Handbook of Polymers for Pharmaceutical Technologies, Biodegradable Polymers

By Vijay Kumar Thakur and Manju Kumari Thakur

Polymers are one of the most fascinating materials of the present era finding their applications in almost every aspects of life. Polymers are either directly available in nature or are chemically synthesized and used depending upon the targeted applications.Advances in polymer science and the introduction of new polymers have resulted in the significant development of polymers with unique properties.  Different kinds of polymers have been and will be one of the key in several applications in many of the advanced pharmaceutical research being carried out over the globe.

This 4-partset of books contains precisely referenced chapters, emphasizing different kinds of polymers with basic fundamentals and practicality for application in diverse pharmaceutical technologies. The volumes aim at explaining basics of polymers based materials from different resources and their chemistry along with practical applications which present a future direction in the pharmaceutical industry. Each volume offer deep insight into the subject being treated.                                     

Volume 1: Structure and Chemistry
Volume 2: Processing and Applications
Volume 3: Biodegradable Polymers
Volume 4: Bioactive and Compatible Synthetic/Hybrid Polymers

• Language: English
• Publisher: Wiley
• Release date: Sep 23, 2015
• ISBN: 9781119041436

Book preview

Chapter 1

Bioactive Polysaccharides of Vegetable and Microbial Origins: An Overview

Giuseppina Tommonaro*,¹, Annarita Poli¹, Paola Di Donato¹,², Gennaro Roberto Abbamondi, Ilaria Finore¹ and Barbara Nicolaus¹

¹National Council of Research of Italy, Institute of Biomolecular Chemistry, Pozzuoli (NA), Italy

²University of Napoli Parthenope, Department of Sciences and Technologies, Napoli, Italy

*Corresponding author: [e-mail]

Abstract

Natural products play a dominant role in the discovery of leads to develop drugs for the treatment of human diseases. In recent years, some bioactive polysaccharides isolated from natural sources have attracted much attention in the field of biochemistry and pharmacology because of their biological activities as anticarcinogenic, anti-inflammatory, immunostimulating, antioxidant agents, etc. The high potential for some of these compounds suggested that they could be developed as drugs. This chapter presents the most relevant findings on the latest research concerning bioactive polysaccharides isolated from vegetables and microbial sources.

Keywords: Exopolysaccharides, antioxidant, anti-inflammatory, bioplastic, microbial source, plants

1.1 Introduction

The bioactive compounds that are synthesized in nature, in order to protect a living organism, have been selected from a wide variety of possibilities until reaching optimal activity after several hundreds of million years. The high potential for some of these products suggested that they could play a dominant role in the discovery of lead compounds for the development of drugs for the treatment of human desease. Recently, some bioactive polysaccharides isolated from natural sources have attracted much attention in the field of biochemistry and pharmacology: polysaccharides or their glycoconjugates were shown to exhibit multiple biological activities, including anticarcinogenic, anticoagulant, immunostimulating, antioxidant, etc.

Nowadays, the increased demand for the exploration and use of natural sources for white biotechnology processes has led to a renewed interest in biopolymers, in particular, in polysaccharides both of vegetable and microbial origins. Polysaccharides are naturally occurring polymers of aldoses and/or ketoses connected together through glycosidic linkages. They are essential constituents of all living organisms and are associated with a variety of vital functions which sustain life. These biopolymers possess complex structures because there are many types of inter-sugar linkages involving different monosaccharide residues. In addition, they can form secondary structures which depend on the conformation of component sugars, molecular weight, inter- and intrachain hydrogen bondings. On the basis of structural criteria, it is possible to distinguish homoglycans and heteroglycans, if they are made up by the same type or by two or more types of monomer units; linear and branched polymers, with different degrees of branching; neutral or charged (cationic or anionic). Moreover, on the basis of their biological role, polysaccharide from vegetables can also be distinguished in structural elements, such as cellulose and xylans, and in energy-reserve polysaccharides such as starch and fructans. In the case of polysaccharides produced by microorganisms, they can be classified into three main groups according to their location in the cell: cytosolic polysaccharides, which provide a carbon and energy source for the cells; polysaccharides that make up the cell walls, including peptidoglycans, techoid acids and lipopolysaccharides, and polysaccharides that are exuded into the extracellular environment in the form of capsules or slime, known as exopolysaccharides (EPSs). Since the latter are completely excreted into the environment, they can be easily collected by cell culture media precipitation by cold ethanol after removal of cells [1]. The elucidation of the polysaccharide structures are very important to clarify the physicochemical and biological properties of these biopolymers and to attribute, and in some cases predict, their biotechnological applications. Several chemical and physical techniques are used to determine the primary structure of these molecules: chemical degradation and derivatization, combined with chromatographic methods and mass spectrometry analysis, are used to determine the sugar composition, their absolute configuration and the presence and the position of possible substituents [2].

Since polysaccharides are biodegradable materials expressing biocompatibility, they could act as versatile tools for applications in biomedical fields such as drug delivery, tissue engineering, bioadhesives, prostheses and medical devices [3–7]. These polymers present several derivable groups on molecular chains that make polysaccharides a good substrate for chemical modification, such as acetylation, sulphation, silanation or oxidation, producing many kinds of polysaccharide derivatives with additional and different properties and bioactivities. The carboxymethyl pullulan conjugated with heparin represents an example of chemical modification for tissue engineering applications. Moreover, considering the presence of hydrophobic moieties in the chain of polysaccharide, the formation of self-assembled micelles can be possible, making natural EPSs like pullulan, dextran, levan or bacterial cellulose ideal candidates for drug solubility and stability [6,8,9].

Bacterial polysaccharides present a real potential in cell therapy and tissue engineering with the advantage, over the polysaccharides from eukaryotes, that they can be totally produced under controlled conditions in bioreactors. Polysaccharides synthesized by microorganisms suggest unique properties and advantages in their exploration and are an attractive alternative of plant, algal and synthetic polysaccharides. They represent a fast renewable resource that could partially compensate the restricted mass of plant polysaccharides. Their production is a matter of days, while plants’ life cycles last for months or years, being that the production cycle is usually seasonal. Microbial polysaccharides are produced by a wide variety of microorganisms from both eukaryotic and prokaryotic groups, including cyanobacteria [10], lactic acid bacteria [11,12], and halophilic bacteria [13–16]. Other microorganisms such as yeast [17] and marine microalga [18,19] have been studied for EPS synthesis. The market price also depends on the infrastructures required for production, which can include bioreactors and maintaining asepsis [20]. The inherent costs of large-scale fermenters are significantly higher in comparison with chemical extraction processes for plant polysaccharides. Recently, the use of cheaper raw materials like agricultural waste or dairy waste has helped to reduce the cost of fermentative production [21–23].

