Polymeric Compounds of Lingonberry Waste: Characterization of Antioxidant and Hypolipidemic Polysaccharides and Polyphenol-Polysaccharide Conjugates from Vaccinium vitis-idaea Press Cake

Lingonberry (Vaccinium vitis-idaea L.) fruits are important Ericaceous berries to include in a healthy diet of the Northern Hemisphere as a source of bioactive phenolics. The waste generated by the V. vitis-idaea processing industry is hard-skinned press cake that can be a potential source of dietary fiber and has not been studied thus far. In this study, water-soluble polysaccharides of V. vitis-idaea press cake were isolated, separated, and purified by ion-exchange and size-exclusion chromatography. The results of elemental composition, monosaccharide analysis, ultraviolet–visible and Fourier-transform infrared spectroscopy, molecular weight determination, linkage analysis, and alkaline destruction allowed us to characterize two polyphenol–polysaccharide conjugates (PPC) as neutral arabinogalactans cross-linked with monomeric and dimeric hydroxycinnamate residues with molecular weights of 108 and 157 kDa and two non-esterified galacturonans with molecular weights of 258 and 318 kDa. A combination of in vitro and in vivo assays confirmed that expressed antioxidant activity of PPC was due to phenolic-scavenged free radicals, nitrogen oxide, hydrogen peroxide, and chelate ferrous ions. Additionally, marked hypolipidemic potential of both PPC and acidic polymers bind bile acids, cholesterol, and fat, inhibit pancreatic lipase in the in vitro study, reduce body weight, serum level of cholesterol, triglycerides, low/high-density lipoprotein–cholesterol, and malondialdehyde, and increase the enzymatic activity of superoxide dismutase, glutathione peroxidase, and catalase in the livers of hamsters with a 1% cholesterol diet. Polysaccharides and PPC of V. vitis-idaea fruit press cake can be regarded as new antioxidants and hypolipidemic agents that can be potentially used to cure hyperlipidemic metabolic disorders.


Introduction
The food industry produces more than one billion tons of waste per year, and the largest part is secondary products generated after mechanic procedures of cleaning, peeling, and pressing of fruits, vegetables, and berries [1]. Recycling of agro-food wastes is the focus of scientists who are interested in eco-friendly technologies for waste processing, which is an important problem of modern life [2]. The problem of fruit processing is of interest to many scientists, but insufficient attention is paid to berry industry waste [3]; at best, this type of waste is composted, and at worst, it is simply thrown away [4]. Among the processed berries, the least attention is paid to hard-skinned berries derived from Vaccinium and Ribes species, which is explained by the impossibility of its transformation into a puree or a homogeneous mass, despite the use of heat treatment methods [5]. Diethylaminoethyl Cellulose (DEAE cellulose) Fractionation of VVPS. Solution of VVPS (2 kg) in water (40 L) was passed through the DEAE cellulose 52 column (10 kg; Sisco Research Laboratories Pvt. Ltd., New Delhi, India) in HCO3-form and eluted by the water, NH4HCO3 solution (0.1%, 0.3%, 0.5%, 1%) and 1% NaOH till negative reaction with phenol-sulfuric acid reagent. All eluates were dialyzed in benzoylated dialysis tubes against distilled water (48 h) and non-dialyzed residues were freeze-dried. The yield of DEAEcellulose fractions was 140 g (DEAE-H2O), 16 g (DEAE-0.1% NH4HCO3), 36 g (DEAE-0.3% NH4HCO3), 40 g (DEAE-0.5% NH4HCO3), 8 g (DEAE-1% NH4HCO3), and 1.62 kg (DEAE-1% NaOH).

