Natural Poly- and Oligosaccharides as Novel Delivery Systems for

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Natural poly- and oligosaccharides as novel delivery systems for plant protection compounds Olga Selyutina, Irina Apanasenko, Salavat Khalikov, and Nikolay E. Polyakov J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02591 • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 28, 2017

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Journal of Agricultural and Food Chemistry

Natural poly- and oligosaccharides as novel delivery systems for plant protection compounds

O. Yu. Selyutinaa*, I. E. Apanasenkoa, S. S. Khalikovb, N. E. Polyakova a

Voevodsky Institute of Chemical Kinetics and Combustion of Siberian Branch of the Russian Academy of Sciences, Institutskaya St., 3, 630090, Novosibirsk, Russia, e-mail: [email protected] b

Nesmeyanov Institute of Organoelement Compounds of Russian Academy of Sciences, Vavilova St., 28, 119334, Moscow, Russia

*

Corresponding author (Tel: +7(383)333-29-47; Fax:+7(383) 330-73-50; E-mail:[email protected])

ACS Paragon Plus Environment


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Abstract

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To increase the bioavailability of plant protection products we have applied new approach

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based on non-covalent association with natural water soluble polysaccharides and

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oligosaccharides as delivery systems (DSs). The mechanochemical technique has been

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applied to prepare the solid state nano-dispersed compositions of antidote 1,8-naphthalic

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anhydride (NA) with arabinogalactan, sodium salt of carboxymethylcellulose, and

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glycyrrhizin as DSs. The effect of DSs on the solubility and the penetration of NA into the

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seeds of barley and wheat has been investigated by various physicochemical techniques. All

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DSs considerably enhance the solubility of NA and improve its penetration into the grain.

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The influence of polysaccharides and oligosaccharides on artificial lipid membrane was

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studied by NMR relaxation method. It was concluded, that the effect of polysaccharides and

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oligosaccharides on the penetration efficacy of plant protection products might be associated

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with the detected solubility enhancement and the affinity of DSs to the surface of cell

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membranes.

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Keywords

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1,8-naphthalic anhydride; antidotes; delivery systems; glycyrrhizic acid; arabinogalactan; sodium salt of

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carboxymethyl cellulose; nuclear magnetic resonance; mechanochemistry.

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Introduction

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Nowadays more than 30 thousand of compositions based on more than 600 active agents are

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involved in protection of plants. But all over the world increasingly high demands are made

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on the safety of chemicals used. Development of new forms of plant protection agents (PPA)

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may results in a more effective and safe products.

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Last decades the tremendous progress was achieved in pharmacology due to applying of

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nanosized drug delivery systems (DDS) in order to enhance the bioavailability and safety of

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the drugs1-6. The idea is based on using water soluble carriers (polymers, micelles and so on)

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to increase the solubility of low soluble drugs and their permeation ability7-19. The use of

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nanotechnology in the agricultural sector has lagged far behind, despite the growth of

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scientific papers on the subject10,

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agriculture started less than a decade ago20. The authors point out the obvious prospects for

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the introduction of nanotechnology in agriculture. Nanotechnological approach allows to

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reduce the use of agricultural inputs (pesticides, herbicides, fertilizers, etc.) by increasing the

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efficiency of its delivery to the plants22. Thus, in the field of plant protection significant

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decrease consumption rates of plant protection products and their harmful effects on the

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environment are indicated as benefits. In the present study we have applied the same

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approach to increase the effectiveness of known antidote 1,8-naphthalic anhydride (anhydride

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of naphthalene-1,8-dicarboxylic acid (NA), Fig. 1). NA helps to accelerate the detoxification

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of herbicides in cereals and increase harvest resistance to them.

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Large amount of experimental data indicates that NA is the most effective agent for

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neutralizing the phytotoxic action of sulfonylureas among the commercial antidotes23.

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Antagonistic effect of NA on plant hormones associated with the implementation of activity

20, 21

. Research on the applications of nanotechnology in

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auxin-like herbicides; it can also restore the synthesis of lipids and inhibit absorption of

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herbicides by plants under seeds treatment24.

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We focused our attention on associates of NA with various delivery systems (arabinogalactan

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(AG), glycyrrhizic acid (GA), and sodium salt of carboxymethyl cellulose (Na-CMC)) which

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demonstrated high effectiveness as the DDS7-19. We compared permeation of NA into the

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seeds in pure form and in the form of associates mentioned above. Also we have made an

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attempt to study the interaction (affinity and penetration) of these delivery systems with

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model lipid membrane.

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Glycyrrhizic acid (GA, glycyrrhizin (Figure 2a)) is a triterpene saponin extracted from the

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licorice root. Glycyrrhizin has a number of properties that make it an attractive delivery

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system. The key feature is the amphiphilicity of glycyrrhizin molecule: the hydrophilic part

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consists of glucose rings, hydrophobic one is a glycyrrhetic acid residue (Fig. 2a). Owing to

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this, glycyrrhizic acid is capable to form micelles in water solution and "host-guest"

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complexes with various hydrophobic molecules7-11.

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Different studies show that formation of GA complexes with drugs results in a significant

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increase in their solubility, enhancement the therapeutic effect and, consequently, reduce the

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therapeutic doses of drug8-12. In addition, previous studies showed increase of the cell

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membrane permeability for small molecules in the presence of GA and increase of lipid

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mobility in model cell membranes (liposomes) after GA treatment12-14.

