Pectin polysaccharides (PSs) were isolated from a bark of
Two species of the genus
However, the larch bark does not find industrial application. Annual volume of waste produced by wood-processing industry and the pulp-and-paper enterprises is more than 30 million m3. It represents a serious environmental challenge because the bark is poorly exposed to biodegradation. At the same time, the chemical compounds of the bark can be used as a source of valuable biologically active substances including polysaccharides.
Earlier we have showed that the content of pectin polysaccharides in the larch bark is about 12% from the weight of absolutely dry raw material (a.d.r.m.) and the larch bark can be promising alternative raw material. The method of pectin isolation has been developed, and common physical and chemical characteristics and membranotropic activity of pectin have been investigated.
It has been established and patented that the larch bark pectin can possess reducing and stabilizing properties in the formation of nanobiocomposites with precious metals ions.
The present work aims at the following. The study of general characteristics of pectin substances (PS) extracted from bark of The study of PS interaction with silver nitrate and elucidation of alteration of pectin structural characteristics during the formation of “pectin-Ag(0)” nanobiocomposite.
Pectin substances were extracted from the bark of
Scheme of pectin substances extraction from larch bark.
PS (50 mg) was dissolved in 20 mL of water. Pektinaza (2 mg, Sigma, USA) aqueous solution was added. The mixture was temperature-controlled at 37°C for 3 h. Then, a reaction mixture was heated for 5 min in water bath at 100°C. Coagulated protein was separated by centrifugation. Obtained supernatant was concentrated up to 5 mL, 96% ethyl alcohol was added (4 volumes). Deposition was separated by centrifugation. Alcoholic supernatant was concentrated and analyzed by paper chromatography (PC).
Paper chromatography was carried out on “Filtrak FN-13” paper with a descending method in n-butanol-pyridine-water system (volume correlations 6 : 4 : 3, resp.). To define carbohydrates, the paper was poured with aniline phthalate and heated at 105°C.
Glucuronic acids content in PS was defined according to the reaction with 3,5-dimethyl phenol in the presence of concentrated H2SO4, protein using Lowry method [
Gas-liquid chromatography (GLC) was carried out on a Hewlett-Packard 4890A (the USA) chromatograph equipped with flame-ionization detector, RTX-1 (0.25 mm × 30 m) capillary column, argon carrier gas, 1 : 60 dumping. Temperature rate: 175°C (1 min)–250°C (2 min), Δ3°/min.
Total acid hydrolysis PS (5 mg) was carried out with the implementation of 2 M trifluoroacetic acid (TFA) (2 mL) which contained
Ion-exchange chromatography PS (100 mg) was carried out on DEAE-cellulose (25 × 2 cm) column. NaCl solutions were used as an eluent with increasing concentration (0.01 M–1 M, 60 mL/h elution’s speed, fractions’ selection by 12 mL). Pick correspondent fractions at the output bents were combined, dialyzed, and lyophilized. As a result, PS 1–4 fractions were obtained. Monosaccharides of each fraction after hydrolysis of PS 1–4 and acetilation were defined by GLC.
Monosaccharides acetilation: each PS 1–4 fraction was dissolved in 1 M ammonia solution (1 mL) and 5 mg of NaBH4 was added. The mixture was kept at room temperature during one day. Then, NaBH4 abundance was destroyed by adding 2-3 drops of concentrated acetic acid; 0.2 mL of dry pyridine and acetic anhydride were added to a dry residue. The mixture was being acetilized at 100°C during 1 h. The solution was evaporated up to pyridine and acetic anhydride abundance removal, first adding 1 mL of toluene and then 1 mL of methanol. The obtained acetate mixture of PS 1–4 polyol fractions was dissolved in 0.2 mL of dry chloroform and moved quantitatively to Appendorf tubes, concentrated up to 0.1-0.2 mL, and analyzed with GLC method.