The overall objective of this chapter is to provide information on these important biopolymers regarding applications in the field of medical industries for their pharmacological activities, including anticarcinogenic, anticoagulant, immunostimulating and antioxidant.

1.2 Anticarcinogenic Polysaccharides

Cancer is a leading cause of death in industrialized countries [24]. Although the mortality share has decreased in the last years, owing to the efforts that have been made in the search for new anticancer drugs and earlier detection, most cancers remain incurable. Chemoprevention represents a strategy used to decrease the incidence of cancer diseases in humans by inhibition of initiation step and spread of carcinogenesis and by improvement of lifestyle [25,26]. Many factors are involved in increasing the risk of cancer, including diet, exposure to radiation, environmental pollutants and tobacco use [27]. Cancer, a malignant neoplasm, is a kind of disease resulting from several causes [28]. Among these, mutations and epigenetic alterations of cancer genes promote the malignant transformation of cancer progenitor cells by disrupting key processes involved in normal growth control and tissue homeostasis [29].

Natural products play a dominant role in the discovery of lead compounds for the development of drugs to treat human diseases, including cancer, because of the variety of their chemical structures and biological activities [30]. Among natural products, polysaccharides also find their application as antitumor compounds (Table 1.1).

Table 1.1 Anticarcinogenic polysaccharides.

1.2.1 Microbial Sources

An active polysaccharide, named marinactan, was purified from the marine bacterium Flavobacterium uliginosum. Marinactan, a heteroglycan consisting of glucose, mannose and fucose (7:2:1 molar ratio), showed 70–90% inhibition of the growth of solid sarcome 180 in mice. Complete regression of the tumor was observed in some treated mice. Moreover, marinactan prolonged the survival period of mice bearing ascites sarcoma180 [31]. Previous papers described the antitumor activity of polysaccharides isolated from other microorganisms such as, for example, the β-(1→;3)-D-glucan, produced by Alcaligenes fecaelis var. myxogenes that showed a remarkable antitumor effect against sarcoma 180 solid tumor, with doses of 5 to 50 mg/Kg i.p. given once a day for 10 days [32]. Schizophyllan, a polymer isolated from the culture filtrate of Schizophyllum commune, was chemically characterized and showed to be formed by repeating units composed of three or four β-(1→;3)-linked D-glucopyranose residues to one of which is attached, through β-(1→;6)-linkage, a side chain consisting of a single β-D-glucopyranose residue. It was tested against four kinds of transplantable tumors in both ascites and solid forms. The most significant results were obtained with 0.5–10 mg/kg doses of schizophyllan on all the subcutaneously implanted tumors, i.e., sarcoma-37, sarcoma-180, Ehrlich carcinoma, and Yoshida sarcoma, accompanied by complete regressions. The treatment failed to inhibit the growth of ascites tumors or to induce prolongation of life span, with the exception of ascites sarcoma-180, moreover no inhibitory effect was observed also on Friend virus disease and spontaneous mammary carcinoma arising in Swiss mice. The mechanism of this action was considered to be host-mediated on the basis of lack of effect in in-vitro contact test [33]. A lipopolysaccharide (serratigen) and a polysaccharide (serratimannan), isolated from Serratia marcescens, red strain No. 51, were assayed for their antitumor activity against solid tumor of sarcoma-180 using ICR mice. Serratimannan showed 63% tumor inhibition and serratigen 38%, at a dose of 150 mg/kg [34].

Recently it has been reported the antitumor activity through Toll-like receptor 4 (TLR-4) of xanthan gum (XG), a complex polysaccharide produced by plant-pathogenic bacterium Xanthomonas campestris pv. Results showed that in-vitro culture with XG induced interleukin-12 (IL-12p40) and tumor necrosis factor-alpha (TNF-α) production from murine macrophages J744.1 and RAW264.7. Moreover, XG stimulated macrophages in a MyD88 mice-dependent manner and was mainly recognized by TLR-4. Oral administration of XG significantly retarded tumor growth and prolonged survival of the mice inoculated subcutaneously with B16Kb melanoma cells. The in-vivo antitumor effects of XG were also dependent on TLR-4, likewise in C3/HeJ mice, which lack TLR-4 signaling, where XG exhibited no effect on the growth of syngeneic bladder tumor, MBT-2. Results suggested that oral administration of XG could be beneficial against cancer diseases [35].

Bacteria can produce exopolysaccharides, secreting them in the surronding medium (released exopolysaccharides, r-EPS) or they can be attached to the bacterial surface (cell-bond exopolysaccharides, c-EPS). A c-EPS was isolated from the supernatant of Lactobacillus plantarum 70810. The chemical characterization revealed that it was a galactan containing a backbone of a-D-(1-→;6)-linked galactosyl, β-D-(1-→;4)-linked galactosyl, β-D-(1-→;2,3)-linked galactosyl residues and a tail end of β-D-(1-→;)-linked galactosyl residues. The c-EPS was assayed for its inhibitory effect on the proliferation of HepG-2, BGC-823 and HT-29 human cancer cell lines. Results indicated moderate antitumor activity against HepG-2 cells (56,34±1.07% of inhibition, 600 mg/mL), whereas a significant inhibitory effect was observed on BCG-823 and HT-29 (61.57±2.07% and 88.34±1.97%, respectively) [36]. Wang et al. also reported the isolation and bioactivity of two exopolysaccharides (r-EPS1 and r-EPS2) released from Lactobacillus plantarum 70810. Results showed that both r-EPSs exhibited antiproliferative effects against the human tumor cell lines Caco-2, BGC-823 and HT-29. The r-EPS2 possessed higher growth inhibition effects on the cancer cell lines used than r-EPS1. The reason could be due to the presence of sulfated group and beta glycosidic bond composition in r-EPS2 [37].

1.2.2 Vegetable Sources

Polysaccharides of vegetable origin have emerged as an important class of bioactive compounds because of their multiple biological properties, including anti-neoplastic effects.

