*4.1. Polysaccharides*

Algae: The following species of red algae were collected at The Peter the Great Bay, Japan Sea, which is near the border between the boreal and tropical zones: *Chondrus armatus* (Gigartinales, Gigartinaceae) and *Tichocarpus crinitus* (S.G. Gmelin) (Gigartinales, Tichocarpoaceae). All algae were harvested at the end of August and identified by Prof. E. Titlynov and T. Titlynova (National Scientific Center of Marine Biology, Far-Eastern Branch of the Russian Academy of Sciences). The selected

seaweeds were in the vegetative form lacking any reproductive organs. The algae were washed with tap water to remove excess of salt. Bleaching of the seaweed was by maintaining the specimen in pure acetone for 3 days prior being dried in the air.

Extraction of CRGs: Dried and milled algae (50 g) were suspended in hot water (1.5 h) and the polysaccharides were extracted at 90 ◦C for 2 h in a water bath. The residue was removed by centrifugation and supernatant poured into ethanol (3 volumes), yielding the crude extract (unfractionated) of polysaccharides. The crude extracts were purified by redissolving in water, concentrated, dialyzed, and freeze-dried, yielding Σ-CRG. Then, the polysaccharides were separated into gelling KCl-insoluble and non-gelling KC1-soluble fractions as described previously [34] and their structures were established according to the published protocol. λ- and k-CRGs from *C. armatus* and κ/β-CRG from *T. crinitus* were obtained. The biologically active food supplement Carrageenan-FE (Pacific Institute of Bioorganic Chemistry Far East Branch of the Russian Academy of Sciences) is composed of κ- and λ-CRGs from *C. armatus* at a ratio 3:1 (*v*/*v*) and is labeled as Σ-CRG. The endotoxin admixture in samples of CRGs was determined by the fluorogenic endotoxin detection assay PyroGene rFC purchased from Lonza (USA) in accordance with test instruction. The level of environmental endotoxin was low: 0.60 EU mL−<sup>1</sup> in doses of 100 mg/mL.

Commercial LPS from *Escherichia coli* 055:B5 (Cat No: L2880, Lot No: 102M4017V, Sigma, St. Louis, MO, USA) was used in the study.

Molecular Weight Estimation: Viscosimetric molecular weight of the polysaccharide sample was calculated using the Mark–Kuhn–Houwink equation: |η| = KmM<sup>α</sup>, where |η| is the intrinsic viscosity and Km and α are empirical constants for carrageenan constituting 3 × 10−<sup>3</sup> and 0.95 at 25 ◦C in 0.1 M NaCl, respectively, according to the literature data for this polymer–solvent system [36]. The viscosity of polysaccharide solution (1–2 mg/mL in 0.1 M NaCl) was measured with a modified Ubbellohde viscometer (Design Bureau Puschino, Russia) with a capillary diameter of 0.3 mm at 25 ◦C, the time of accuracy being within ±0.1 s. The intrinsic viscosity of the CRGs sample was calculated by the extrapolation of the dependence ln (η)rel/C to infinite dilution using the least squares method.

#### *4.2. Atomic Force Microscopy (AFM)*

LPS was dissolved in distilled de-ionized water at concentration of 0.05 mg/mL; CRG samples were at concentrations of 0.1 mg/mL. The LPS–CRG mixture were prepared at the same concentrations. The CRG solution was mixed with LPS solution (2:1 *w*/*w*). Aliquots (12 μL) of the aqueous solutions of complex and their initial component were deposited onto freshly cleaved mica and dried at 37 ◦C for 24 h or at 70 ◦C for 30 s (for LPS). The morphology of CRG, LPS, and their mixtures was studied in air by AFM (Solver P47) in the tapping contact mode using a tip with the radius of 10 nm.

#### *4.3. IL-10 and TNF-*α *Inducing Activity on the Human Blood Cells*

The blood processing was performed using procedure of De Groote et al. Peripheral blood was collected by vena puncture into sterile siliconized tubes containing 30 IU of lithium heparinate per 5 mL tube diluted 1:5 in sterile Medium 199 (Sigma-Aldrich, Saint Louis, MO, USA) containing 300 mg/<sup>L</sup> of glutamine (Gibco, Life Technology, Darmstadt, Germany) and 50 μg/mL of gentamicin. Diluted blood (0.1 mL) was transferred into sterile polypropylene plates and then incubated with the LPS, carrageenans, or with LPS and carrageenan (370 ◦C, 5% CO2). After 24 h, the supernatants were collected and frozen followed by cytokine determination using a specific ELISA kit ("Cytokine", Saint-Petersburg, Russia). The study protocol was approved by the medical ethics committee of the local hospital (Vladivostok, Russia). Informed consent was obtained from all subjects who participated in the study. All donors were free of medicine administration for 14 days prior to blood sampling. Blood was drawn from the antecubital vein of normal healthy human volunteers and anticoagulated in plastic tubes (Greiner Bio-One International AG, Kremsmuenster, Austria) with 30 IU lithium heparinate used as an anticoagulant.