PS (5 mg) partial acidic hydrolysis was carried out with 0.05 M TFA (2 mL) which contained
PS (5 mg) partial acidic hydrolysis was carried out with 0.01 M TFA (2 mL) which contained
IR spectra were registered on a FT-IR (RAM II) Brukker Vertex 70 spectrometer in pellets with KBr.
13C NMR spectra were recorded on a Bruker DRX 500 instrument (Germany) using 3–5% carbohydrate solution in D2O at 55 and 70°C; the internal standard was DMSO-d6.
Synthesis of PS-g1 and PS-g1 nanobiocomposites “pectin-Ag(0)” was carried out according to the [
UV-Vis spectra of nanobiocomposite aqueous water solutions were recorded on a Perkin Elmer Lambda 35 instrument in ultraviolet and visible areas.
Pectin substances are abundant in land and water plants, as well as some freshwater algae [
Unique physicochemical properties of pectin make it indispensable material in medical, food, and cosmetic industries as gelling agent, thickener, stabilizer, and dietary fiber. Since recently, it is extensively used as a matrix carrier for biologically active components in drugs. Pectins exert diverse physiological activities such as immunomodulating, hepatoprotective, anticarcinogenic, and antimetastatic, that allow them to be applied as drugs and biologically active food additives.
The pectins are typically isolated from economically valuable pants, for example, citrus, apple, sugar beet, and sunflower head pith. Other plants such as amaranth, small mallow, duckweed, SILENE, and coffee beans, have been reported as potential sources of pectins [
The methods for isolation of pectin polysaccharides from plant tissues are numerous. Among the classic ones is hydrolysis extraction of dry raw material particles of certain sizes [
Pectin substances were extracted from the bark of
Table
Absorption band maximums in IR spectra of PS and their assignment.
Frequency ( |
Assignment |
---|---|
3460 |
|
3260 |
|
2962, 2872 |
|
2573 |
|
1730 |
|
1640 |
|
1540 |
|
1380–1450 |
|
1331 |
|
1265 |
|
1150 |
|
1095 |
|
1027 |
|
890 |
|
766, 629, 528 | Pulse vibrations of pyranose ring |
To determine the sugar composition of PS the total acid hydrolysis has been carried out. Monosaccharide identification in hydrolysate has been performed by gas-liquid chromatography. It has been shown that the sample consists of galacturonic acid and monosaccharides of arabinose, galactose, rhamnose, glucose, mannose, and (in minor quantity) xylose (Table
Monosaccharide compositions determined by GLC analysis of PS and PS-s samples.
Sample | Content, % | ||||||
---|---|---|---|---|---|---|---|
Gal A | Rha | Ara | Xyl | Man | Glc | Gal | |
PS | 38.4 | 1.6 | 6.8 | traces | 2.5 | 3.7 | 18.4 |
PS-s | 87.86 | 0.97 | 2.75 | traces | 0.73 | 0.82 | 3.02 |
Partial acid hydrolysis of PS by 0.05 M TFA gives polysaccharide PS-s representing a linear polysaccharide with insignificant amount of side chain subunits. It is also confirmed by the decrease of the relative content of neutral monosaccharides of PS-s in comparison with their content in initial polysaccharide PS (GLC analysis of polyols peracetates of the corresponding sugars in the studied substances, Table
Values of chemical shifts (CSs) of carbon atoms in 13C NMR spectrum of PS-s (Table
Chemical shifts values of carbon atoms in D-galacturonic acid residues in the 13C NMR spectrum of PS-s (
Residue | C-1 | C-2 | C-3 | C-4 | C-5 | C-6 | C-6–(OCH3) | –O |
---|---|---|---|---|---|---|---|---|
|
101.9 | 68.9 | 72.1 | 79.2 | 73.4 | 176.2 | 172.2 | 54.4 |
|
100.7 | 69.4 | 69.8 | 79.4 | 72.2 | 175.2 | — | — |
|
103.4 | 72.4 | 74.3 | 78.3 | 76.0 | 175.2 | — | — |
The signal of anomeric carbon atom at 101.9 ppm indicates both the (1
Ion exchange column chromatography of PS on DEAE cellulose by 0.01–0.2 M sodium chloride aqueous solutions has yielded four polysaccharide fractions, PS 1–4, whose chemical characteristics are summarized in Table
Chemical characterization of PS sample after DEAE-cellulose fractioning.