Chemopreventive effects of plant polysaccharides (Aloe barbadensis Miller APS, Lentinus edodes LPS, Ganoderma lucidum GPS and Coriolus versicolor CPS) were evaluated using different biomarkers involved in chemical carcinogenesis. Biomarkers used for the initiation stage of cancer were: a) DNA adduct formation (B[a]P-DNA adducts); b) 8-hydroxydeoxyguanosine (8-OH-dG), representing oxidative DNA damage; and c) induction of glutathione S-transferase (GST) activity. Biomarkers for the promotion stage of cancer were: a) phorbol myristic acetate (PMA)-induced tyrosine kinase (TK) activity increase in human leukemia cells (HL-60); b) PMA-induced ornithine decarboxylase (ODC) activity elevation in Balb/3T3 cells; and c) free radical formation in PMA-induced HL-60 cells. APS was the most active in inhibition of B[a]P binding to DNA in mouse liver cells and it significantly decreased the oxidative DNA damage. CPS also was active in the reduction of oxidative DNA damage. GPS was found to be the most effective in the induction of glutathione S-transferase. APS inhibited either phorbol myristic acetate (PMA)-induced ornithine decarboxylase activity in Balb/3T3 cells and PMA-induced tyrosine kinase activity in human leukemic cells. Therefore, plant polysaccharides could be considered as novel agents in the prevention and promotion of cancer diseases [38].

In a recent review, Cao has reported several bioactivities (immunostimulating, antidiabetic, antioxidant, antitumor, and others) of tea (Camelia sinensis) extracts [39]. A polysaccharide (TSPS) isolated from water extract of tea seeds consisted of rhamnose, xylose, arabinose, glucose and galactose, GalA, GulA (4.9:1.7:11.1:27.2:14.0:3.4:1, molar ratio) showed the inhibition (38.44%) of the growth of K562 cells (human myelogenous leukemia) at a concentration of 50 microg/mL [40]. From green tea a glycan was also isolated with an average molecular weight of 8.3x10⁵ Da, containing rhamnose, arabinose, xylose, mannose, galactose, and glucose (1.06:2.31:5.17:0.91:3.06:4.24, molar ratio). It exhibited a weak concentration-dependent antitumor activity against the SKOV-3 cells (human adenocarcinoma). Chen and coworkers investigated the influence of tea carbohydrates on biochemical parameters in hepatocellular carcinoma induced in animals. Their results showed that the tea carbohydrates could inhibit tumor growth and decreased microvessel density in tumor tissue [41]. There was an interesting study by He et al. concerning the inhibitory effect of selenium-containing tea polysaccharides (Se-GTPs) extracted from a Chinese variety of tea, Ziyang green tea, against human MCF-7 breast cancer cells. Se-GTPs induced a concentration-dependent inhibition of cell growth with an IC50 of 140.1 microg/mL by inducing MCF-7 cancer cells to undergo G2/M phase arrest and apoptosis [42].

The bioactive polysaccharide LBP-1 from Lilii bulbus, was isolated by hot water extraction, ethanol precipitation and lyophilization. It was a glucan having an average molecular weight of 30.5 KDa. It inhibited the growth of Lewis lung carcinoma in mice after its intraperitoneal administration at doses of 50–200 mg/Kg. Furthermore, it significantly increased the production of serum cytokines, macrophage phagocytosis and splenocytes proliferation in mice, suggesting an antitumor activity through an immunomodulatory effect [43].

Two acidic polysaccharide fractions were isolated from the hot water extract of Cymbopogon citratus, a plant commonly known as lemongrass. The two polysaccharides were tested for their ability to inhibit the growth of human cancer cell lines Siha (cervix carcinoma) and LNCap (prostate carcinoma) by MTT citotoxicity assay. Results showed that the cytotoxic effect occurred in a time- and concentration-dependent manner. It is noteworthy that the mortality of cancer cells increased while prolonging the incubation time as well as the polysaccharide concentration. Further analysis explained a possible mechanism of action of polysaccharide fractions. In fact, an apoptotic process was observed in treated cells when compared to the untreated cells. This result was confirmed by the analysis of mitochondrial potential (Ψm). In treated cells, there was a loss of mitochondrial potential (Ψm) due to mitochondrial depolarization, which is considered as the initial and irreversible step of apoptosis [44]. The apoptotic cascade activation was also reported in another paper in which it has been demonstrated that the polysaccharide fraction CP-1 isolated from Coix lachryma-jobi L. seeds induced the apoptosis of A549 cells (human non-small cell lung cancer) in a concentration-dependent manner. CP-1 inhibited the proliferation of cancer cells in a time- and concentration-dependent manner, observing a cell viability of 64.23% at a concentration of 300 mg/mL for 72 h. Further analysis showed a cell cycle arrest in S phase and the induction of apoptosis [45].

From Portulaca oleracea, a known vegetable used in folk medicine in several countries, a crude polysaccharide fraction POL-P was isolated, from which the polysaccharide POL-P3b was further purified and tested for its anticancer activity against HeLa cells and in U14-bearing mice. POL-P3b exhibited an antiproliferative effect in a concentration-dependent manner on HeLa cells with IC50 values of 1225.32, 489.17 and 407.23 microg/mL at 24, 48 and 72 h, respectively. Moreover, the in-vivo study performed on U14-bearing mice showed that 50–200 mg/kg of POL-P3b significantly inhibited tumor growth in a dose-dependent manner [46].

1.3 Anti-inflammatory/Immunostimulating Polysaccharides

Inflammation is a complex and well-coordinated response of the innate and adaptive immune system following infection or injury. This process is characterized by a vascular response and recruitment of circulating leukocytes, defined initially by polymorphonuclear granulocytes followed by monocytes, which differentiate locally into macrophages [47].

Host defense mechanisms are divided into two distinct, but inextricably linked, pathways. The innate immune response mounts a rapid response to injury. It detects a broad range of molecular patterns that are commonly found on pathogens but are foreign to mammals, called pathogen-associated molecular patterns (PAMPs), and thus lacks the exquisite immune response [48]. Macrophages express a set of pattern recognition receptors, including various scavenger receptors and Toll-like receptors, whose ligands include PAMPs such as lipopolysaccharides (LPS), surface phosphatidylserine, and aldehyde-derivatized proteins, as well as modified forms of a classical risk factor for atherosclerosis, low-density lipoproteins (LDL) modified by oxidation or glycation [49]. Ligation of scavenger receptors can lead to endocytosis and lysosomal degradation of the bound ligands, while engagement of Toll-like receptors results in activation of NF-kB and mitogen-activated protein kinase (MAPK) pathways [50]. Ligation of Toll-like receptors can also heighten phagocytosis, production of reactive oxygen species (ROS), and release of cytokines, autacoids, and lipid mediators that coordinate and amplify the local inflammatory response [51].