Chemical Composition of VVPS and DEAE Cellulose Fractions.
Ready-to-use kits for spectrophotometric assays were applied to measure the total carbohydrate content (High Sensitivity Carbohydrate Assay Kit, BioVision Inc., Milpitas, CA, USA; cat. No K2049-100), uronic acids (D-Glucuronic/D-Galacturonic Acid Assay Kit, Megazyme, Bray, Ireland; cat. No K-URONIC), starch (Total Starch Assay Kit, Megazyme; cat. No K-TSTA-100A), protein (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, MA, USA), and phenols (Phenolic Compounds Assay Kit, Sigma-Aldrich; cat. MAK365). Arabinogalactan-protein complexes content was estimated colorimetrically with Yariv reagent as described by Lamport et al. [ 1 ] and ash content determined by AOAC Official Method SM 942.05 using muffle furnace ignition at 600°C [ 2 ].   The water extract was filtered through industrial filter CUNO™ Self Cleaning Metal Edge Filter Cartridge (200 µm; 3M Purification Inc., Meriden, CT, USA) and concentrated 20 times in vacuo, and the residue was mixed with 95% ethanol (1:4). The mixture was left for 24 h, and the crude precipitate was filtered through the cellulose membrane followed by drying, at 50 • C. The yield of crude precipitate was 3.2 kg (5.8% of press cake dry weight). Dry crude precipitate (3 kg) was suspended in 60 L of distilled water, the temperature of the mixture maintained at 60 • C for 1 h, and the resulting solution was filtered through two sequential columns with polyamide (10 kg; Sigma-Aldrich, cat. No. 02395) and a cation-exchanging column (KU-2-8, H + -form; Eco-Vita, St. Petersburg, Russia; 20 kg) eluted with 100 L of distilled water (dephenolization and demineralization step). The water eluate was reduced in vacuum, at 30 • C, to 3 L, and the residue was vigorously mixed with a chloroform-n-butanol mixture (4:1) in the ratio of 1:1 and centrifuged (6000 rpm, 30 min) (Sevag deproteination step 1). Organic solvents were removed under vacuum, and the water residue was incubated with protease from Streptomyces griseus (type XIV, ≥3.5 units/mg; 1 unit per 1 mL of polysaccharide solution; Sigma-Aldrich, cat. No. P5147), at 35 • C, for 3 days (pronase deproteination step 2), and the Sevag deproteination step was repeated. The final solution was dialyzed in benzoylated dialysis tubes (cut-off of  (48 h), and the non-dialyzed residue was freeze-dried to give V. vitis-idaea total polysaccharide fraction (VVPS) as a light-brownish powder in a yield of 2.04 kg (3.7% of press cake dry weight).

Elemental Composition
A 2400 Series II elemental analyzer (Perkin Elmer, Waltham, MA, USA) was used for analysis of carbon, hydrogen, oxygen, and nitrogen contents in the polysaccharides.

Monosaccharide Composition
The monosaccharide composition of polysaccharides was studied after trifluoroacetic acid (TFA) hydrolysis, followed by 1-phenyl-3-methyl-5-pyrazolone (PMP) labeling and HPLC with ultraviolet detection separation (HPLC-UV) as previously described with modifications [35]. Polysaccharide samples (10 mg) were subjected to the hydrolysis procedure with 1 mL of 2 M TFA using sealed ampoules incubated, at 120 • C, for 2 h. After cooling, the mixture was centrifuged (6000× g, 15 min) and evaporated in vacuo to remove TFA, and the residue was dissolved in 1 mL of distilled water. The hydrolyzed samples (60 µL) were mixed with 25 µL of 1.5 M NaOH (in water) and 80 µL of 0.5 M PMP (in methanol), incubated at 70 • C (2 h), cooled, and neutralized with 70 µL of 0.5 M HCl. The samples were purified by adding double chloroform (1 mL), followed by vigorous agitation (30 s) and centrifugation (3000× g, 10 min). Finally, the organic phase was removed, and the aqueous layer was analyzed by HPLC-UV. HPLC-UV separation of PMP-labeled sugars was performed using a MiLiChrom A-02 microcolumn chromatograph (Econova, Novosibirsk, Russia) coupled with a UV detector and microcolumn ProntoSIL-120-5-C18 AQ (75 mm × 1 mm × 1 µm; Metrohm AG, Herisau, Switzerland) eluted in gradient mode. A solution of 100 mM CH 3 COONH 4 (pH 6.9) was eluent A, acetonitrile was eluent B, and the following gradient program was used: 0-20 min for 20-26% B. The parameters of column temperature, injection volume, and flow rate were 35 • C, 1 µL, and 150 µL/min, respectively. Chromatograms were recorded at 250 nm. Bidistilled water stock solutions (1 mg/mL) of reference monosaccharides of mannose, ribose, rhamnose, glucose, galactose, xylose, arabinose, fucose, galacturonic acid, and glucuronic acid were prepared and PMP-labeled in the same manner before analysis ( Figure S1). The calibration curves were created by plotting the peak area vs. the concentration levels. All analyses were performed in triplicate.

