**3. Larch bark pectins**

## **3.1. Physicochemical and biological properties of pectins**

Pectin substances are present in the majority of land and water plants, and in some freshwater algae [80]. Being an important component of cell walls, they are involved in ion exchange, water metabolism and cell wall structure formation. They stimulate seed germination and germ growth, provide turgor, etc.

The unique physicochemical properties of pectin make it indispensable in medical, food and cosmetic industries as a gelling agent, thickener, stabilizer and dietary fibre. Recently, it has become widely used as a matrix carrier for biologically active components in drugs. Pectins have physiological activities of their own (immunomodulating, hepatoprotective, anticarcinogenic, antimetastatic, etc.) making them applicable as medical preparations and biologically active food supplements.

Industrial demand for pectins in Russia is estimated at 2000 t/year, of which 10% is for the fragrance and cosmetic industries, 15% goes to medicine and pharmaceuticals, and 75% is for the food industry [81]. However, this demand is generally met by imported production. There are recent innovative Russian developments that are ecologically harmless and economically viable (there is no need to utilize aggressive acid media and to support treatment facilities), therefore having a low cost price. The raw material for pectin is the marc of citrus fruit, apple, sugar beet and sunflower head pith. There are proposals for using other plants as raw materials, such as amaranth, small mallow, duckweed, silene, coffee beans, etc. [82-86].

The bark of *L. sibirica* Ledeb. and *L. gmelinii* (Rupr.) Rupr., having a 12% pectin content, is a promising alternative raw material. At our laboratory, we are conducting systematic studies of the structure and properties of pectin from these larch species to determine a suitable technology for its industrial production.

## **3.2. Isolation of pectin substances from larch bark**

166 The Complex World of Polysaccharides

Material and reagent

**Table 1.** Criteria of variant estimation

**3. Larch bark pectins** 

FR-UF-OR-MF

**3.1. Physicochemical and biological properties of pectins** 

germination and germ growth, provide turgor, etc.

biologically active food supplements.

technology for its industrial production.

coffee beans, etc. [82-86].

FR-UF-MF**-**OR FR-OR-UF-MF

Productivity 1 1 1 1 1 1 **7** 7

consumption 3 3 3 3 3 3 **1** <sup>1</sup> Steel intensity 1 1 1 1 1 1 **7** 7 Laboriousness 3 5 3 5 7 7 **2** 1 Manufacturability 2 1 2 5 7 6 **6** 4 **Total 10 11 10 15 19 18 23 20** 

Pectin substances are present in the majority of land and water plants, and in some freshwater algae [80]. Being an important component of cell walls, they are involved in ion exchange, water metabolism and cell wall structure formation. They stimulate seed

The unique physicochemical properties of pectin make it indispensable in medical, food and cosmetic industries as a gelling agent, thickener, stabilizer and dietary fibre. Recently, it has become widely used as a matrix carrier for biologically active components in drugs. Pectins have physiological activities of their own (immunomodulating, hepatoprotective, anticarcinogenic, antimetastatic, etc.) making them applicable as medical preparations and

Industrial demand for pectins in Russia is estimated at 2000 t/year, of which 10% is for the fragrance and cosmetic industries, 15% goes to medicine and pharmaceuticals, and 75% is for the food industry [81]. However, this demand is generally met by imported production. There are recent innovative Russian developments that are ecologically harmless and economically viable (there is no need to utilize aggressive acid media and to support treatment facilities), therefore having a low cost price. The raw material for pectin is the marc of citrus fruit, apple, sugar beet and sunflower head pith. There are proposals for using other plants as raw materials, such as amaranth, small mallow, duckweed, silene,

The bark of *L. sibirica* Ledeb. and *L. gmelinii* (Rupr.) Rupr., having a 12% pectin content, is a promising alternative raw material. At our laboratory, we are conducting systematic studies of the structure and properties of pectin from these larch species to determine a suitable

Possible algorithms

FR-MF-UF-OR

FR-MF-OR-UF

UF-FR-OR-MF UF-FR-MF-OR

FR-OR-MF-UF

Criteria

There are a number of methods to isolate pectin polysaccharides from plant tissues, including hydrolysis extraction of dry raw material particles of certain sizes [87] using hot water, organic and inorganic acid solutions as well as salts, alkali or their mixtures as extracting solutions. Basic parameters of the pectin isolation process, such as raw material pre-processing, hydromodulus, temperature, extraction duration, medium рН and precipitator used, can all be varied depending on characteristics of the raw material [88]. We studied the influence of the following combinations of the basic parameters upon yield and product quality:

