**2. Larch Arabinogalactan**

154 The Complex World of Polysaccharides

**Figure 1.** *Larix sibirica* Ledeb.

manufactured in Russia.

production of pectin in Russia.

and cosmetic industries. Arabinogalactan is a perspective matrix for obtaining on its basis metal-, sulpho-, amino- and other derivatives due to the reactive hydroxyl and aldehyde groups contained in its molecule. The ability of arabinogalactan to form water-soluble stable substances with inorganic nanoparticles and low-molecular medical substances (MS)

In the USA, arabinogalactan has been extracted from the wood of *L. occidentalis* Nutt. and *L. laricina* (Du Roi) by K. Koch for more than 40 years as a commercial product. Effective immunity-modulating and prebiotic biologically active food additives have been developed to improve quality of human life. Application of arabinogalactan in agriculture as fodder additive allows the greater efficiency of animal industries. Arabinogalactan is not currently

The bark of larch does not however have industrial application. Annually, wood-processing industries and pulp-and-paper enterprises waste more than 30 million m3 in volume. It has become a serious environmental problem because the bark is badly exposed to biodegradation. At the same time, the chemical compounds of the bark can be a source of valuable biologically active substances, including polysaccharides. The creation of medical,

Larch bark contains about 7–12% of pectin polysaccharides, based on the weight of absolutely dry raw material. Pectin is acid polysaccharide–glycogalacturonane, and is contained in practically all plants. It is obtained from diverse sources that differ in their chemical structure due to distinctions in the qualitative structure of carbohydrates and their quantitative parities. Pectin substances promote digestive processes, and help organisms to resist many diseases such as atherosclerosis, diabetes, cancer, etc. There are two basic sources of pectin production in Germany and Denmark. However, there is no industrial

food and other useful products on the basis of polysaccharides is also possible.

provides serious prospects for the development of materials with unique properties.

## **2.1. Physicochemical and biological properties of Arabinogalactan**

Larch wood is distinctive for its high content of water soluble polysaccharide arabinogalactan, reaching up to 35% by weight of dry wood [1]. This valuable substance has been studied since the 1950s [1-6]. The physicochemical and biological properties of AG from the wood of *L. occidentalis* Nutt. and Siberian larch species *L. sibirica* Ledeb. and *L. gmelinii* (Rupr.) Rupr. are the most explored. The wood of Siberian larches contains up to 10–15% of AG [2,7] and is a reliable source of industrial raw material for AG production.

The AG macromolecule from larch wood has a highly branched structure. Its main chain consists of -(13) linked galactose residues (Figure 2). Approximately one half of the side chains (in *L. occidentalis* Nutt., *L. sibirica* Ledeb. and *L. gmelinii* (Rupr.) Rupr.) is formed of - (16)-linked dimers of galactopyranose; galactopyranose monomers comprise about a quarter; and the remainder contains the major part of the polysaccharide's arabinose in aggregates of two or more monomers [1,8,9]. Arabinose fragments mainly occur as side chains consisting of 3-O-substituted -L-arabinofuranose residues and terminal residues of -L-arabinopyranose, -D-arabinofuranose and -L-arabinofuranose [8,9]. However, arabinose fragments have also been found in the main chain [1]. Glucuronic acid fragments in AG from wood of various larch species is low in content. As for AG from the above species, no glucuronic acid fragments have been detected in the purified samples [6].

Monosaccharide composition and molecular mass (MM) of AG macromolecules differ among the species and also varies within single species. It has been established that the composition of AG macromolecules is dependent on the conditions under which it is isolated from larch wood and on the purification procedure [9,10] as well as on molecular weight [1,11]. AG macromolecules have low molecular weights (13–20 kDa, according to HPLC data) and a narrow molecular weight distribution (degree of polydispersity 1.1–2.3) [10].