Sample* | Yield, % | Content, % | |||||||
---|---|---|---|---|---|---|---|---|---|
GalpA | Protein | Monosaccharides | |||||||
Rha | Ara | Xyl | Man | Glu | Gal | ||||
PS-1 | 12.1 | 5.67 | 6.9 | traces | 18.26 | 1.54 | 2.53 | 5.95 | 52.92 |
PS-2 | 5.9 | 29.12 | 7.3 | 0.53 | 11.65 | 1.02 | 2.71 | 8.81 | 30.83 |
PS-3 | 17.0 | 65.93 | 5.7 | 1.91 | 4.45 | 0.75 | 1.18 | 1.12 | 9.42 |
PS-4 | 37.0 | 79.87 | 3.6 | 0.35 | 0.93 | 0.18 | 0.24 | 0.21 | 1.06 |
Note: *PS-1 isolated with use of 0.01 M NaCl solution, PS-2—0.1 M NaCl solution, PS-3 and PS-4—0.2 M NaCl solution.
In the fractions PS-1 and PS-2, galactose and arabinose are predominant (18.26/52.96% and 11.65/30.83%, resp.), thus, they belong to acidic arabinogalactans. Acidic character of PS is caused bythe presence of D-galacturonic acid residues, the content of which in PS-1 is 5 times less than in PS-2, while in PS-3 and PS-4 it is a major monosaccharide that allows PS to pectins to be assigned. Content of neutral monosaccharides in PS-4 is minimum as compared to other fractions (3 mass %).
All the fractions contain protein substances that are not eliminated by gel filtration (Table
Amino-acid composition of PS proteins has been studied. Major components of proteins are glutamic acid (6%) and aspartic acid (2.8%), while total content of amino acids with aliphatic side chains (glycine, alanine, valine, isoleucine, leucine) is 9% (Figure
Amino acid composition of PS proteins.
As mentioned above, full acid hydrolysis of PS with 2 M TFA has delivered galacturonan (PS-s). A milder treatment of PS with 0,01 M TFA gives PS-g.
According to the 13C NMR data, PS-g is a pectin polysaccharide. The spectrum contains typical signals of galacturonic acid residues, namely, pronounced signals of anomeric carbon atoms at 100.4 and 100.9 ppm, and signals of the carboxyl carbon atoms at 171.4, 166.5, and 53.7 ppm, the latter two being of carbon atoms in uronic acid residues methoxylated by C-2 and C-3 atoms (Table
Chemical shifts of carbon atom signals in 13C NMR spectrum of PS-g.
Residue | C1 | C2 | C3 | C4 | C5 | C6 | –O |
---|---|---|---|---|---|---|---|
|
100.4 | 68.9 | 70.8 | 78.9 | 72.2 | 171.4 | — |
2-Me- |
100.9 | 166.5 | 69.6 | 78.9 | 73.8 | 171.4 | 53.7 |
3-Me- |
100.9 | 68.9 | 166.5 | 78.9 | 73.8 | 171.4 | 53.7 |
|
99.7 | 77.6 | 70.8 | 82.5 | 68.9 | — | 17.9 |
|
104.64 | 71.7 | 74.1 | 69.6 | 76.1 | 62.0 | — |
|
104.38 | 71.7 | 73.8 | 69.6 | 74.3 | 70.8 | — |
|
104.64 | 71.7 | 82.5 | 69.57 | 74.1 | 71.4 | — |
|
108.6 | 80.7 | 78.9 | 84.9 | 62.0 | — | — |
|
101.1 | 69.6 | n.d. | n.d. | n.d. | — | |
|
108.6 | 80.7 | 84.9 | 83.2 | 67.8 | — | — |
|
108.0 | 84.9 | 77.6 | 83.2 | 67.8 | — | — |
Note. n.d.: not determined.