The other major limb of host defenses, the adaptive immune response, mounts more slowly, and furnishes a more finely focused response mechanism that requires the recognition of specific molecular structures and depends on the generation of large numbers of antigen receptors (i.e., T-cell receptors and immunoglobulins) by somatic rearrangement processes in blast cells [49]. When T-cells recognize a foreign antigen presented to them, they initiate responses that precisely target an antigen, including a direct attack against the antigen-bearing cell by cytotoxic T-cells, stimulation of antibody production by B-cells and induction of a local inflammatory response. T-cells can differentiate into at least two subtypes of T helper (Th) cells. Th1 cells elaborate a number of cytokines; among them, interferon-gamma (IFN-γ) prominently coordinates crosstalk between innate and adaptive limbs of the immune and inflammatory responses by stimulating the macrophage to increase its production of a broad gamut of mediators, including autacoids, ROS, lipid species, and proinflammatory cytokines [52]. Th2 cells can stimulate humoral immunity by elaborating a number of cytokines that, in turn, induce B-cell maturation into antibody-producing plasma cells and promote B-cell class-switching to increase production of immunoglobulin E (IgE) antibodies. Th2 cells can also aid recruitment and activation of mast cells, another effector of allergic responses and contributor to chronic inflammation in a variety of tissues and disease states. In addition to these specialized proinflammatory responses, Th2 cells can dampen the inflammatory response by elaborating cytokines with anti-inflammatory properties such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) [53].

The resolution of inflammation occurs mainly via clearance of apoptotic cells by phagocytosis, a mechanism by which tissue is protected from harmful exposure to the inflammatory and immunogenic contents of dying cells. The resolution of inflammation is also due to production of anti-inflammatory mediators, such as IL-10 and TGF-β, in the lesion by tissue macrophages that phagocyted apoptotic cells. Disorder of apoptosis leading to leukocyte survival, defective clearance of apoptotic cells as well as inappropriate macrophage activation have been suggested to contribute, at least in part, to the development of chronic inflammation [54].

Natural polysaccharides also displayed very interesting anti-inflammatory/immunomodulating properties (Table 1.2).

Table 1.2 Anti-inflammatory/immunostimulating polysaccharides.

1.3.1 Microbial Sources

A novel exopolysaccharide EPS-1, containing tetrasaccharide repeating units formed by sugars with a mannano-pyranosidic configuration, was isolated from a halophilic and thermotolerant Bacillus licheniformis strain. EPS-1 was tested for its effect on the production of different cytokines (IFN-α, IL-12; IFN-γ, TNF-α, IL-18) involved in the immune response in human peripheral blood mononuclear cells (PBMC) during the HSV-2 virus infection. These results showed that EPS-1 affected cytokines production in a dose-dependent manner. At a concentration of 300 microg/mL, it stimulated IFN-α production, while IL-18 production was not increased. Moreover, EPS-1, at a concentration of 100 microg/mL, induced the production of IL-12 at considerable levels; IFN-γ and TNF-α were also detected. The immunomodulatory activity of EPS was correlated to evaluation of its antiviral effect [55]. A similar study was published later in a paper by Arena et al., in which the immunomodulatory and antiviral effects of an exopolysaccharide EPS-2, isolated from a strain of Geobacillus thermodendrificans, were described. EPS-2 also stimulated the cytokine production in PBMC cells in a concentration-dependent manner, like EPS-1. A high level of IL-12, IFN-γ, TNF-α and IL-18 was revealed after EPS-2 treatment [56].

From a strain of B. licheniformis (8-37-0-1) a bioactive esopolysaccharide EPS was purified and characterized. Chemical and spectroscopic analysis indicated that this EPS was a levan carrying a (2→;6)-linked backbone, with a single β-D-fructose at the C-1 position every seven residues. It showed, in a concentration-dependent manner, a prominent effect on the proliferation of mouse splenocytes in-vitro for concentration ranging from 50 to 800 microg/mL [57].

1.3.2 Vegetable Sources

Polysaccharides of the pectin type were extracted and isolated from the leaves of Sutherlandia frutescens (syn. Lessertia frutescens). All fractions obtained after pectinase digestion were assayed for their immunomodulatory activity by complement fixating test based on the haemolysis inhibition of antibody-sensitized sheep red blood cells (SRBC) by complement from human sera [58]. Results showed that all fractions had immunomodulating properties; in particular the fraction with the highest amount of xylose (Z100W-II-I.A) was the most active polysaccharide with an ICH50 < 0.5 microg/mL. Previous study on plant polysaccharides has already described the correlation between the amount of xylose and immunomodulatory properties of polysaccharides [59]. A lot of plant polysaccharides of the pectic type having immunostimulating activity are described in the literature. For papers published until 2005, almost all bioactive polysaccharides are described in the article by Paulsen and Barsett [60].

Active polysaccharides were also extracted from the leafs and stem cell walls and mucilage of Dendrobium huoshanense, Orchidaceae, an herbal plant used in Chinese traditional medicine [61]. Polysaccharides from leafs and stems showed a monosaccharide composition with Xyl, Ara, Man, Glc, Gal, and GalA with small amount of Rha, Fuc, GlcA and 4-O-methyl GalA. Polysaccharide from mucilage contained glucomannan in β-(1-→;4)-D-Glcp and β-(1-→;4)-D-Manp linkages with partially acetylated mannosides at the 2- and 3-positions. Among isolated polysaccharides, HPS-1B23 extracted from the stems of D. huoshanense possessed a marked stimulating function on IFN-γ and TNF-α production by in-vitro culture of splenocytes and macrophages, respectively [62]. The immunostimulating effect of HPS-1B23 was also evaluated in mouse small intestine, spleen and liver after oral administration of polysaccharides. In particular, to evaluate the immune response in small intestine, the proliferation of marrow cells was assessed. The proliferation of such cells increased, in a dose-dependent manner, in response to the oral administration of different concentrations of polysaccharide (50, 100, and 200 mg/kg). In the spleen the secretion of IFN-γ, also in a dose-dependent manner, was significantly stimulated by polysaccharide administration (at 200 mg/kg). The level of IL-4 in the spleen did not change after the same treatment. Instead, in the liver the oral administration of polysaccharides stimulated the secretion of both IFN-γ and IL-4 at a dose of 200 mg/kg, together with a significant proliferation of hepatocytes [63]. Polysaccharide extracted from mucilage of D. huoshanense exhibited an effect in murine splenocytes. It induced the production of several cytokines, including IFN-γ, IL-10, IL-6, and IL-1α, and hematopoietic growth factors GM-CSF and G-CSF in mice splenocytes [64]. Further experiments were performed in mice and human cells in-vitro on polysaccharides isolated from D. huoshanense to point up the potentiality of these biopolymers in a therapeutic approach to some immune disease [65].