Experiment 1: 0.5% ammonium oxalate solution (hydromodulus 1:5)

Experiment 2: 0.5% oxalic acid solution (1:5)

Experiment 3: equimolar mixture of 0.5% oxalic acid and 0.5% ammonium oxalate solutions (1: 5)

Experiment 4: 0.25% sodium hydroxide solution (1:5)

Experiment 5: similar to experiment 3 (1:7)

Experiment 6: similar to experiment 3 (1:10)

In all the experiments, extraction process were the same (at 80 °С for 2 h of constant stirring).

The pectin samples obtained were white or light cream-coloured powders, tasteless and with no smell (Figure 6).

**Figure 6.** A laboratory sample of larch bark PS

Table 2 sets out the yield (% of weight of absolutely dry bark, a.d.b.) and composition data of pectin substances obtained in experiments 1–6.

The highest yields were observed with a weak alkali solution, but the ash content was too high (16.66%), which affected gelling ability [89]. The lowest ash content was found in the preparations isolated using an equimolar mixture of ammonium oxalate and oxalic acid (5– 5.6%).


**Table 2.** Yield and elemental composition of pectin substances in larch bark

The optimal hydromodulus was observed in experiments 1–4 (hydromodulus 1:5) with yield increasing by 1.5 times. Hydromodulus provides insufficient penetration of extracting agent, lower than 1: 5. A 1:10 rise of hydromodulus (experiment 6) had no essential effect upon the yield and qualities of the product. Thus, the equimolar mixture of ammonium oxalate and oxalic acid used as an extracting solution at hydromodulus 1:7 (experiment 5) was the most effective in isolating pectin substances from larch bark, leading to a 2.7% yield of absolutely dry bark mass with ash content of 5.2%.

Raw material pre-processing by solvents of increasing polarity (hexane, ethyl acetate and water) resulted in both enzyme deactivation and elimination of the impurities, therefore increasing the extracting solution's ability to access the plant cell walls. Notably, the preextracted substances are valuable for medicine [90] and the leather industry [91].

We experimentally compared the yields of pectins isolated with and without raw material pre-processing in the conditions described above (see Figure 7). It was shown that prior elimination of impurities leads to higher yield of the product (about 1.5 times), clearly due to higher availability of pectin substances.

**Figure 7.** PS yield dependence on: a) raw material pre-processing, and b) extraction temperature

The PS yield increased as the extraction temperature rose, reaching a maximum at 80 °С. The data obtained were in good correspondence with the literature on classic pectin isolation [89]: raising temperature causes partial hydrolysis of protopectin. Thus, pectin yield increases while at temperatures higher than 80 °С the superstructure of pectin substances is broken. This is also confirmed by the dependence of the molecular weights of the resulting pectins on extraction temperatures (see Figure 8.).

Kinetic studies, particularly those concerning the pectin hydrolysis extraction process, have a particular interest. Pectin yields vs. extraction times are charted in Figure 9. A major part of PS is transferred into the extract within 1 h of extraction, after which there is no significant increase of yield.

**Figure 8.** PS molecular weight (M.W.) dependence on extraction temperature

**Figure 9.** Dependence of PS yield on extraction time

168 The Complex World of Polysaccharides

% of a.d.b. Medium рН Composition, %

1 0.77 6.95 30.28 6.66 7.89 2 0.97 1.90 31.17 5.75 8.34 3 1.64 3.90 32.05 5.42 5.58 4 5.81 11.97 36.55 4.51 16.66 5 2.71 2.86 34.24 6.30 5.20 6 2.93 2.84 31.48 6.82 5.08

The optimal hydromodulus was observed in experiments 1–4 (hydromodulus 1:5) with yield increasing by 1.5 times. Hydromodulus provides insufficient penetration of extracting agent, lower than 1: 5. A 1:10 rise of hydromodulus (experiment 6) had no essential effect upon the yield and qualities of the product. Thus, the equimolar mixture of ammonium oxalate and oxalic acid used as an extracting solution at hydromodulus 1:7 (experiment 5) was the most effective in isolating pectin substances from larch bark, leading to a 2.7% yield

Raw material pre-processing by solvents of increasing polarity (hexane, ethyl acetate and water) resulted in both enzyme deactivation and elimination of the impurities, therefore increasing the extracting solution's ability to access the plant cell walls. Notably, the pre-

We experimentally compared the yields of pectins isolated with and without raw material pre-processing in the conditions described above (see Figure 7). It was shown that prior elimination of impurities leads to higher yield of the product (about 1.5 times), clearly due

**Figure 7.** PS yield dependence on: a) raw material pre-processing, and b) extraction temperature

the resulting pectins on extraction temperatures (see Figure 8.).