Molecular Weight Determination
A gel permeation-high performance liquid chromatography (GP-HPLC) procedure was used for the molecular weight determination. Experiments were performed on an LCMS 8050 liquid chromatograph coupled with a photodiode array detector (Shimadzu, Columbia, MD, USA) using a Shim-pack Diol-150 column (250 mm × 7.9 mm × 5 µm; Shimadzu) at a column temperature of 25 • C. The eluent was a 10 mM phosphate-buffered solution (pH 7.0). The injection volume was 1 µL, and the elution flow was 1 mL/min. Isocratic elution was applied, and chromatograms were integrated at 190 nm. A series of dextrans (10-410 kDa; Sigma-Aldrich) were used to create a calibration curve. The polysaccharide sample was dissolved in 10 mM phosphate-buffered solution (pH 7.0), centrifuged (6000× g), and filtered through a 0.22-µm PTFE syringe filter before injection into the HPLC system for analysis. All analyses were performed in duplicate.

Linkage Analysis
For linkage analysis, 10 mg of the polysaccharide was methylated by methyl iodide, followed by hydrolysis of the permethylated product using 90% formic acid and 2 M TFA, NaBH 4 reduction, and acetylation with acetic anhydride [36]. Partially methylated alditol acetates were analyzed by gas chromatography-mass spectrometry using a 5973N gas chromatograph mass spectrometer (Agilent Technologies, Santa-Clara, CA, USA) equipped with a 6890N mass selective detector, a diffusion pump, and an HP-Innowax capillary column (Agilent Technologies; 30 m × 250 µm × 0.50 µm) within a programmed temperature range of 150 to 250 • C, at a rate of 2 • C/min, with helium as the carrier gas (flow rate of 1 mL/min) [37]. The temperature of the transfer line and ion source was 280 • C. The sample injection volume was 1 µL with a split ratio of 50:1, and the scanning range was m/z 30-400. All analyses were performed in triplicate. Alkaline hydrolysis of the polysaccharides was performed as previously described using 10% potassium hydroxide solution heating, 80% H 2 SO 4 neutralization, and liquidliquid extraction with ethyl acetate [38]. Degradation products were analyzed by highperformance liquid chromatography with photodiode array detection and electrospray ionization triple quadrupole mass spectrometric detection (HPLC-PDA-ESI-tQMS) using an LC-20 Prominence liquid chromatograph coupled with an SPD-M30A photodiode array detector (wavelength range 200-600 nm), and an LCMS 8050 triple quadrupole mass spectrometer (Shimadzu, Columbia, MD, USA) with two-eluent gradient elution [39]. The management of the LC-MS system was realized by LabSolution workstation software equipped with an internal LC-MS library. The final identification of metabolites was performed after an integrated analysis of retention time, ultraviolet spectra, and mass spectra in comparison with reference standards and literature data. The relative content of phenolic acids was calculated using calibration curves created using reference substances (ferulic acid, sinapic acid), methanolic solution (1-100 µg/mL) analysis, and by building concentration-peak area graphs. Contents of diferulic and triferulic acids were calculated as ferulic acid equivalents, and disinapic acid was measured as sinapic acid equivalents. All quantitative analyses were performed five times, and the data were expressed as the mean value ± standard deviation (S.D.).

In Vitro Assays
The cholesterol binding capacity of polysaccharides was measured by the method of Nagaoka et al. [45] and an enzymatic Amplex™ red cholesterol assay kit (Thermo Fisher Scientific, Waltham, MA, USA; No. A12216). The bile acid binding properties were studied using the Kim and White assay [46] with an enzymatic bile acid assay kit (Sigma-Aldrich; No. MAK309). The fat binding capacity of polysaccharides was estimated gravimetrically using the Jin et al. assay [47], and pancreatic lipase inhibition was studied spectrophotometrically using p-nitrophenol palmitate as a substrate [48]. All analyses were performed three times, and the data were expressed as the mean ± S.D.