Biological activity of AG in higher plants is directly linked to their structural characteristics, such as length of galactan chain, structure of side chains, molecular weight and ability to form intermolecular associates [12,13]. Larch AG is characterized by low toxicity, showing neither acute poisoning for doses of 5 g/kg nor chronic poisoning for doses of 500 mg/kg per day [14]. Diverse biological activity of AG includes immunomodulatory, prebiotic, hypolipidemic, gastro- and hepatoprotective, mitogenic, antimutagenic and antiviral effects, etc. There are reports on the inhibitory and destructive action of AG against certain types of malignant tumours [6,15-22]. Moreover, it has a good solubility in cold water, uniquely low viscosity of concentrated aqueous solutions, an ability to bind fat and retain liquid and dispersive capacity, etc. All these benefits are in high demand in medicine and veterinary science as well as in food and cosmetic industries [5,6]. There are a number of biologically active food supplements which incorporate AG [23-25]. In medicine, an ophthalmic composition (eye drops/contact lens care solution) has been developed [26]. Membranotropicity caused by galactose fragments and realized through receptor mediated endocytosis makes AG a promising drug carrier to increase absorbability and selectivity of medical substances that are characterized by low bioavailability [27-36]. Applications of AG in photodynamic diagnostics, in oncological disease therapy and in gene therapy (targeted delivery of functional genes) are currently being explored [37-39]. The unique properties of AG are prominent among the known polysaccharide carriers of medical substances [40,41].

**Figure 2.** Structural fragment of AG macromolecule

Significant interest for medicine is raised by the products of AG modification [6]. Introducing diverse functional groups into the AG macromolecule makes it a manifold synthone to obtain a wide range of new biologically active substances. Oxidation is a promising way to functionalise AG. Methods of selective oxidation developed in classical carbohydrate chemistry [42] have been used to develop functionalized AG products [43,44]. Among others, oxidative destruction with simultaneous introduction of carboxylic groups into the macromolecule under the action of hydrogen peroxide in aqueous medium has been carried out [45]. It has been revealed that oligomeric products show anti-inflammatory and antiulcer activities. Reactions of AG and oxidized AG with some known MS give intermolecular complexes [46-48]. Such complexes of AG with 5-aminosalicylic acid show high antiulcer activity, while complexes incorporating 4-aminosalicylic or isonicotinic acid hydrazide demonstrate an antituberculosis effect. It has been established that conjugates of AG and products of its modification increase the physiological effect of MS and decrease their toxicity [28,46-48]; for instance, a conjugate of AG (9kDa) 9-β-Darabinofuranosyladenine-5'-monophosphate was 25-fold more active than the parent compound 9-β-D-arabinofuranosyladenine (araA) in decreasing the amount of hepatitis B virus, and the toxicity of the complex preparation is much lower than araA [28]. To increase reactivity of AG with MS, the synthesis of conjugates proceeds by bromination, phosphorylation, amination, formation of hydrazides or reaction with NaBH4 [27,33,34].

156 The Complex World of Polysaccharides

**Figure 2.** Structural fragment of AG macromolecule

neither acute poisoning for doses of 5 g/kg nor chronic poisoning for doses of 500 mg/kg per day [14]. Diverse biological activity of AG includes immunomodulatory, prebiotic, hypolipidemic, gastro- and hepatoprotective, mitogenic, antimutagenic and antiviral effects, etc. There are reports on the inhibitory and destructive action of AG against certain types of malignant tumours [6,15-22]. Moreover, it has a good solubility in cold water, uniquely low viscosity of concentrated aqueous solutions, an ability to bind fat and retain liquid and dispersive capacity, etc. All these benefits are in high demand in medicine and veterinary science as well as in food and cosmetic industries [5,6]. There are a number of biologically active food supplements which incorporate AG [23-25]. In medicine, an ophthalmic composition (eye drops/contact lens care solution) has been developed [26]. Membranotropicity caused by galactose fragments and realized through receptor mediated endocytosis makes AG a promising drug carrier to increase absorbability and selectivity of medical substances that are characterized by low bioavailability [27-36]. Applications of AG in photodynamic diagnostics, in oncological disease therapy and in gene therapy (targeted delivery of functional genes) are currently being explored [37-39]. The unique properties of AG are prominent among the known polysaccharide carriers of medical substances [40,41].