In the 13C NMR spectrum of PS-g sample 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 166.42 and 171.33 ppm were found to have a ratio of 1 : 5. The total integral intensity of signals for anomeric C-1 atoms for rhamnose and total integral intensity of signals of anomeric atoms of s were equal to each other, that is, they had the same ratio for rhamnose galacturonan residue in the chain. According to data [
Thus, according to the 13C NMR spectral data, linear fragment of pectin polysaccharide PS-g is rhamnogalacturonan (RG-I), where D-galacturonic acid residues in pyranose form with
Further 13C NMR studies of PS-g sample shows that arabinogalactan subunits are present in rhamnogalacturonan as side chains. In the 13C NMR spectrum of PS-g sample, in the region of anomeric carbon signals, apart from the signals of anomeric carbons in galacturonopyranosyl residues of the galactan core, appear the signals at 101.1, 104.38, 104.64, and 108.6 ppm. According to [
Hence, according to spectral data for PS-g fragment of the pectin polysaccharide from larch bark, highly branched arabinogalactan is detected as side chains consisting of linear chains with
It is known that the reactivity of polysaccharide molecule is due to terminal sugars, mainly localized in side chains [
Arabinogalactan is a prevailing polysaccharide of larch wood; hence, its presence in the polysaccharides bark is owing to biogenetic reasons. It has been reported that arabinogalactan is present in the pectin substances of cell walls of plants both as associable bonding and accompanying component [
Significant interest to nanosize metals is caused by their high technological potential as important magnetic materials, catalysts, nonlinear-optical medium, and biologically active agents. So, for example, it has been established that silver nanoparticles possess rare combination of valuable features, that is, unique optical properties due to surface plasmon resonance, highly developed surface, catalytic activity [
One of the widespread approaches to the synthesis of metal nanoparticles involves the reduction reaction of metal ions in a polymeric solution. As a rule, the high-molecular compound (polysaccharide) employed in this case acts as a protective polymeric screen ensuring both the size of metal nanoparticles and stabilization of the nanobiocomposite formed [
Another approach to the nanocomposites synthesis is based on the nanocomposites self-organization, where the polymers play a role of reducing agent and nanostabilizing medium [
Study of the Ag(I) redox reaction and Ag(0) nanoparticles formation and stabilization in pectin polysaccharide matrix was carried out depending on the reaction conditions, in particular, pH value, initial reagents ration (metal salt/pectin), and reaction time.
The absorption spectra of pectin and silver nitrate aqueous solutions at different reaction time are depicted in Figure
Absorption spectra of a mixture of aqueous solutions of pectin (0.5%) and silver nitrate (0.1%) in a ratio of 1 : 1 depending on (a) reaction duration: 1 min (1), 24 h (2), 48 h (3), 72 h (4), 96 h (5); (b) pH value: 3.5 (1), 7 (2), 9.7 (3), 11.5 (4); (c) reaction duration at pH 11.5: 1 min (1), 30 min (2), 60 min (3), 180 min (4), 24 h (5).
Thus, the increase in rate of Ag(I) reduction with pectin at elevated pH value allows one to assume the direct participation of OH− in this reaction.