In Vivo Assays
The experimental hyperlipidemia was reproduced using the recommendations of Cheng et al. [49] with modification. Fifty male Golden Syrian hamsters (weight of 70-72 g; BioNursery Stezar, Vladimir, Russia) were housed one per cage with a 12 h light/dark cycle (humidity of 50-55%), and regular rodent cholesterol-free chow (Asortiment-Agro Company, Sergiev Posad, Russia) and free access to food and water were provided. After two weeks of adaptation, the animals were weighed and divided into five groups (n = 10): (1) the normal diet group; (2) the group with a 1% cholesterol supplementation diet; (3) the group with a 1% cholesterol supplementation diet + simvastatin; (4) the group with a 1% cholesterol supplementation diet + DEAE-1% NaOH-f2 polysaccharide; (5) the group with a 1% cholesterol supplementation diet + with 1% cholesterol diet + DEAE-1% NaOH-f3 polysaccharide. The diets of all groups were switched to a high-fat diet except for the normal diet group over a 3-month period. The general composition of the high-fat diet was 10% lard, 10% yoke powder, 1% cholesterol, and 79% regular rodent cholesterol-free chow. For the next six months, the animals were orally supplemented with simvastatin (10 mg/kg/day; group 3), DEAE-1% NaOH-f2 polysaccharide (250 mg/kg/day; group 4), and DEAE-1% NaOH-f3 polysaccharide (250 mg/kg/day; group 5). Animals in groups 1 and 2 received 0.9% NaCl solution. The experimental procedure was authorized by the Institute of General and Experimental Biology's Ethical Committee (protocol No. LM-0324, 27 January 2012) before starting the study and was conducted under the internationally accepted principles for laboratory animal use and care. Serum concentrations of total cholesterol, triglycerides, high-density lipoprotein-cholesterol, and low-density lipoproteincholesterol were measured enzymatically using Sigma-Aldrich cholesterol assay kit (No. MAK436), serum triglyceride determination kit (No. TR0100), and HDL and LDL/VLDL quantitation kit (No. MAK045), and the malondialdehyde level was measured using a malondialdehyde colorimetric assay kit (Elabscience Biotechnology, Inc., Houston, TX, USA; No E-BC-K025-S). Liver superoxide dismutase, glutathione peroxidase, and catalase were determined using Sigma-Aldrich SOD assay kit (No. 19160), glutathione peroxidase assay kit (No. MAK437), and catalase assay kit (No. MAK100). All analyses were performed five times, and the data were expressed as mean values ± S.D.

Statistical and Multivariate Analysis
Statistical analyses were performed by one-way analysis of variance, and the significance of the mean difference was determined by Duncan's multiple range test. Differences at p < 0.05 were considered statistically significant. The results are presented as the mean ± S.D. The linear regression analysis and generation of calibration graphs were conducted using Advanced Grapher 2.2 (Alentum Software, Inc., Ramat-Gan, Israel).

Yield and Chemical Composition of V. vitis-idaea Press Cake Polysaccharides (VVPS) and DEAE-Cellulose Fractions
Hot water extraction is able to isolate polysaccharides from the press cake V. vitisidaea, which is also typical for other vacciniums such as blueberry, cranberry [25], and Manitoba lingonberry [29]. The total yield of V. vitis-idaea polysaccharide fraction (VVPS) after hot water extraction, ethanol precipitation, polyamide column dephenolization, cationexchange demineralization, two steps of deproteination, and dialysis was 3.7% of dry press cake weight ( Table 1). The total carbohydrate level in VVPS was measured to be 92.89%, which included uronic acids at 37.62% and starch at 2.04%. Non-carbohydrate constituents were proteins at 1.67%, phenolics at 2.96%, and ash at 1.56%. The positive test with Yariv reagent implied the presence of arabinogalactan-protein complexes (AGP) estimated as 0.29%. The early study of Vaccinium berry polysaccharide, indicating the presence of watersoluble polymers in V. ashei, yielded 2.7% of dry berry weight and contained 37% of uronic acids [50]. The purified water-soluble complex polysaccharide from the Northern manitoba lingonberry had 2.1% yield and showed 36% of total carbohydrates, 4.7% of proteins, and 3.5% of phenolics [29]. DEAE-cellulose separation of VVPS resulted in the preparation of six polymer fractions eluted sequentially with water, ammonium bicarbonate (0.1-1%), and sodium hydroxide (1%) ( Table 1). The neutral fraction DEAE-H 2 O that eluted first with water showed 7% yield (of VVPS weight), high starch content (25.36%), the highest content of proteins (3.85%), and AGP (3.44%) and no phenolics.