#### *4.4. Animals and Diets*

The work was carried out in accordance with "Directives 2010/63/EU of the European Parliament and the Council of the European Union for the Protection of Animals used for Scientific Goals" and approved by the Federal Scientific Center of biodiversity FEB RAS Animal Care and Use Committee.

Mature male CD-1 mice were obtained from G.B. Elyakov Pacific Institute of Bioorganic Chemistry, FAR RAS (Vladivostok, Russia). The mice with body mass of 22–24 g kept in standard conditions of the vivarium at a controlled temperature 20–22 ◦C and ambient humidity 60–65%. Light were maintained on an artificial 12 h light–dark cycle. Each experimental group consisted of eight animals, each mouse in the cage had a floor area of 70 cm2, which corresponds to international standards. The mice were provided with water standard feed compliant GOST R 50258-92 (CJSC ProKorm, Russia) ad libitum.

## *4.5. Experimental Design*

This part of the study consisted of estimating the influence of di fferent types of carrageenans on the physiological state of mice intoxicated *E. coli* LPS.

The mice were randomly allocated into six groups. All animals except the control group were given intraperitoneally LPS solution in dose 1 mg/kg body mass (0.1 mg/mL, pH 7.0) for five days. The control group received intraperitoneally 0.2 mL saline. The mice of the control and LPS groups were given only standard feed, whereas mice of other groups daily administrated CRGs suspensions in dose 100 mg/kg body mass (4 mg/mL, pH 7.0) 1 h before LPS injection through gastric gavage. At the end of experiment, mice were killed by decapitation, and their inner organs were removed and weighed. The blood samples were centrifuged at 1.200× *g* for 15 min. Relative organ masses of liver, thymus, thyroid, and adrenals were calculated as organ mass (mg)/body mass (g). Serum corticosterone concentrations were determined by the fluorometric method [57]. The glycogen content in liver was estimated with the anthrone reagen<sup>t</sup> [58]. The adenosine triphosphate (ATP) and lactate levels in liver were measured enzymatic spectrophotometric methods used test-system with nicotinamide coenzymes NADF and NAD•H, respectively [59,60]. Statistical analysis was performed using Student's t-criterion for unpaired data, and p-values of less than 0.05 were considered significant. All data presented as mean ± standard deviation.

#### *4.6. Medico-Biological Study of Food Supplement Carrageenan-FE*

Ethical approval: The medico-biological study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). Informed consent was obtained from all subjects who participated in the study.

The medical-clinical study of food supplementation Carrageenan-FE was carried out on the basis of the permission of the State Committee for Standardization of the Russian Federation (No.035/002158) and the consent of the ethics committee of the Central Research Institute of Epidemiology of the Federal Service on Customers Rights protection and human Well-being Surveillance (Moscow, Russia).

The food supplement Carrageenan-FE (Pacific Institute of Bioorganic chemistry, Far East Branch of the Russian Academy of Sciences, Vladivostok, Russia) is composed of two structural types of CRG. The supplement meets the requirements for food supplements and is recommended as an additional source of food fiber.

Patients diagnosed with enteric infection diseases and healthy volunteers were selected for current clinical trial based on an analysis out-patient medical records obtained from Ethical Committee in the framework of the Central Research Institute of Epidemiology of the Federal Service on Customers Rights Protection and Human Well-being Surveillance. After the patients were selected, they were enrolled in the current clinical trial to test the e ffects of the CRG-food supplement. The study of the therapeutic e ffect of CRG-FE in patients with enteric infection diseases with *Salmonella* etiology was carried out at a clinical infectious hospital (Moscow, Russia).

Dynamic examination of 42 patients (men and women, average age 31 years) with severe intoxication syndrome was conducted. Patients came to the hospital for 1–3 days with signs of intoxication: body temperature of up to 38–39 ◦C, chills, headache, frequent (10–15 times per day) loose stools, nausea, and repeated vomiting. Patients (22) received per os 150 mg of Carrageenan-FE administered together with 150 mL of Ringer solution to three times per day. In the second group, patients received 150 mL of the same solution (standard therapy). In the control group were healthy patients. The results were evaluated by clinical data and research parameters of hemostasis and immunological status. Hemostasis was characterized by the following parameters: prothrombin time, thrombin time, activated partial thromboplastin time, fibrinogen level, and platelet aggregation activity. To assess the state of immune system, relative and absolute number of lymphocytes, T lymphocytes (CD3+), and B-lymphocytes (CD19+), as well as immunoregulatory subpopulation of T-cells helpers (CD4+) and T cytotoxic lymphocytes (CD8+) were calculated. Investigations were carried out by flower cytometer EPICS XL (Beckman Coulter) using monoclonal antibodies IO-Test (double label). Functional activity of venous blood neutrophils was determined via spontaneous and stimulated with the test NBT (NBT-test) and expressed as conventional units.