Yield PS, % a.d.b.

The PS yield increased as the extraction temperature rose, reaching a maximum at 80 °С. The data obtained were in good correspondence with the literature on classic pectin isolation [89]: raising temperature causes partial hydrolysis of protopectin. Thus, pectin yield increases while at temperatures higher than 80 °С the superstructure of pectin substances is broken. This is also confirmed by the dependence of the molecular weights of

20 40 60 80 100

t, ºC

a)

b)

extracted substances are valuable for medicine [90] and the leather industry [91].

**Table 2.** Yield and elemental composition of pectin substances in larch bark

of absolutely dry bark mass with ash content of 5.2%.

to higher availability of pectin substances.

С Н Ash

Experiment Yield,

We also studied the influence of type of precipitator used upon the yield and qualities of the product. For this purpose, pectin extract was prepared from larch bark by treating it with an equimolar mixture of 0.5% ammonium oxalate and 0.5% oxalic acid (hydromodulus 1:7) at 80 °С for 2 h. The extract was concentrated in a circulation vacuum evaporator until it reached one third of its original volume. One half of the concentrate was precipitated with acetone and the other half with ethanol. Precipitators were added in equal quantity, dropwise while continuous stirring was applied. The precipitate was vacuum-filtered, dissolved in 100 ml of distilled water by heating to 40–50 °С when necessary, and then again precipitated and filtered. The final precipitates were washed with the same precipitator and then with diethyl ether, dried in the air and then in a drier at 50 °С, cooled to room temperature in a desiccator and measured to determine yield. It is noteworthy that the dropwise addition of precipitator into the extract increased yield by 0.5% compared to the usual precipitation procedure. We established that acetone is less selective, and ethanol therefore gives a purer product. Purity of the pectin preparation obtained can also be estimated based on galacturonic acid content [92]: for larch bark pectins precipitated by acetone and ethanol, the result was 69.77 and 78.12%, respectively.

Thus, the optimal procedure for isolating pectin substances from larch bark involves pretreatment by hexane, ethyl acetate and water, extraction by an equimolar mixture of 0.5% oxalic acid and ammonium oxalate solutions at hydromodulus 1:7 and an extraction temperature of 80 °С for 1 h, and precipitation by ethanol. The method has been patented [93] and used for preparing the samples for physicochemical and application studies.

## **3.3. Characterization of pectin substances**

Pectinase enzyme hydrolysis of PS samples isolated from larch bark by the above method, and further analysis of hydrolysis products by paper chromatography (PC), have shown an essential destruction of PS with formation of free D-galacturonic acid.

Table 3 sets out the main maxima of absorption bands in the IR spectra of PS and their assignment, proving the PS pectin nature of the samples [94].


**Table 3.** Absorption band maxima in IR spectra of PS and their assignments

Thus, enzyme hydrolysis and IR spectroscopy data prove that the polysaccharide isolated from larch bark refers to the pectin group.

The monosaccharide composition of PS was determined by total acid hydrolysis with trifluoroacetic acid (TFA). Monosaccharide identification of PS was performed using gas– liquid chromatography (GLC) and the sample was shown to consist of galacturonic acid, protein compounds and monosaccharides of arabinose, galactose, rhamnose, glucose, mannose and (in minor quantities) xylose. Dominant monosaccharides were galactose and arabinose, in a ratio of 2.7: 1.

The degree of homogeneity for PS was determined by ion exchange chromatography on DEAE cellulose with sodium chloride aqueous solutions. Four fractions were detected (Table 4). In the fractions PS-1 and PS-2, arabinose and galactose were predominant (18.26/52.96% and 11.65/30.83%, respectively); thus, they refer to acidic arabinogalactans. The acidic nature of PS was developed with D*-*galacturonic acid residues with the PS-1 proportion five times less than in PS-2, while in PS-3 and PS-4 it was a major monosaccharide, and thus they refer to pectins. The content of neutral monosaccharides in PS-4 was minimal compared to other fractions (3% mass). All the fractions contained protein compounds that were not eliminated by gel filtration. It seems likely that the protein and polysaccharide compounds were strongly aggregated, or that their molecular weights were close to each other.