Elemental Composition
The basic elements of polysaccharides are carbon, hydrogen, and oxygen, and the general formula is C x H y O z . Fraction VVPS showed contents of C, H, and O at 39.08%, 6.14%, 54.52%, respectively, which is typical for carbohydrate polymers, and the composition of the DEAE-cellulose fractions varied in the ranges of 38.94-39.96% (C), 6.10-6.67% (H), and 52.81-54.57% (O) ( Table 2). Nitrogen content was at a zero level (DEAE-0.5% NH 4 HCO 3 , DEAE-0.5% NH 4 HCO 3 ), low (DEAE-0.3% NH 4 HCO 3 , DEAE-1% NaOH), or varied from 0.26% (VVPS) to 0.62% (DEAE-H 2 O), which agreed with the previous chemical composition data. To better visualize the elemental composition results, we used a Van Krevelen diagram [51] to plot the atomic O/C ratio as a function of atomic H/C ratio ( Figure 2).
Plant polysaccharides traditionally include hexoses, pentoses, hexuronic acids, and desoxyhexoses as structural blocks that are located in the Van Krevelen diagram in different places, creating a "monosaccharide triangle" reflecting extreme points of composition for the possible carbohydrate polymer. Because the monosaccharide composition of the polysaccharides varied in a wide range, the elemental composition data also varied. As shown, VVPS and DEAE-cellulose fractions were inside the triangle, whereas the DEAEcellulose fraction eluted with water, 0.1%, and 0.3% NH 4 HCO 3 were closer to the "neutral angle" of hexose (pentose), demonstrating low acidic monosaccharide content. The VVPS and DEAE-cellulose fractions eluted with 0.5% NH 4 HCO 3 and 1% NaOH were in a lower position, reflecting the presence of uronic acids. The DEAE-cellulose fraction eluted with 1% NH 4 HCO 3 was in the middle position away from the triangle side Hexose(Pentose)-Hexuronic Acid, which may be due to an increased content of desoxyhexoses.

Bioactivity of VVPS and DEAE-Cellulose Fractions of V. vitis-idaea Press Cake
Polysaccharides of various Vaccinium species are known antioxidants with a potency to inactivate free radicals [52], chelate ferrous ions [50], and bind bile acids [25], making them good antioxidative and lipid-lowering agents. The primary cause of antioxidant potential for plant polysaccharides is phenolic constituents covalently bonded to carbohydrate chains [55], while uronic aids are capable of binding metal ions, bile acids, and cholesterol [56], providing the acidic polysaccharides with bioactivity. Given the diverse chemical properties of V. vitis-idaea press cake polysaccharides, we assumed potency for both VVPS and DEAE-cellulose fractions due to the high level of phenolics and uronic acids.
Comparative data of VVPS activity and Trolox used as a reference antioxidant demonstrated the better potential of Trolox in scavenging DPPH • , ABTS +• , O 2 •− , OH • , Cl • , and H 2 O 2 inactivation, but the total polysaccharide fraction VVPS was an effective scavenger of NO molecules and chelator of Fe 2+ ions. Interestingly, three commercially available polysaccharides (i.e., pectin from citrus peel, starch, and arabinogalactan) were inactive in scavenging all free radicals, and only pectin demonstrated at least some activity in scavenging NO, H 2 O 2 inactivation, and good Fe 2+ chelation.
Analysis of DEAE-cellulose fractions showed DEAE-H 2 O, DEAE-0.1% NH 4 HCO 3 , and DEAE-0.3% NH 4 HCO 3 as inactive and DEAE-0.5% NH 4 HCO 3 and DEAE-1% NH 4 HCO 3 as poorly active. Only fraction DEAE-1% NaOH showed an antioxidant effect comparable to that of VVPS, which indicated the leading role of DEAE-1% NaOH in VVPS antioxidant activity. This fraction has the highest phenolic content, which may explain its excellent antioxidant activity.
Pectin and arabinogalactan demonstrated good fat binding potential with values of 186.85 and 156.14 g/100 g, respectively, and cholestyramine was inactive. A similar pattern was found for cholesterol binding by polysaccharides. Fractions VVPS and DEAE-1% NaOH were the most active (57.02 and 68.37 mg/g, respectively) but were less intensive binders than cholestyramine (93.11 mg/g). Inhibition of pancreatic lipase was detected only for VVPS and DEAE-1% NaOH polysaccharides that had IC 50 values of 6.24 and 5.33 mg/mL, respectively, exceeding the activity of cholestyramine (IC 50 14.02 mg/mL). Thus, polysaccharide fraction VVPS from V. vitis-idaea press cake and its active constituent DEAE-1% NaOH showed good in vitro hypolipidemic potential.
Previously, freeze-dried dietary berries showed in vitro bile acid binding with a value ranging from 0.43 µmol/100 g (cranberries, Vaccinium macrocarpon) to 0.73 µmol/100 g (blueberry, Vaccinium spp.) owing to the high polysaccharide content (particularly dietary fibers) [25]. The bile acid binding potentials of dietary fruits were from 0.21 µmol/100 g for nectarines (Prunus persica) to 0.90 µmol/100 g bananas (Musa paradisiaca) [61]. Known polysaccharides with bile acid binding activity were also isolated from Abelmoschus esculentus [62], Kadsura coccinea [63], and Laminaria japonica [64], and in all cases, the presence of uronic acids was determined as a basic principle for effect manifestation [65]. The lipid lowering effect of Inonotus obliquus and Volvariella volvacea polysaccharides was associated with their fat and cholesterol binding [66,67], providing the hypolipidemic activity of polymers. In addition, the pancreatic lipase inhibition plays an important role in the lipid lowering effect of plant food and polysaccharide-derived products. Pectic polysaccharides with varied molecular weight and methoxylation degree are effective pancreatic lipase inhibitors, which is related to a reduction in the surface of the lipid droplet exposed to the enzyme [68], but neutral polysaccharides are also good inhibitors as in the case of Dictyophora indusiata polymers [69]. Summarizing the data, the polysaccharide fraction VVPS of V. vitis-idaea press cake and its component DEAE-1% NaOH are possible effective antioxidants and potential hypolipidemic agents that need further separation for the isolation of homogenous polymers and their chemical investigation, followed by an in vivo study of bioactivity.