An artificial co-infection model by using pathogen-free embryonated eggs injected with subgroup B avain leucosis virus (ALV-B) and Bordetella avium (B.avium) was used to evaluate the immunoregulatory effects of a polysaccharide isolated from Taishan Pinus massoniana pollen (TPPPS). Results showed that TPPPS injection increased the lymphocyte ratio and the production of B. avium antibodies in the TPPPS group compared with the non-TPPPS group. Moreover, TPPPS stimulated the secretion of IFN-γ and IL-2, thus promoting cellular immunity [66]. Earlier papers already described the immunomodulatory effect of TPPS [67–69], thus all results indicated the potential of TPPPS for its use as immunoregulator.

Four polysaccharides (FCp-1, FCp-2, FCp-3 and FCp-4) were isolated from citrus fruits (Citrus medica L. var. sarcodactylis) after hot-water extraction and ethanol precipitation. Among all, only the FCp-3, a polysaccharide with a molecular weight of 177.1 KDa, showed an immunological activity evaluated by splenocyte and thymocyte proliferation assay. FCp-3 displayed a significant splenocyte proliferation at a dose of ≥ 25 microg/mL, and showed a moderate effect on thymocyte proliferation at the same dose [70].

A water-soluble polysaccharide (SCPP11) was isolated from Schisandra chinensis (Turcz.) Baill, belonging to the family of Magnoliaceae, and well-known in traditional Chinese herbal medicine. At a dose of 50 mg/kg, SCPP11 caused an increase in thymus index and IL-2 and TNF-a levels in serum - (tumor-bearing mice) and a substantial increase in both phagocytosis and NO in RAW264.7 in-vitro, in a dose-dependent manner [71].

The immunomodulatory effect of a polysaccharide (HCP-2) isolated from Houttuynia cordata is described in [76]. Previous papers described the bioactivities of water extract of H. cordata [72–74], but few pharmacological studies on isolated polysaccharides have been described [75]. HCP-2 increased the secretions of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), macrophage inhibitory proteins (MIP-1α and MIP-1β), and RANTES (regulated on activation, normal T cell expressed and secreted) in human peripheral blood mononuclear cells (PBMCs) [76].

Two polysaccharides (TOP-1 and TOP-2) isolated from Taraxacum officinale exhibited anti-inflammatory activity by reducing expression of inducible oxide synthase (iNOS) and tumor necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 cells. In cells treated with TOPs the inhibition of phosphorylation of inflammatory transcription factor, nuclear factor (NF)κB, and its upstream signaling molecule, PI3K/Akt, was observed. Then, TOPs exerted their anti-infammatory effect through the inhibition of NFκB expression [77]. In a previous paper the inhibitory effect of a polysaccharide from tomato wastes on NFκB expression was already described [78]. In particular, PS(1) inhibited, in a concentration-dependent manner, nitrite and ROS production as well as inducible nitric oxide synthase (iNOS) protein expression in J774 macrophages stimulated with LPS for 24 h. Moreover, incubation of cells with PS(1) determined a significant decrease of nuclear factor-κB (NF-κB)/DNA binding activity, which was correlated with a marked reduction of iNOS mRNA levels. These results show that PS(1) inhibits NF-κB activation and iNOS gene expression by preventing the ROS production and suggest a role for this compound in controlling oxidative stress and/or inflammation.

1.4 Antiviral Polysaccharides

During the last few years many antiviral compounds approved for clinical use have increased to more than 30 new drugs [79]. Because these drugs often do not have the expected efficacy or might be not well-tolerated, there is a great demand for new antiviral drug development. A great variety of natural products derived from plants, micro-algae, bacteria, fungi and others represent a very promising approach in the screening and development of new antiviral compounds.

Pathogenesis is the process by which virus infection leads to disease. Pathogenic mechanisms include implantation of the virus at a body site (the portal of entry), replication at that site, and then the spread and multiplication within sites (target organs) where disease or shedding of virus into the environment occurs. Factors that determine whether infection and disease occur are the many virulence characteristics of the infecting virus. Viruses cannot synthesize their genetic and structural components, and so they rely almost exclusively on the host cell for these functions. Their parasitic replication therefore robs the host cell of energy and macromolecular components, severely impairing the host’s ability to function and often resulting in cell death and disease. Pathogenesis at the cellular level can be viewed as a process that occurs in progressive stages, leading to cellular disease. An essential aspect of viral pathogenesis at the cellular level is the competition between the synthetic needs of the virus and those of the host cell. Since viruses must use the cell’s machinery to synthesize their own nucleic acids and proteins, they have evolved various mechanisms to subvert the cell’s normal functions to those required for production of viral macromolecules and eventually viral progeny. Most viruses have an affinity for specific tissues, that is, they display tissue specificity or tropism. This specificity is determined by selective susceptibility of cells, physical barriers, local temperature and pH, and host defenses [80].

Among the natural compounds, polysaccharides represent a class of natural products with a significant antiviral activity, suggesting that they could be explored as novel antiviral agents (Table 1.3).

Table 1.3 Antiviral polysaccharides.