\* PS-1 isolated with use of 0.01М NaCl solution, PS-2 – 0.1М NaCl solution, PS-3 and PS-4 – 0.2М NaCl solution

**Table 4.** Chemical characterization of PS sample after DEAE-cellulose fractioning

The amino acid composition of PS proteins was studied. The major components of PS were glutamic acid (6%) and aspartic acid (2.8%), while total content of amino acids with aliphatic side chains (glycine, alanine, valine, isoleucine, leucine) was 9% (Figure 10).

**Figure 10.** Amino acid composition of PS proteins

170 The Complex World of Polysaccharides

**3.3. Characterization of pectin substances** 

Thus, the optimal procedure for isolating pectin substances from larch bark involves pretreatment by hexane, ethyl acetate and water, extraction by an equimolar mixture of 0.5% oxalic acid and ammonium oxalate solutions at hydromodulus 1:7 and an extraction temperature of 80 °С for 1 h, and precipitation by ethanol. The method has been patented

Pectinase enzyme hydrolysis of PS samples isolated from larch bark by the above method, and further analysis of hydrolysis products by paper chromatography (PC), have shown an

Table 3 sets out the main maxima of absorption bands in the IR spectra of PS and their

Frequency (ν, cm-1) Assignment

3460 ν(ОН), ν(Н2О) 3260 ν(NН) 2962, 2872 ν(СН3) 2573 ν(ОН), 1730 ν(С=О) в СООН 1640 δ(ОН)<sup>λ</sup> 1540 δ(NН)

1380–1450 δ(С-СН3), ν(С-О) pyranose rings 1331 δ(ОН) in pyranose rings 1265 ν(С-О) in esters 1150 ν(С-О-С) 1095 ν(С-С) 1027 ν(С-ОН)

890 δ(С1-Н) in glucopyranose ring 766, 629, 528 pulse vibrations of pyranose ring

Thus, enzyme hydrolysis and IR spectroscopy data prove that the polysaccharide isolated

The monosaccharide composition of PS was determined by total acid hydrolysis with trifluoroacetic acid (TFA). Monosaccharide identification of PS was performed using gas– liquid chromatography (GLC) and the sample was shown to consist of galacturonic acid, protein compounds and monosaccharides of arabinose, galactose, rhamnose, glucose, mannose and (in minor quantities) xylose. Dominant monosaccharides were galactose and

[93] and used for preparing the samples for physicochemical and application studies.

essential destruction of PS with formation of free D-galacturonic acid.

**Table 3.** Absorption band maxima in IR spectra of PS and their assignments

from larch bark refers to the pectin group.

arabinose, in a ratio of 2.7: 1.

assignment, proving the PS pectin nature of the samples [94].

Thus, we isolated PS from the bark of *L. sibirica* Ledeb. and *L. gmelini* (Rupr.) Rupr., pretreated with ethyl acetate and hot water. Enzyme hydrolysis with pectinase and IR spectroscopy were employed to prove the presence of pectin polysaccharides in the samples isolated. DEAE-cellulose chromatography revealed that PS includes four fractions, two of which are acidic arabinogalactans and the other two belong to the pectin group.

## **3.4. Structural study of main chain of larch bark pectin**

Acid hydrolysis of PS by 2М TFA results in galacturonan PVG-1. The high value and positive sign of the rotation angle of +245.3º (*с* 0.1, H2O) suggest α-D-configuration of Dgalactopyranosyluronic acid residues.

Values of chemical shifts (CS) of carbon atoms in the 13С NMR spectrum of PVG-1 (Table 5), compared to other data, [95] corresponded to those for carbon atoms in D-galacturonic acid residues in pyranose form which compose the linear fragment of pectin molecules (pectin core). The presence of an anomeric carbon atom signal at 101.9 ppm indicated both (14) bonding between D-galacturonic acid residues and α-configuration of С-1 anomeric atoms. Signals at 176.2 ppm were assigned to the С-6 atom and indicated a free carboxyl group in D-galacturonic acid residue. Additionally, there were galacturonic acid residues esterified by methoxyl in the PVG-1 molecule, according to signals with CS at 172.2 ppm (С-6-ОСН3) and 54.4 ppm (-ОСН3). The ratio of the integrated signal intensity of carbon atoms observed in methoxyl and carboxyl groups suggests a high degree of galacturonan methoxylation. The 13С NMR spectrum also showed signals at 76.1 and 74.9 referring to the С-3 carbon atom substitute in (…4)-α-D-GalрA-(1…) galacturonic acid residue in the galacturonan molecule (the non-substituted atom has CS at 72.1 ppm).