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Arabinogalactan (AG, Fig. 2b) is a natural polysaccharide consisting of arabinose and

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galactose fragments. AG demonstrates strong binding with different hydrophobic biologically

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active molecules which results in significant increase of their solubility and oxidative

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resistance15-17. AG also increases the permeability of cell membranes for small molecules18. 5



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So, GA and AG are natural compounds which are known to form complexes with different

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drugs and to influence on cell permeability25-28. We hope that they could be used as effective

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delivery systems (DS) for plant protection agents also. For preparation of water soluble solid-

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state nanodispersed compositions, the mechanochemical technique was applied. This

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approach permits solid-state preparation of water soluble compositions of insoluble

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compounds in one technological step without use of any organic solvents27.

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Materials and methods

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Materials

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1,8-Naphthalic anhydride (Shenzhen Sunrising Industry Co., Ltd. China, purity > 96.0%) was

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used in the present study. Arabinogalactan of the Siberian larch, glycyrrhizic acid and sodium

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salt of carboxymethyl cellulose (Na-CMC) (CEKOL 700, pharmacopeia purity,

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http://cpkelco.com/products/cellulose-gum.1) were used for mechanochemical modification

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of NA.

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Liposomes were formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC,

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Avanti Polar Lipids, purity > 99%).

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Preparation of NA compositions

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The method of water soluble complex preparation used in the present work was a

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mechanochemical treatment of the solid mixture of NA crystals with polysaccharides or

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oligosaccharides powder. Typical mechanochemical reactions are those activated by co-

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grinding or milling of powder materials27-29. In the present study, we used electro-

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mechanochemical equipment. Co-grinding of solid materials results in penetration of NA

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molecules into the polymer without use of any organic solvents. This approach allowed us to 6


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prepare the water soluble composites in one technological step. For preparation of NA

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complexes pre-mixed powders of 1,8-naphthalic anhydride and delivery systems (AG, Na-

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CMC, GA) with mass ratios 1:9, 1:2 and 1:9, respectively, were loaded into a caprolon

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cylinder of the roller mill LE-101 with metal balls. The cylinder was filled on 65%,

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activation modulus was 1:15. Total mass of metal balls was 600 g, ball diameter was about

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12-15 mm. Then physical mixture was activated for 7 hours. The rotation rate of cylinder

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was in range 60-80 rpm. The described procedure resulted in nano-dispersed powder

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composition of NA with delivery systems. The particle size (dispersion composition) of the

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particles was determined using a laser particle size analyzer Microsizer-20la ("BA Instalt",

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St. Petersburg).

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Solubility analysis was performed using HPLC technique (Agilent 1200; column Hypersil

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5µ HyPURITY Elite C18 (150×4.6 mm); eluent acetonitrile/water 1:1, 1 mL/min, T = 30

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°C; detection at 230 and 340 nm). Blue Ribbon Quantitative Filter Paper was used for fine

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precipitates separation.

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Preparation of grain samples

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Experiments were done with wheat “Omsk 36” and spring barley “Acha”. Barley and wheat

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grains (1.5 g) were placed in tube with 0.1 mL of NA complex dispersions or with water

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dispersion of NA. Equal amounts of NA were used for all experiments. Then the tube was

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rotated for 15 minutes for homogeneous putting of agents on samples. Then grains were

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dried for 24 hours. After that 0.5 g of grains was washed with acetone to obtain solution of

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NA remained on the surface of grains. Then washed grains were milled and mixed in

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acetone for 24 hours to extract NA from the inside of grains. Grain solids were removed by

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centrifugation. 7



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Another 0.5 g of treated grains was placed in Petri dish in humid atmosphere for 3 days for

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germination. Then washing and extract of NA were prepared in the way described above.

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Washed and extracted samples in acetone were analyzed by means of NMR. 1H NMR

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spectra were recorded on Bruker Avance III 500 MHz spectrometer.

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Each NMR sample contains extract from ~50 grains. All experiments were repeated twice

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and resulted values were calculated as an arithmetic mean, and resulted error was calculated

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as mean value of least squares errors, the differences were considered statistically significant

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at p < 0.01 using a t-test.

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Preparation of liposome samples

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Powder components were pre-dissolved in chloroform. After removing the solvent, the dry

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lipid film was hydrated with D2O. The final concentration of lipid was 10 mM. The

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suspension was then sonicated (about 37 kHz, 1 hour) to obtain unilamellar liposomes. NMR

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spectra were recorded for samples of 0.6 mL of vesicle suspension supplemented with 4 mM

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PrCl3 in 5-mm NMR tubes. After addition of PrCl3, the liposome suspension was

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supplemented with different delivery systems (GA, AG, Na-CMC).

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NMR study

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1

H NMR spectra were recorded on Bruker Avance III 500 MHz spectrometer, spin-spin

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relaxation time T2 was measured by the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence.

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Deuterated solvent (D2O (99.9% D), Acetone-D6 (99.8% D), Aldrich) were used as received.

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The study of lipid mobility

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Spin-spin and spin-lattice relaxation times (T1 and T2) are sensitive to molecular motions.

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The T1 relaxation parameter is sensitive to high frequency lipid motions such as trans-gauche

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methylene isomerization, whereas the T2 time is sensitive to low frequency (e. g., large

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amplitude chain wagging) lipid motions30,

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functional groups (-N+(CH3)3-group in lipid polar head and terminal CH3-groups in

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hydrophobic tails) were measured for characterization of delivery systems influence. The

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spin-spin relaxation time T2 is closely related to the mobility of the molecule and is inversely

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proportional to the rotational correlation time. Thus, using T2 data, one can form conclusions

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about changes of environment or state (free / bound) of molecules. The ability to resolve lipid

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resonances in the inner and outer headgroup region using shift-reagent and in the terminal

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methyl groups has allowed us to study the effect of delivery systems on lipid motions from

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the surface to the center of the membrane. Measurement temperature was 300 K.

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Statistical analysis

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independent experiments, and the differences were considered statistically significant at p

Natural Poly- and Oligosaccharides as Novel Delivery Systems for

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