1.4.1 Microbial Sources

The antiviral activity linked to the immunoregulatory effect of bacterial polysaccharides was reported by Arena et al. in 2006 and 2009 [55,56]. In the first paper, the antiviral effect of a novel exopolysaccharide EPS-1 produced by a strain of thermotolerant Bacillus licheniformis, isolated from a hot spring at Vulcano Island (Italy), was reported. EPS-1 inhibited HSV-2 replication in PBMC (human peripheral blood mononuclear cells) by upregulating the expression of proinflammatory cytokines [55]. In a later paper, an antiviral exopolysaccharide EPS-2, produced by a strain of Geobacillus thermodenitrificans, was reported. EPS-2 treatment in PBMC, at a concentration of 200 and 300 microg/mL, affected HSV-2 replication in a concentration-dependent way, increasing the inflammatory response [56].

From the marine bacterium Pseudoalteromonas sp. AM, an exopolysaccharide (EPS) was isolated which exhibited significant antiviral activity towards Herpes simplex (HSV-I). The effect resulted in the inhibition of virus replication of 60% when HSV-I was treated with 10% of EPS [81]. A similar result was reported in a previous paper of Matsuda et al. They described the antiviral effect, evaluated in RPMI 8226 cells infected with HSV-1 virus, of a sulfated polysaccharide produced by a marine Pseudomonas species WAK-1 strain [82].

A wide screening to evaluate the antiviral activity of polysaccharide compounds isolated from the cyanobacterium Arthrospira platensis on different viruses was performed by Rechter et al. [83]. These authors reported the results obtained by using specific assays for the quantification of in-vitro viral replication. The polysaccharide fractions, containing spirulan-like molecules, showed a marked inhibition of human cytomegalovirus, herpes simplex virus type 1, human herpesvirus type 6 and human immunodeficiency virus type 1. On the contrary, weak or no inhibition was detected for Epstein-Barr virus and influenza A virus [83]. The in-vitro antiviral activity of polysaccharide calcium spirulan produced by Arthrospira platensis (formerly Spirulina platensis) was previously described [84–86].

From a terrestrial cyanobacterium Nostoc flagelliforme, an acidic polysaccharide, nostoflan, was isolated. Nostoflan showed a very strong antiviral effect against HSV-1, HSV-2, human cytomegalovirus and influenza A virus, but no activity was noticed against adenovirus and coxsackie virus. These results suggested that nostoflan exerted the antiviral activity against enveloped viruses whose cellular receptors are carbohydrates [87].

Many sulfated polysaccharides have proved to possess a very strong antiviral activity [88,89]. The antiviral effect of sulfated polysaccharides is due to their structural features and not only to their charge density and chain length. In fact, the in-vivo efficacy demonstrated their capacity to inhibit the attachment of the virion to the surface of host cell [90]. Mercorelli et al. reported the achievement of some sulfated derivatives of E. coli K5 capsular polysaccharides and their potency as antiviral agents against HCMV (human cytomegalovirus) [91]. These derivatives were structurally related to cellular heparin sulfate and their antiviral effect against enveloped and nonenveloped viruses (HIV, HSV-1 and HSV-2, human papillomaviruses) was previously reported [92–94].

1.4.2 Vegetable Sources

Two polysaccharides, P1 and P2, isolated from Azadirachta indica leaves, and their respective chemically sulfated derivatives, P1S and P2S, were evaluated for their activity in the replication of polivirus type 1 in HEp-2 (epithelial cells of human larynx carcinoma) cell cultures. Such compounds exhibited a strong antiviral activity with an IC50 of 80 microg/mL, 37.5 microg/mL, 77.5 microg/mL and 12.1 microg/mL for P1, P1S, P2 and P2S, respectively. The best antiviral effect was detected when the polysaccharides were added simultaneously to viral infection and a reduction of the activity was observed when they were added after the infection [95].

From the green leafy part of Welsh onion (Allium fistulosum L.) a fructan was isolated displaying an in-vivo anti-influenza A virus activity after oral administration in mice. Despite the observed in-vivo effect, the fructan did not exhibit in-vitro efficacy in MDK cells infected with influenza A virus. The antiviral mechanism could be dependent on the host immune system [96].

Pectic arabinogalactans with unusual β-(1-β6)-linked D-Galp residues were isolated from leaves of Stevia rebaudiana (Asteraceae family). Their anti-Herpes simplex virus type-1 (HSV-1) activity was evaluated on infected Vero cells treated with different concentrations of polysaccharides and incubated for 72 h. Results showed that the crude polysaccharides exhibited antiviral activity against HSV-1 in-vitro [97].

Two glycosaminoglycans, DIP30 and DIP60, consisting of more than four different monosaccharides, were extracted from Duchesneae indicae. DIP30 was composed of mannose, rhamnose, galacturonic acid, glucose and galactose, while DIP60 was constituted by rhamnose, glucuronic acid, galacturonic acid and galactose. Both polysaccharides were tested for their antiviral effect in human embryonic lung fibroblast (HELF) infected with varicella zoster virus (VZV). Results showed an interesting anti-VZV activity with EC50 values of 265.2±35.4 microg/mL and 325.6 ±42 microg/mL for DIP30 and DIP60, respectively [98].

A sulfated derivative (SPLCf) of a galactomannan, previously obtained from the aqueous extract of the Caesalpinia ferrea seeds [99], was evaluated for its activity in herpes simplex virus (HSV) and poliovirus (PV) replication in infected HEp-2 cells (human larynx epithelial cells carcinoma). SPLCf exhibited an inhibitory effect on HSV-1 and PV-1 replication with IC50 values of 405 microg/mL and 1.73 microg/mL, respectively. Moreover, the SPLCf displayed the best antiviral activity against HSV-1 and PV-1 when added concomitantly with viral infection. The antiviral effect of SPLCf could be due to its polyanionic nature, interfering in the step of HSV adsorption, as well as, to its effect on virus particles and on the expression of viral proteins. The antiviral activity of SPLCf on PV was stronger, interfering with early steps of virus replication (adsorption and penetration) and in the synthesis of polyprotein [100]. Another paper reported the achievement of sulfated derivatives of glucans (P444, P445 and P446) extracted from rice (Oryza sativa) with the aim to evaluate them for antiviral activity compared to non-sulfated glucan (SIG). P444, P445 and P446 were significantly active against human cytomegalovirus (HCMV) replication in primary human fibroblast. The IC50 for P444, P445 and P446 ranged over 2.44±0.58 microg/mL, 2.52±0.21 microg/mL and 6.54±0.21 microg/mL, respectively. The non-sulfated glucan, SIG, showed a significantly different IC50 (12.48±0.60 microg/mL). In order to evaluate the selectivity of antiviral effect, all compounds were further tested against a panel of human and animal viruses, representatives of the families Herpesviridae human [HCMV], mouse [MCMV], and guinea pig [GPCMV] cytomegaloviruses as well as human herpes simplex virus type-1 [HSV-1]), Poxviridae (vaccinia virus), Adenoviridae (human AdV-2), and Orthomyxoviridae (human influenza A/WSN/33 virus). The best activity was found against HCMV, while low level or no activity was detected against the other viruses. Results demonstrated the selectivity of these sulfated polysaccharides against cytomegaloviruses [101].