**Table 5.** Chemical shifts of signals of galacturonic acid carbon atoms in 13С NMR spectrum of PVG-1

Thus, according to spectral and chromatographic data, linear polysaccharide from larch bark has a structure of homogalacturonan consisting of (…4)-α-D-GalрA-(1…)-linked fragments D-galacturonic acid has partially etherified by methoxyl groups with branching points at С-3 atom of galacturonopyranosyl residue.

#### **3.5. Structural study of side branches of larch bark pectin**

Partial acid hydrolysis of PS with 0.01М TFA for 3 h resulted in galacturonan PVG-2. According to 13С NMR data, it was a pectin polysaccharide. The spectrum contained both typical signals of galacturonic acid residues, namely pronounced signals of anomeric carbon atoms at 100.4 and 104.4 ppm, and signals of carboxyl carbon atoms at 171.4, 166.5 and 53.7

ppm, the latter two being signals of carbon atoms in uronic acid residues methoxylated by the C-2 and/or C-3 atoms (Table 6).. Intensities and spectral positions of signals at 68.9, 70.8, 78.9 and 72.2 ppm corresponded to data in the literature for α-D-GalрA residues connected by 14 bonds. There is a ratio of 1:5 between integral signal intensities of carboxyl and methoxyl carbon atoms, which suggests a high degree of PS methoxylation.

galactopyranosyluronic acid residues.

4)-α-D-GalрA- (1

Thus, we isolated PS from the bark of *L. sibirica* Ledeb. and *L. gmelini* (Rupr.) Rupr., pretreated with ethyl acetate and hot water. Enzyme hydrolysis with pectinase and IR spectroscopy were employed to prove the presence of pectin polysaccharides in the samples isolated. DEAE-cellulose chromatography revealed that PS includes four fractions, two of

Acid hydrolysis of PS by 2М TFA results in galacturonan PVG-1. The high value and positive sign of the rotation angle of +245.3º (*с* 0.1, H2O) suggest α-D-configuration of D-

Values of chemical shifts (CS) of carbon atoms in the 13С NMR spectrum of PVG-1 (Table 5), compared to other data, [95] corresponded to those for carbon atoms in D-galacturonic acid residues in pyranose form which compose the linear fragment of pectin molecules (pectin core). The presence of an anomeric carbon atom signal at 101.9 ppm indicated both (14) bonding between D-galacturonic acid residues and α-configuration of С-1 anomeric atoms. Signals at 176.2 ppm were assigned to the С-6 atom and indicated a free carboxyl group in D-galacturonic acid residue. Additionally, there were galacturonic acid residues esterified by methoxyl in the PVG-1 molecule, according to signals with CS at 172.2 ppm (С-6-ОСН3) and 54.4 ppm (-ОСН3). The ratio of the integrated signal intensity of carbon atoms observed in methoxyl and carboxyl groups suggests a high degree of galacturonan methoxylation. The 13С NMR spectrum also showed signals at 76.1 and 74.9 referring to the С-3 carbon atom substitute in (…4)-α-D-GalрA-(1…) galacturonic acid residue in the galacturonan

Residue С-1 С-2 С-3 С-4 С-5 С-6 С-6-

**Table 5.** Chemical shifts of signals of galacturonic acid carbon atoms in 13С NMR spectrum of PVG-1

Thus, according to spectral and chromatographic data, linear polysaccharide from larch bark has a structure of homogalacturonan consisting of (…4)-α-D-GalрA-(1…)-linked fragments D-galacturonic acid has partially etherified by methoxyl groups with branching

Partial acid hydrolysis of PS with 0.01М TFA for 3 h resulted in galacturonan PVG-2. According to 13С NMR data, it was a pectin polysaccharide. The spectrum contained both typical signals of galacturonic acid residues, namely pronounced signals of anomeric carbon atoms at 100.4 and 104.4 ppm, and signals of carboxyl carbon atoms at 171.4, 166.5 and 53.7

70.1 76.1 74.9 (ОСН3) -ОСН<sup>3</sup>

79.2 73.4 176.2 172.2 54.4

which are acidic arabinogalactans and the other two belong to the pectin group.