Compound (No. Figure 5) ESI-MS, [M + H] + , m/z ESI-MS/MS, m/z
mpound Figure 5) Interestingly, the degradation polymers DEAE-1% NaOH-f1-d and -f2-d formed after elimination of arabinose from DEAE-1% NaOH-f1 and -f2, showed no hydroxycinnamates after alkaline cleavage and likely only occurred if phenolic acids were linked with arabinose residues. This is not unusual because feruloylated arabinose oligomer chains were previously found in the pectin fraction of spinach [77], diferuloyl fragments were found in Zea mays cell walls [78,79], and sinapoylated polysaccharides were b c
Interestingly, the degradation polymers DEAE-1% NaOH-f1-d and -f2-d formed after elimination of arabinose from DEAE-1% NaOH-f1 and -f2, showed no hydroxycinnamates after alkaline cleavage and likely only occurred if phenolic acids were linked with arabinose residues. This is not unusual because feruloylated arabinose oligomer chains were previously found in the pectin fraction of spinach [77], diferuloyl fragments were found in Zea mays cell walls [78,79], and sinapoylated polysaccharides were detected in radish seedlings [80]. Acidic polymers DEAE-1% NaOH-f3 and -f4 released no phenolics after alkaline treatment.
The obtained data showed that homogenic components of the bioactive polysaccharide fraction of V. vitis-idaea press cake include four polymers, two of which are polyphenolpolysaccharide conjugates as neutral arabino-3,6-galactans esterified by hydroxycinnamoyl fragments and the other two are pectic-like polysaccharides. Although studies have been conducted on the primary structure of polymers, additional spectral studies need to identify the fine structure of polysaccharides isolated from V. vitis-idaea. Looking back at previous information about Vaccinium berry polysaccharides, only bilberry (V. myrtillus) [53] and rabbiteye blueberry (V. ashei) [50] have been mentioned as a source of bioactive polymers of pectic nature without fine structure determination; thus, it is still too early to speculate about genus features in the polysaccharide composition.
Investigation of the in vitro hypolipidemic activity demonstrated higher bile acid binding potentials for DEAE-1% NaOH-f1 and -f2 polymers of 7.83 and 8.26 µmol/100 g, respectively, vs. 1.85 µmol/100 g for DEAE-1% NaOH-f3 and 1.90 µmol/100 g for DEAE-1% NaOH-f4 (Table 5). Both active polymers were also inhibitors of pancreatic lipase with IC 50 values of 4.27 and 3.86 mg/mL for DEAE-1% NaOH-f1 and -f2, respectively, whereas DEAE-1% NaOH-f3 and -f4 were inactive. Acidic polysaccharides DEAE-1% NaOH-f3 and -f4 showed fat binding potentials (308.75 and 315.61 g/100 g, respectively), and the cholesterol binding activities of the four homogenic polymers were similar in the range of 59.27-73.92 mg/g. These results mean that the four components of active hypolipidemic fraction DEAE-1% NaOH also have potential to bind bile acids, fat, and cholesterol and inhibit pancreatic lipase, each in its own way. The polyphenol-polysaccharide conjugates showed good bile acid binding and inhibition of pancreatic lipase; however, the acidic nonphenolized polysaccharides were binders of fat, whereas the cholesterol binding activity was at a similar level. The removal of phenolic fragments from DEAE-1% NaOH-f1 and -f2 resulted in significant reduction in the in vitro hypolipidemic activity of the DEAE-1% NaOH-f1-d and -f2-d polymers (Table S2), which indicates the importance of phenolics as active sites of the neutral arabinogalactans.