1.5 Antioxidant Polysaccharides

The state called oxidative stress is the result of an imbalance between the pro-oxidants and antioxidants, on behalf of the first one. The level of pro-oxidants increases when the production of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) greatly raises or when the antioxidant defenses decrease. The increasing level of pro-oxidants, and therefore of free radicals, is the result of exposure to environmental or pathological agents (UV rays, toxic chemicals, cigarette smoking, atmospheric pollutants) [102]. Included in the ROS and RNS species are non-radical species such as hypochlorous acid (HOCl), ozone (O3), nitrous acid (HNO2), singlet oxygen (¹O2), hydrogen peroxide (H2O2) and lipid peroxide (LOOH); free radical species like peroxyl (RO2·), superoxide (O2·‒), alkoxyl (RO·), hydroxyl (OH·), hydroperoxyl (HO2·), nitric oxide (NO·), peroxyl (ROO·), nitrogen dioxide (NO2·), and lipid peroxyl (LOO·); and also radicals that are formed during the normal cellular metabolism [103,104]. The free radicals are a highly reactive chemical species and are capable of damaging the cellular biomolecules (nucleic acids, proteins, lipids), leading to several disease conditions (neurodegenerative, cancer, cardiovascular pathologies) [105]. Antioxidants are compounds able to scavenge free radicals, preventing the initiation and propagation steps, avoiding the biomolecule damages and the oxidative stress. In the last few years, the interest towards antioxidants from natural sources has greatly increased. In particular, plants, fruits and vegetables represent a rich source of new antioxidants and health-promoting compounds as potential therapeutic agents. Natural antioxidants are classified as vitamins, phenolic compounds, tannins and volatile compounds. Recently, polysaccharides have also emerged as an important class of bioactive antioxidant compounds, able to reduce free radical generation and prevent a diseased state (Table 1.4).

Table 1.4 Antioxidant polysaccharides.

1.5.1 Microbial Sources

A polysaccharide fraction (EPS) was extracted from the cell-free medium of a culture of Paenibacillus polymyxa (previously named Bacillus polymyxa) EJS-3, an endophytic bacterium strain isolated from the root tissue of Stemona japonica (Blume) Miquel, a traditional Chinese medicine [106,107]. The crude EPS was further purified by chromatography of DEAE-52 and Sephadex G-100, obtaining two pure EPSs, EPS-1 and EPS-2, with molecular weights of 1.22x10⁶ and 8.69x10⁵ Da, respectively. Their monosaccharide composition was: mannose, fructose and glucose in a molar ratio of 2.59:29.83:1 and 4.23:36.59:1, respectively. Both crude and purified EPSs were tested for their antioxidant activity in-vitro by scavenging activities on superoxide and hydroxyl radicals. Results showed that the reducing power of crude EPS was higher than purified EPS-1 and EPS-2, but all samples showed a lower activity than ascorbic acid. The scavenging effect of all EPS fractions was dependent on the concentrations. Also, in this case, the antioxidant activity of crude EPS was higher than purified EPSs, showing an inhibition of superoxide radical of 74.38% at a concentration of 1 mg/mL. At the same concentration, purified EPSs showed an inhibition of 12.11%. In the scavenging activity on hydroxyl radical, crude EPS and EPS-1 and EPS-2 exhibited an inhibition of 87.58%, 76.73% and 68.55%, respectively, at a concentration of 1 mg/mL [108]. Based on these results, further experiments were performed with the aim to test the antioxidant activity of EPSs in-vitro and in-vivo. In particular, EPS-1 (the main fraction, 53.6%) was further tested in-vivo by using the D-galactose (D-Gal)-induced aging mice model. Results of in-vivo experiments showed that EPS-1 administration increased the activities of antioxidant enzymes (SOD, CAT and GSH-Px), decreased malondialdehyde (MDA) levels and improved the total antioxidant capacity in the livers and serum of D-Galinduced aging mice [109].

Two water-soluble extracellular polysaccharides, named ETW1 and ETW2, were isolated from a marine bacterium Edwardsiella tarda. The two mannan exopolysaccharides, with a MW of 29 and 70 kDa, were investigated for their antioxidant activity by using in-vitro assays based on hydroxyl, DPPH radical scavenging and lipid peroxidation inhibition. The scavenging capacities of both exopolysaccharides on DPPH and hydroxyl radicals, and lipid peroxidation inhibition were concentration dependent. The inhibition capacities of ETW1 and ETW2 on DPPH and hydroxyl radicals, and lipid peroxidation were 87.66–76.54%, 88.96–76.85% and 78.93–71.48%, respectively, at a concentration of 8.0 mg/mL. The reason for which ETW1 was more active than ETW2 could be related to the difference in the molecular weights [110].

An acidic exopolysaccharide, with an estimated molecular weight of 8.83x10⁵Da, was isolated from marine bacterium Pseudomonas PF-6 [111]. This EPS, that belonged to a β-type heteropolysaccharide with a pyran group, exhibited a scavenging ability on DPPH, hydroxyl and superoxide radicals. At a concentration of 0.1 mg/mL the scavenging effect on DPPH radical was 79.81%, less than Vitamin C (Vc) used as the standard. In the hydroxyl radical scavenging assay, at a concentration of 0.6 mg/mL, EPS showed an inhibition value of 92.12%, stronger than Vc (47.96%). In superoxide anion scavenging assay, EPS exhibited an IC50 value of 0.149 mg/mL, comparable to IC50 of Vc (0.147 mg/mL) [112].