**3.4. Structural study of main chain of larch bark pectin** 

molecule (the non-substituted atom has CS at 72.1 ppm).

100.9 68.9

points at С-3 atom of galacturonopyranosyl residue.

**3.5. Structural study of side branches of larch bark pectin** 


**Table 6.** Chemical shifts in signals of carbon atoms in the 13С NMR spectrum of PVG-2

In the 13С NMR spectrum of PVG-2 samples there were upfield signals at 17.9 and 18.13 ppm belonging to C-6 atoms in terminal rhamnose residues and in polysaccharide chains, respectively. The integral intensities of these signals and those of C-2 and/or C-3 and C-6 carbon atoms for galacturonan residues at 166.5 and 171.4 ppm were found to have a ratio 1:5. The total integral intensity of signals for anomeric С-1 atoms for rhamnose and the total integral intensity of signals of anomeric atoms of galacturonan residues were equal to each other, *i.e.* they had the same ratio for rhamnose and galacturonan residue content in the chain. According to data in the literature, signals at 99.7, 77.6, 70.8, 82.5, 68.9 and 17.9 ppm are assigned to С-1, С-2, С-3, С-4, С-5 and СН3 carbon atoms in 2,4)- α-L-Rhap-(1 residues.

Thus, according to 13С NMR spectral data, linear fragments of pectin polysaccharide isolated from larch bark are rhamnogalacturonans where D-galacturonic acid residues in pyranose form with an α-configuration of their anomeric centre are connected 1–4 by glycosidic bonds. One fifth of galacturonan residues associated with the С-6 atom were esterified by methoxyl groups. The ratio between 2,4-substituted rhamnopyranosyl and galacturonosyl residues (1:5), thus, the main chain structure of the pectin polysaccharide was highly branched at the С-4 atoms of rhamnopyranosyl residues.

Further 13С NMR spectrum analysis of the PVG-2 sample showed that arabinogalactan fragments are present in rhamnogalacturonan as side chains. Concerning signals of anomeric carbon atoms, the 13С NMR spectrum of the PVG-2 sample showed that there are

signals at 101.1, 104.38, 104.64 and 108.6 ppm, as well as signals of anomeric carbon atoms in galacturonopyranosyl residues of the galactan core. According to [9], intensities and values of CS can be assigned to signals of anomeric carbon atoms in -L-Arap, α-L-Araf and -D-Galр residues. The most upfield of the signals mentioned ( 101.1 ppm) belong to terminal - L-Arap residues. Signals at 104.38 and 104.64 ppm belong to С-1 in -D-Galр residues while CS values of С-2, С-3, С-4, С-5 and С-6 atom signals are calculated according to the official data for -D-galactopyranosyl residues. Bonding at the С-3 and С-6 positions of -Dgalactopyranose was proven by downfield shifts of these signals at 8.7 and 8.8-9.4 ppm, respectively, due to glycosylation of these atoms as compared to their positions in nonsubstituted 13,6 linked -D-Galр residues. Signals at 108.6 ppm, like those at 80.7, 78.9, 84.9 and 62.0 ppm, are terminal α-L-arabinofuranose. The anomeric atoms of arabinose and galactose are monosaccharides integrated at a ratio of 1:2.

Hence, according to spectral data for the PVG-2 fragment of the pectin polysaccharide from larch bark, highly branched arabinogalactan was detected as side chains consisting of linear chains with 3,6)--D-Galр-(1 residues with branching at С-6 atoms. Side chains of arabinogalactan fragments contain terminal arabinose, both in pyranose and in furanose form, as well as 2,5)-α-L-Araf-(1 and 3,5)-α-L-Araf-(1 residues as intermediate fragments.

## **3.6. Larch bark pectin peculiarities and implementation fields**

It has been determined that larch bark pectin substances possess immunomodulatory, antineoplastic, gastroprotective and antitoxic action [96-98]. In order to understand larch bark pectin's physiological and pharmacological action, we have started research focussed on examining its membrane-acting action. The vacuoles of isolated cell plants and their membranes were found to be an appropriate object for our research. The influence of pectin on membranes and the peculiarities of their barriers were estimated according to the change of destruction dynamics in isolated vacuoles in comparison with the control. The results are depicted in Figure 11. It has been established that implementation of pectin aqueous solutions leads to their protective action on vacuolar membranes, exceeding the control threefold. Thereby, the experiments proved that larch bark pectin possesses a membrane stabilizing activity.