In the alkaline medium, polysaccharides are known to undergo diverse transformations [
13C NMR spectroscopy data (Tables
Chemical shifts (
Residue | C-1 | C-2 | C-3 | C-4 | C-5 | C-6 | –OCH3 |
---|---|---|---|---|---|---|---|
|
100.3 | 69.5 | 70.2 | 78.0 | 72.2 | 176.8 | |
6-Me- |
100.3 | 69.5 | 70.0 | 78.0 | 74.1 | 174.7 | 55.4 |
|
99.7 | 77.6 | 71.0 | 82.5 | 72.8 | 18.0 | |
|
104.7 | 71.7 | 74.1 | 69.5 | 76.6 | 62.5 | |
|
105.1 | 71.7 | 74.1 | 69.5 | 72.7 | 70.3 | |
|
105.1 | 71.7 | 82.7 | 70.1 | 72.7 | 71.7 | |
|
108.6 | 80.7 | 78.9 | 84.9 | 62.0 | — |
The comparison of 13C NMR spectra of PS-g1 and PS-g has shown that PS-g1 represents a partially destructed acidic polysaccharide. This is evidenced from the presence (13C NMR) of characteristic signals of anomeric carbons at 100.3 ppm in the
The structure of PS-g2 differs from that of PS-g1 by the content of Ag(0) (72%). According to 13C NMR data (Table
Chemical shifts (
Residue | C-1 | C-2 | C-3 | C-4 | C-5 | C-6 |
---|---|---|---|---|---|---|
|
104.8 | 71.7 | 74.2 | 69.6 | 76.6 | 62.5 |
|
104.8 | 71.7 | 74.2 | 69.6 | 72.8 | 70.1 |
|
104.8 | 71.7 | 82.8 | 70.1 | 74.9 | 71.7 |
Thus, the comparison of structural characteristics of the initial pectin with those of “pectin-Ag(0)” nanobiocomposites PS-g1 and PS-g allows one to assume that in the reaction studied the pectin polysaccharide acts a reducing agent and also undergoes destructive changes. Pectin carboxy-groups and other groups (–OH, =O) are contained in structure of side chains of RG-I participate in formation of zero-valence silver participates.
The destruction of PS-g1 backbone in rhamnosyl residue is observed upon the addition of larger quantities of Ag(I) to the reaction. As a result, RG-I is abstracted, the main chain of which is less destructed by this moment and stabilizes the Ag(0) nanoparticles.
Efficiency of stabilization functions has been estimated by influence of quantitative ratio of the initial pectin substances taken in reaction: metal salt (mmol)/pectin (1 g) on the content of Ag(0) nanoparticles and their sizes in formed nanobiocomposites “pectin-Ag(0).” It has been shown that in area 0.1–2.5 mmol AgNO3/1 g of pectin the content of zero-valent silver in received nanobiocomposite is indirectly proportional relationship of quantity the salt taken in treatment (Figure
Influence of a ratio of initial metal salt/pectin on the content of Ag(0) in formed nanobiocomposites.
Diffractogram of precipitate formed in the reaction medium at use of initial components in area from 2.5 up to 6.5 mmol of AgNO3/1 g of pectin.
The content of Ag(0) nanoparticals in nanobiocomposite “pectin-Ag(0)” can be increased up to 72% using the most nanobiocomposite as an initial component. It should be noted that thus the size of nanoparticals in nanobiocomposite increases and is from 4 up to 27 nm.
In conclusion, the interaction of aqueous solutions of pectin with silver nitrate affords “pectin-Ag(0)” nanobiocomposites, where pectin plays reducing and stabilizing roles. The reaction rate essentially increases when pH values of the reaction mixture are close to alkaline ones. The ratio of the starting reactants influences the content of Ag(0) in the nanobiocomposites and their sizes: the more is Ag(I) per 1 g of pectin, the lower amount of Ag(0) nanoparticles is formed.
It has been established (13C NMR) that when 0.1–2.5 mol of AgNO3 and 1 g of pectin are employed in the reaction, the PS structure is preserved. The increase of Ag(I) amount up to 6.5 mmol leads to the pectin destruction with abstracting the side fragments and destruction of rhamnogalacturonan core. Under ratio of AgNO3/pectin equaling 6.5 mmol/1 g, the formation of the nanocomposite is stopped due to the total destruction of pectin polysaccharide.
Thus, in the reaction of “pectin-Ag(0)” nanobiocomposites formation, pectin acts as having reducing and stabilizing functions and regulates the sizes of Ag(0) nanoparticles.