:678, 1964; Fedorov, V. A., Tolmachev, A. M., Panchenkov, G. M., Zh. Fiz. Khim, 38:1248, 1964; Wolf, F., Foertig, H., Kolloid Z.-Z. Polymere, 206:48, 1965, Sherry, H. S., Adv. Chem. Ser., 101:350, 1971, Brooke, N. M., Rees, L. V. C., Adv. Chem. Ser, 101:405, 1971:Barrer, R. M., Klinowski, J., Phil. Trans 285:637, 1977). In the conditions of balance, the following applies: (Schwuger, M. J., Smolka, H. G, Tenside Detergents, 13:305, 1976):zB×Aza(aq)+zA×Bzb(s)⇄zB×Aza(s)+zA×Bzb(aq)where zA and zB are charge numbers (“valences”) of replaceable cations A and B, while aq and s denote the solution, i.e., the firm phase (zeolite).
Natural zeolites are impurified with various admixtures, therefore we used a synthetic zeolite with strictly defined characteristics in the preparation of this invention.