Significant interest for medicine is raised by the products of AG modification [6]. Introducing diverse functional groups into the AG macromolecule makes it a manifold synthone to obtain a wide range of new biologically active substances. Oxidation is a promising way to functionalise AG. Methods of selective oxidation developed in classical carbohydrate chemistry [42] have been used to develop functionalized AG products [43,44]. Among others, oxidative destruction with simultaneous introduction of carboxylic groups into the macromolecule under the action of hydrogen peroxide in aqueous medium has been carried out [45]. It has been revealed that oligomeric products show anti-inflammatory and antiulcer activities. Reactions of AG and oxidized AG with some known MS give Mechanochemical activation is another promising method of AG modification, in which the target products are obtained in one stage without the use of solvents. Mechanochemical treatment can give rise to numerous physicochemical transformations of AG macromolecules, which are associated, first, with the breaking and formation of valence bonds and, second, with the disturbance and origination of weak intermolecular interactions (disordering, conformational rearrangements, etc.). As a result, the polysaccharide can change its biological activity, toxicity and pharmacological properties. HPLC and quantitative 13C NMR spectroscopy has established [49] that mechanochemical treatment of AG isolated from Siberian larch wood changes its molecular weight distribution, monosaccharide composition and degree of branching due to partial destruction of the macromolecules and subsequent recombination of their fragments. The extent of these changes depends on the activation conditions. The IR and 13C NMR spectra have not shown any functionalization of AG macromolecules in the conditions studied. Toxicpharmacological study has established that a mechanochemically activated AG sample has the same LD50 (more 5000 mg/kg) than the starting AG. As for the effect of mechanochemically activated AG upon the central nervous system, it has demonstrated anxiolytic activity similar to sibason in a dose of 20 mg/kg tested in laboratory animals. Meanwhile, single intravenous injection of the substance, in a dose of 3.5 mg/kg, slightly but statistically significantly decreases arterial pressure (by 6%) in normotensive rats without affecting electrocardiogram parameters and heart rate. Thus, mechanochemically activated AG is a promising drug carrier [49].

Combined mechanochemical activation of MS with AG (pharmacon clathration) is even more effective in improving safety and bioavailability of drugs [50-53]. Essentially (up to 50 times) increased solubility of MS and dramatically decreased therapeutic doses of the same efficiency are reported for clathrates of poorly soluble anti-inflammatory, psychotropic and hypotensive drugs [51,52]; for instance, clathrates of AG with nifedipine containing a 10 times lower dose than the starting MS show pronounced hypotensive and antiarrhythmic effects [52]. Additionally, the side effects of MS in clathrates are decreased, for instance in the ulcerogenity of nonsteroidal anti-inflammatory drugs. HPLC, 13C NMR and IR spectroscopic studies have shown the absence of any chemical reaction between AG and MS at pharmacon clathration [51,53]. The X-ray phase study and thermal analysis prove the destruction of the crystal structure of MS and its dispersion within the AG matrix. The

polysaccharide macromolecules are cleaved similarly to the mechanochemical activation of AG alone [49].

*L. sibirica* Ledeb. and *L. gmelinii* (Rupr.) Rupr. contain much bioflavonoid dihydroquercetin (DHQ, taxifolin) and their diverse biological activity is well studied. It is an officinal drug with a wide range of therapeutic action and is also the basis for a number of efficient medical preparations and food supplements [54]. Complexes of AG and DHQ combining these unique properties are very promising. Such complexes have been obtained by pharmacon clathration [54] and have shown essentially improved solubility (up to 38 times) in comparison with starting DHQ and untreated AG/DHQ mixture. No chemical reaction between AG and DHQ takes place, as in clathrates [51]. According to HPLC data, AG in AG/DHQ clathrates has a narrower molecular weight distribution in comparison with pure mechanochemically activated AG, due to a decrease in both high- and low-molecular fractions, and thus DHQ stabilizes the polysaccharide macromolecules in mechanochemical treatment.