**Figure 11.** Influence of pectin upon isolated vacuole half-lives (Т1/2)

In order to broaden larch bark pectin implementation fields, we carried out research into its implementation as a reducer and stabilizer of noble metal particles in nanosized state. Supramolecular structure peculiarities, optical activity, carboxyl and abundance of hydroxyl groups, and polymeric pectin molecule stabilizing effect provided significant potential in nanobiocomposite formation processes in metals with a polysaccharide matrix ("pectin – metal (0)").

174 The Complex World of Polysaccharides

stabilizing activity.

galactose are monosaccharides integrated at a ratio of 1:2.

**3.6. Larch bark pectin peculiarities and implementation fields** 

**Figure 11.** Influence of pectin upon isolated vacuole half-lives (Т1/2)

signals at 101.1, 104.38, 104.64 and 108.6 ppm, as well as signals of anomeric carbon atoms in galacturonopyranosyl residues of the galactan core. According to [9], intensities and values of CS can be assigned to signals of anomeric carbon atoms in -L-Arap, α-L-Araf and -D-Galр residues. The most upfield of the signals mentioned ( 101.1 ppm) belong to terminal - L-Arap residues. Signals at 104.38 and 104.64 ppm belong to С-1 in -D-Galр residues while CS values of С-2, С-3, С-4, С-5 and С-6 atom signals are calculated according to the official data for -D-galactopyranosyl residues. Bonding at the С-3 and С-6 positions of -Dgalactopyranose was proven by downfield shifts of these signals at 8.7 and 8.8-9.4 ppm, respectively, due to glycosylation of these atoms as compared to their positions in nonsubstituted 13,6 linked -D-Galр residues. Signals at 108.6 ppm, like those at 80.7, 78.9, 84.9 and 62.0 ppm, are terminal α-L-arabinofuranose. The anomeric atoms of arabinose and

Hence, according to spectral data for the PVG-2 fragment of the pectin polysaccharide from larch bark, highly branched arabinogalactan was detected as side chains consisting of linear chains with 3,6)--D-Galр-(1 residues with branching at С-6 atoms. Side chains of arabinogalactan fragments contain terminal arabinose, both in pyranose and in furanose form, as well as 2,5)-α-L-Araf-(1 and 3,5)-α-L-Araf-(1 residues as intermediate fragments.

It has been determined that larch bark pectin substances possess immunomodulatory, antineoplastic, gastroprotective and antitoxic action [96-98]. In order to understand larch bark pectin's physiological and pharmacological action, we have started research focussed on examining its membrane-acting action. The vacuoles of isolated cell plants and their membranes were found to be an appropriate object for our research. The influence of pectin on membranes and the peculiarities of their barriers were estimated according to the change of destruction dynamics in isolated vacuoles in comparison with the control. The results are depicted in Figure 11. It has been established that implementation of pectin aqueous solutions leads to their protective action on vacuolar membranes, exceeding the control threefold. Thereby, the experiments proved that larch bark pectin possesses a membrane Synthesis of nanobiocomposites was carried out using the redox reactions of PS with silver nitrate. Nanobiocomposite samples 0.5 "pectin – Ag(0)" up to 72% content of silver were obtained in different reaction conditions. It was discovered that the effectiveness of the reaction to create a silver nanoparticle flow depends on medium spectrum pH. The spectra of the mixtures of pectin and silver nitrate water solutions versus time reaction are depicted in Figure 12a. It was determined that, with a reduction of pH to 3.5, the Ag(I) reaction proceeds very slowly. This is demonstrated by the appearance of a link in the absorption spectrum in the range of λ 280–470 nm only 24 h after the beginning of the reaction (Fig. 12a). The wide maximum low intensity link was indicated by the formation of silver metal primary centres. Despite this, the reaction speed of the reduction was so slow that even 96 h was not enough to create fully recovered Ag(0) centres. With pectin and Ag(I) interactions in reaction mixtures beginning at a pH of 7, a symmetric bond at λmax 420 nm can be observed in the electron spectra at the start of reaction by proving the formation of Ag(0) nanoparticles (Fig.12b, line 2). Even so, it takes about 24 h for the full silver cation conversion which was experimentally evaluated according to the absorption bond intensity growth. Ag(I) reduction with pectin at pH 11–12 proceeded swiftly immediately after mixing of the components (Figure 12b, line 4 and Figure 12c line 1) and finished within 30 min. Reduction under these conditions was also accompanied by variations in the particle size of Ag(0), as shown by the shift in the Plasmon pick position into the short-wave region at 10 nm (Figure 12c).