To progress from in vitro to in vivo experiments, we chose polymer DEAE-1% NaOH-f2 as an example of high-yielded polyphenol-polysaccharide conjugates and DEAE-1% NaOH-f3 as high-yielded non-phenolized pectic polysaccharide from V. vitis-idaea press cake to treat experimental animals on the standard and high-fat diet ( Figure 6). The reference substance was simvastatin, a known hypolipidemic drug, at a dose of 10 mg/kg/day [81]. The high-fat diet with 1% cholesterol resulted in animal body weight gain from 81 ± 4 g at the beginning of the test to a final weight of 195 ± 10 g (vs. 80 ± 4 g → 141 ± 7 g in the standard diet group). The changes in serum lipid profile involved a reduced level of cholesterol (6.29 mmol/L vs. This was an indication that the high-fat diet with 1% cholesterol leads to hyperlipidemia associated with antioxidant misbalance. Application of simvastatin lowered the animals' body weights and reduced serum lipid markers to sustainable levels close to those of the standard diet group, but the antioxidant effect was medium, which was not unexpected and has been previously demonstrated [82]. Both polysaccharides of V. vitis-idaea press cake DEAE-1% NaOH-f2 and -f3 demonstrated similar serum lipid lowering effects in the high-fat-diet animals against cholesterol (decreased by 33-38%), triglycerides (decreased by 29-35%), low-density lipoprotein-cholesterol (decreased by 48-53%), and high-density lipoprotein-cholesterol (decreased by 9-18%). This contrasted with the antioxidant potential when polymer DEAE-1% NaOH-f2 was more active than DEAE-1% NaOH-f3. The serum MDA value in the DEAE-1% NaOH-f2 group was 63% lower than in the high-fat diet group, and in the DEAE-1% NaOH-f3 group, we found a 36% reduction. The levels of SOD, GPX, and catalase after application of DEAE-1% NaOH-f2 increased by 60.9%, 186%, and 233%, respectively, compared with those in the high-fat diet group, while DEAE-1% NaOH-f3 resulted in 15%, 37%, and 128% boosts of enzymatic activity, respectively. This means that polysaccharides and polyphenol-polysaccharide conjugates of V. vitis-idaea are capable of being antioxidant and hypolipidemic agents in both in vitro and in vivo assays. (42 U/mg protein vs. 121 U/mg protein in the standard diet group). This was an indication that the high-fat diet with 1% cholesterol leads to hyperlipidemia associated with antioxidant misbalance. Application of simvastatin lowered the animals' body weights and reduced serum lipid markers to sustainable levels close to those of the standard diet group, but the antioxidant effect was medium, which was not unexpected and has been previously demonstrated [82]. Both polysaccharides of V. vitis-idaea press cake DEAE-1% NaOH-f2 and -f3 demonstrated similar serum lipid lowering effects in the high-fat-diet animals against cholesterol (decreased by 33-38%), triglycerides (decreased by 29-35%), low-density lipoprotein-cholesterol (decreased by 48-53%), and high-density lipoprotein-cholesterol (decreased by 9-18%). This contrasted with the antioxidant potential when polymer DEAE-1% NaOH-f2 was more active than DEAE-1% NaOH-f3. The serum MDA value in the DEAE-1% NaOH-f2 group was 63% lower than in the high-fat diet group, and in the DEAE-1% NaOH-f3 group, we found a 36% reduction. The levels of SOD, GPX, and catalase after application of DEAE-1% NaOH-f2 increased by 60.9%, 186%, and 233%, respectively, compared with those in the high-fat diet group, while DEAE-1% NaOH-f3 resulted in 15%, 37%, and 128% boosts of enzymatic activity, respectively. This means that polysaccharides and polyphenol-polysaccharide conjugates of V. vitis-idaea are capable of being antioxidant and hypolipidemic agents in both in vitro and in vivo assays. serum malondialdehyde level (MDA) (f), liver superoxide dismutase (SOD) (g), liver glutathione peroxidase (GPX) (h) and liver catalase (i) in hamsters with a normal diet (N), with a 1% cholesterol diet (C), with a 1% cholesterol diet + simvastatin (10 mg/kg/day; S), with a 1% cholesterol diet + DEAE-1% NaOH-f2 polysaccharide (250 mg/kg/day; F2), with a 1% cholesterol diet + DEAE-1% NaOH-f3 polysaccharide (250 mg/kg/day; F3) before the feeding period (-0) and after a 6-month feeding period (-6). i-p < 0.05 vs. 1% cholesterol diet group (C-group); ii-p < 0.05 vs. 1% cholesterol diet + simvastatin group (S-group).