The most recent application of AG as a stabilizing polymer matrix in hybrid nanosized materials is based on iron oxides, cobalt, copper, nickel, ferrites and zero-valence metals such as silver, palladium and platinum [18,55-57]. Metal content in nanocomposite samples depends on synthetic conditions and on the type of metal ion used, varying in the range of 0.1–21.0%. In the case of metal oxide nanocomposites, AG shows properties of a nanostabilizing matrix, while in the composites of noble metals it reduces metal to a zerovalence state and stabilizes the metal nanoparticles formed. Nanocomposites based on AG retain high biological activity. Ferroarabinogalactans show synergy between the pronounced antianaemic activity of the ferric core and the unique membranotropic and immunomodulatory properties of AG. Parenteral administration of ferroarabinogalactan normalizes quantitative and qualitative characteristics of the erythrocyte system and iron depot level in animals (white rats) [18]. The original synthetic method of ferroarabinogalactan retains both membranotropic and immunomodulatory properties of AG. Studies of the natural effects of immunomodulators, together with the investigation of specific immunity to plague, have revealed that ferroarabinogalactan activates peritoneal macrophages in guinea pigs, in comparison with the animals' cells being immunized only with vital plague vaccine.

Antibiotic resistance of microorganisms has led to a new interest in silver preparations. The most efficient are preparations of ultradispersed silver. Highly dispersed (nanosized) particles increase bactericidal activity. It has been established that silver-containing nanocomposites with AG possess high antimicrobial activity against gram-negative enterobacteria (*Escherichia coli, Salmonella typhimurium, Candida albigans, Bacillus subtilis* and *Staphyllococcus aureus*) [57].

Thus, the method for synthesis of nanocomposites with available polysaccharide AG is an easy way to synthesize universal materials. The AG-based nanobiocomposites synergistically combine the properties of the stabilizing natural polysaccharide matrix and the nanocore materials. They are applicable as nanosized water-soluble enantioselective catalysts, magnet-controlled medical substances, materials for coherent and nonlinear optics, high-sensitivity optical markers, universal antimicrobial preparations, etc. The use of AG as a bioactive polysaccharide matrix participating in the processes of receptor-induced endocytosis leads to new approaches to therapy for metal deficiency states and to the development of new biomaterials of target action, which are in high demand in medicine and biology, both as controlled composite materials and as new water-soluble biodegradable metal-containing drugs.

158 The Complex World of Polysaccharides

AG alone [49].

treatment.

with vital plague vaccine.

*Staphyllococcus aureus*) [57].

polysaccharide macromolecules are cleaved similarly to the mechanochemical activation of

*L. sibirica* Ledeb. and *L. gmelinii* (Rupr.) Rupr. contain much bioflavonoid dihydroquercetin (DHQ, taxifolin) and their diverse biological activity is well studied. It is an officinal drug with a wide range of therapeutic action and is also the basis for a number of efficient medical preparations and food supplements [54]. Complexes of AG and DHQ combining these unique properties are very promising. Such complexes have been obtained by pharmacon clathration [54] and have shown essentially improved solubility (up to 38 times) in comparison with starting DHQ and untreated AG/DHQ mixture. No chemical reaction between AG and DHQ takes place, as in clathrates [51]. According to HPLC data, AG in AG/DHQ clathrates has a narrower molecular weight distribution in comparison with pure mechanochemically activated AG, due to a decrease in both high- and low-molecular fractions, and thus DHQ stabilizes the polysaccharide macromolecules in mechanochemical

The most recent application of AG as a stabilizing polymer matrix in hybrid nanosized materials is based on iron oxides, cobalt, copper, nickel, ferrites and zero-valence metals such as silver, palladium and platinum [18,55-57]. Metal content in nanocomposite samples depends on synthetic conditions and on the type of metal ion used, varying in the range of 0.1–21.0%. In the case of metal oxide nanocomposites, AG shows properties of a nanostabilizing matrix, while in the composites of noble metals it reduces metal to a zerovalence state and stabilizes the metal nanoparticles formed. Nanocomposites based on AG retain high biological activity. Ferroarabinogalactans show synergy between the pronounced antianaemic activity of the ferric core and the unique membranotropic and immunomodulatory properties of AG. Parenteral administration of ferroarabinogalactan normalizes quantitative and qualitative characteristics of the erythrocyte system and iron depot level in animals (white rats) [18]. The original synthetic method of ferroarabinogalactan retains both membranotropic and immunomodulatory properties of AG. Studies of the natural effects of immunomodulators, together with the investigation of specific immunity to plague, have revealed that ferroarabinogalactan activates peritoneal macrophages in guinea pigs, in comparison with the animals' cells being immunized only