**Figure 12.** Absorption spectra of mixtures of aqueous solutions of pectin (0.5%) and silver nitrate (0.1%) in a ratio of 1:1 depending on: а) reaction duration: 1 min (1), 24 h (2), 48 h (3), 72 h (4), 96 h (5); b) medium рН: 3.5 (1), 7 (2), 9.7 (3), 11.5 (4); c) reaction duration at рН 11.5: 1 min (1), 30 min (2), 60 min (3), 180 min (4), 24 h (5)

Radiographic phase analysis of obtained nanobiocomposites of "pectin-Ag(0)" demonstrated it to be a mixture of radioamorphous and crystalline phases. There was a

wide halo with maximum intensity at d ~ 0.46 nm in 2θ angle intervals from 8 to 60E (Figure13a) in a radioamorphous phase diffraction pattern typical of a pectin source. There were quite intensive but broadened lines typical of metallic silver (Figure 13b) during silver loading in the diffraction patterns of reaction products against a background of pectin reflection. The calculation of silver unit-cell parameters showed that in their quantity in the provided samples was lower than for massive silver and changed from 0.4036 to 0.4050 nm ( 0.0008 nm). Moreover, the average size of the coherent-scattering region (CSR) was calculated according to Selyakov–Sherarar's formula [99] to be in the range of 3 nm. The data obtained demonstrated that, in the samples of Ag (0), the persistence of nanosized particles was stabilized by an amorphous phase with pectin.

**Figure 13.** Diffraction patterns of pectin sample (a) and of the "pectin-Ag(0)" nanobiocomposite sample (b)

"Pectin – Ag (0)" nanobiocomposite scanning electron microscopy (Figure 14) showed that the analysed samples contain particles considerably smaller than 100 μm.

**Figure 14.** Electron microphotography of "pectin – Ag(0)" sample

Microphotography analysis of nanobiocomposites, obtained by the use of transmission electron microscopy, demonstrated that there are isolated silver particles of null valency in globular form (Figure 15a), of a size within the range from 4 to 17 nm (predominance (up to 80%) at 6–7 nm, Figure 15b).

(b)

wide halo with maximum intensity at d ~ 0.46 nm in 2θ angle intervals from 8 to 60E (Figure13a) in a radioamorphous phase diffraction pattern typical of a pectin source. There were quite intensive but broadened lines typical of metallic silver (Figure 13b) during silver loading in the diffraction patterns of reaction products against a background of pectin reflection. The calculation of silver unit-cell parameters showed that in their quantity in the provided samples was lower than for massive silver and changed from 0.4036 to 0.4050 nm ( 0.0008 nm). Moreover, the average size of the coherent-scattering region (CSR) was calculated according to Selyakov–Sherarar's formula [99] to be in the range of 3 nm. The data obtained demonstrated that, in the samples of Ag (0), the persistence of nanosized

**Figure 13.** Diffraction patterns of pectin sample (a) and of the "pectin-Ag(0)" nanobiocomposite sample

"Pectin – Ag (0)" nanobiocomposite scanning electron microscopy (Figure 14) showed that

Microphotography analysis of nanobiocomposites, obtained by the use of transmission electron microscopy, demonstrated that there are isolated silver particles of null valency in globular form (Figure 15a), of a size within the range from 4 to 17 nm (predominance (up to

the analysed samples contain particles considerably smaller than 100 μm.

**Figure 14.** Electron microphotography of "pectin – Ag(0)" sample

80%) at 6–7 nm, Figure 15b).

particles was stabilized by an amorphous phase with pectin.

**Figure 15.** Transmission electron microphotography, a), and size distribution graph of "pectin-Ag(0)" nanobiocomposite sample, b)

Thereby, "pectin-Ag(0)" nanobiocomposite formation takes place as a result of the interaction of pectin water solutions with Ag(I). Process speed increases significantly with variation within the alkaline pH range of the medium. The initial component proportion influences the results of the reaction: the more Ag(I) that falls per 1g of pectin, the less the quantity of Ag(0) particles that is created in a nanosized condition. Using pectin implements reduction and stabilizing functions, and also adjusts the sizes of obtained Ag(0) nanoparticles.