Polysaccharides of dietary origin are known hypolipidemic agents that normalize the lipid profile and antioxidant level of high-fat-diet animals [83]. The sources of bioactive polymers are fruits of pumpkin (Cucurbita pepo, C. moschata) [84,85], Chinese wolfberry (Lycium barbarum) [86], jujube or red date (Ziziphus jujuba) [87], Cherokee rose (Rosa laevigata) [32], and Japanese cornel (Cornus officinalis) [88]. In most cases, polysaccharides were polygalacturonates that reduced serum parameters (such as total cholesterol, high/low density lipoprotein cholesterol, and triglycerides) by inhibitory effects on the absorption of the bile acids and cholesterol [89], inhibition of the lipase activity [90], fat and cholesterol binding capacity and reduction in the accumulation of lipids and fecal fat and cholesterol contents [91], modulation of the gene expression of fatty acid synthesis [92], and increased formation of short chain fatty acids in the feces and regulation of lipid metabolism pathways [93]. At the same time, the regulation of observed antioxidant status and improvement of the oxidative stress [55] involves scavenging of free radicals and reduction in liver enzymes [88].
Antioxidant and hypolipidemic potential of polysaccharides and polyphenol-polysaccharide conjugates are markedly linked with structural specifics as molecular weight, elemental and monosaccharide composition, glycosidic linkage, and nature of conjugated polyphenolics [55,83]. The value of phenolic content in carbohydrate polymers is a crucial marker of radical-scavenging ability, nitric oxide (II) and hydrogen peroxide inactivating potential [94], as well as pancreatic lipase inhibition [95]. Additionally, high uronic content is an important factor of metal-chelating activity [96] and binding of bile, fat and cholesterol [83]. The homogenic water-soluble polymers of V. vitis-idaea press cake characterized by a high phenolics (polyphenol-polysaccharide conjugates DEAE-1% NaOH-f1 and DEAE-1% NaOH-f2) and uronic content (polysaccharides DEAE-1% NaOH-f3 and DEAE-1% NaOH-f4) and, therefore, the antioxidant and hypolipidemic activity of homogenic polymers, were caused by the various structural factors. Finally, considering the everincreasing volumes of waste production by lingonberry processing factories, the press cake of V. vitis-idaea can become a promising feedstock for bioactive polymers manufacturing.

Conclusions
This is the first report of lingonberry (Vaccinium vitis-idaea L.) fruit press cake polymeric compounds characterization. The results of our study indicate the heterogeneity of V. vitisidaea polysaccharides with a dominance of acidic polymers and polyphenol-polysaccharide conjugates which were neutral arabinogalactans esterified with hydroxycinnamates. This series of in vitro and in vivo studies suggest that polysaccharides normalize the lipid profile and antioxidant status of high-fat-diet hamsters. These findings support the idea of practical use of wastes from food processing as a source for antioxidant and hypolipidemic agent manufacturing in the pharmaceutical industry.