Antibiotic resistance of microorganisms has led to a new interest in silver preparations. The most efficient are preparations of ultradispersed silver. Highly dispersed (nanosized) particles increase bactericidal activity. It has been established that silver-containing nanocomposites with AG possess high antimicrobial activity against gram-negative enterobacteria (*Escherichia coli, Salmonella typhimurium, Candida albigans, Bacillus subtilis* and

Thus, the method for synthesis of nanocomposites with available polysaccharide AG is an easy way to synthesize universal materials. The AG-based nanobiocomposites synergistically combine the properties of the stabilizing natural polysaccharide matrix and the nanocore materials. They are applicable as nanosized water-soluble enantioselective At present, healthy diet is a question of public policy in all developed countries due to the undisputed role of food in public health, working capacity of people, adaptation, child growth and longevity. The increasing popularity of healthy diets has made manufacturers pay more and more attention to functional food, i.e. medically fortified food products. Food supplements and enriched products are becoming increasingly popular. Such products are functionalized by both natural substances (pectin, inulin, gum arabic, etc.) and semisynthetic compounds (lactulose, polydextrose, resistant starches, chitosan, etc.).

Larch arabinogalactan adds nutrition and function to beverages, snack foods, nutrition bars, and more [6,58]. Not only does AG function as a prebiotic fibre and immunity enhancer, it also retains moisture, enhances mouthfeel and bulk, and improves shelf stability. Because of AG's low-viscosity profile and emulsification-enhancing properties, the most immediate applications include refrigerated and non-refrigerated beverages, and beverage mixes. Interest has also been expressed in snack foods, bars, ready-to-eat cereals, yogurt/dairy products and baked goods. Commercially available larch AG-containing products include beverages and nutrition bars.

Not only does AG add nutrition, it also provides functional benefits. An independent food laboratory confirmed that the inclusion of AG improved white pan bread make-up, external symmetry and internal grain scores. Fat-free flour tortillas with AG showed better handling, taste and aroma than the control. AG is a low-calorie additive for artificial sweeteners. It delivers mouthfeel, taste and bulking attributes that are most like sugar.

In confectionery and baked goods, AG lowers water activity and aids flavour and oil retention. AG can be used in browning compositions for uncooked foods, in seasoning powders to improve flow and reduce hygroscopicity, and in starch-containing foods to inhibit swelling.

Recent clinical investigations of AG have demonstrated not only benefits to gastrointestinal health and immunity, but also a significant reduction in serum cholesterol, glucose and insulin levels [58,59]. This opens the door to potential heart health label claims and provides an option for consumers looking for foods that are beneficial in terms of body weight, blood glucose or blood insulin control.

AG benefits, as determined by human and animal clinical trials, have been observed as low as 1.5 g/day, or more specifically 20 mg/kg of body weight. AG has been found to be totally safe as a food ingredient. On average, finished products containing a minimum of 60 mg/kg of body weight or about 4.5 g/day is recommended for foods [58]. Using arabinogalactan additives isolated from Siberian larch, the authors of one study [60] examined the soft wheat flour quality and quantity of gluten, physical properties of the dough, and quality of finished bread, depending on the quantity of the added polysaccharide. The addition of 1% of arabinogalactan to flour causes a significant improvement in the qualitative indices of bread. In this case, AG is totally consumed in the course of bread making because it is utilized by yeast. It is recommended that bread quality can be improved when the flour incorporates 1% mass of AG. When 2–3% of AG is added to flour, the AG content decreases. An excess of AG inhibits yeast growth, which leads to a decrease in bread quality.

The optimum compositions for AG-enriched bakery goods and pastry have been proposed [61,62]. It has been shown that when AG is added to flour in proportions of 1–5% by weight, bakery products are rich in dietary fibre of prebiotic and immunostimulating action, while their energy density is lower due to the decreased amount of sugar in the recipes. The AGenriched bakery and pastry products have a medical effect [59]. Production technology for AG-enriched prebiotic cultured milk products has been developed [63]. The efficiency of AG in veterinary medicine has been proven [64-67].