A solvent-tolerant strain of Bacillus licheniformis UD061 was the producer of antioxidant exopolysaccharides. The production of these EPSs was improved with UV and DES mutagenetic treatments and organic solvent stress treatments [113]. The in-vitro antioxidant effect of EPSs was evaluated by using reducing power, superoxide anion scavenging and hydroxyl radical scavenging assays. Results demonstrated that the crude EPSs exhibited strong scavenging activities on superoxide and hydroxyl radicals in-vitro, showing inhibition values of 42.55% and 50.91%, respectively. The antioxidant power of EPSs did not change significantly before or after the optimization of the production process [114].

1.5.2 Vegetable Sources

Three polysaccharide fractions, named APF1, APF2 and APF3, were extracted from the roots of Angelica sinensis. The main monosaccharides of APFs were identified as arabinose, glucose, rhamnose, galactose and galacturonic acid, as well as trace of mannose and glucuronic. APFs were tested for their antioxidant activity against hydrogen peroxide (H2O2)-mediated oxidative stress in isolated mouse peritoneal macrophages. Among all fractions, APF3 was the most active in the inhibition of (H2O2)-caused decrease of cell viability, malondialdeyde (MADA) formation and lactate dehydrogenase (LDH) leakage at a concentration of 100 microg/mL. Moreover, at the same concentration, APF3 reduced (H2O2)-caused decline of superoxide dismutase (SOD) activity and glutathione (GSH) depletion. Further experiments were done with the aim of establishing whether the antioxidant activity of APFs was the result of the inhibition of intracellular ROS generation and/or NO production in H2O2-injuried macrophages. Results showed that the antioxidant effect of polysaccharide fractions was associated with an effective inhibition of the intracellular ROS production and, for the first time, it was found that APFs also attenuated excess NO generation [115].

Four polysaccharide fractions (PAVF I, II-a and III) were extracted from the fruit calyx of Physalis alkekengi var. francheti and their chemical compositions were determined. The crude polysaccharide fractions (FCPs) and all purified fractions (PAVF I, II-a and III) were evaluated for their antioxidant activity by using hydroxyl radical assay (.OH), superoxide radical assay and DPPH assay. All tested compounds showed a dose-dependent radical-scavenging activity. Among all, PAVF I had the best scavenger activity on DPPH radical, hydroxyl radical and superoxide anion, and its activity was more pronounced than Vc used as the standard [116].

Isatis tinctoria, a known traditional herb that comes from the roots of woad, has been used for its medicinal properties in traditional Chinese medicine [117,118]. From the roots of I. tinctoria a polysaccharide (IRPS) was extracted that exhibited a significant ABTS radical scavenging ability in-vitro, in a concentration-dependent manner with the maximum percentage of inhibition 64.3% at a concentration of 0.3 mg/mL [119].

The antioxidant effects of a polysaccharide (ASP) extracted from Acanthopanax senticosus were evaluated in-vitro by hydroxyl and superoxide radicals scavenging assays, and in-vivo in alloxan-induced diabetic mice. In in-vitro assays, ASP at different concentrations (0.1–1.6 mg/mL) showed a potent scavenging ability in a concentration-dependent manner. Also, in an in-vivo experiment, ASP treatment significantly reduced, in a dose-dependent manner, the levels of lipid peroxidation markers (thiobarbituric acid reactive substances and lipid hydroperoxides) and increased the level of superoxide dismutase (SOD) and catalase (CAT) activities [120].

A crude polysaccharide fraction (GBEP) was obtained by hot water extraction of Ginko biloba exocarp. GBEP was fractionated by a DEAE Sepharose fast flow anionexchange column obtaining five fractions, one neutral polysaccharide (GBEP-N) and four acidic polysaccharides (GBEP-A1, GBEP-A2, GBEP-A3 and GBEP-A4). The crude polysaccharide (GBEP) was evaluated for its in-vitro antioxidant activity by DPPH, hydroxyl radical-scavenging, superoxide anion-scavenging, power-reducing assays. Results displayed an undoubted in-vitro antioxidant activity in a concentration-dependent manner in a range from 0.1 mg/mL to 1 mg/mL [121].

From the leaves of Bruguiera gymnorrhiza, a plant belonging to the family Rhizophoraceae, bioactive polysaccharides (BGPs) were extracted and chemically characterized. The BGPs were tested for their in-vitro antioxidant activity by means of superoxide anion radical, ABTS radical and hydroxyl radical scavenging assays. Results showed a significant antioxidant activity at a concentration of 5 mg/mL with values of radical inhibitions of 62.4%, 62.2% and 63.3% in superoxide anion, ABTS and hydroxyl radical assays, respectively [122].

Ultrasonic extraction (UE) is a new technology by which means it is possible to accelerate the extraction of bioactive plant metabolites in a simple and efficient way. This methodology was used to extract bioactive polysaccharides (PNSPs) from seeds of Pharbitis nil, an annual climbing herb belonging to the family Convolvulaceae. The chemical analysis of PNSPs showed contents of sugar, uronic acid and proteins of 83.6±1.61, 21.8±1.25 and 16.4±0.88% (w/w), respectively. The antioxidant activity of PNSPs was evaluated in-vitro by using ABTS and DPPH radical scavenging assays. At a concentration of 5 mg/mL, PNSPs exhibited values of radical inhibitions of 100% and 89.6% in ABTS and DPPH assays, respectively [123].

1.6 Other Biotechnological Applications

During the last two decades, a significant development has been made for new advances in the design of biodegradable polymeric materials for biomedical application. This application requires specific materials with appropriate biological, chemical, biomechanical, physical and degradation properties. Current research is directed towards the use of natural biodegradable polymers for their application in tissue engineering (bone and cartilage tissue) and as controlled release drug delivery [124,125,8].

Polysaccharides are the most plentiful natural biopolymers and represent the largest group of polymers produced in the world. They can be found in microorganisms, animals and plants, performing several basic biological functions. Natural polysaccharides are biocompatible, nontoxic and biodegradable. Such features make them eligible for biomedical and pharmaceutical purposes such as drug delivery, inert diluent for drugs and for implants in tissue engineering [126].

Two marine polysaccharides, HE800 and GY785, were tested for their mechanical and biological properties with the aim to build new scaffolds for bone and cartilage engineering. The two EPSs were produced by Vibrio diabolicus, a microorganism isolated from a deep-sea hydrothermal vent polychaete annelid Alvinella pompejana [127]. They were incorporated into an injectable silylated hydroxypropylmethylcellulose-based hydrogel (Si-HPMC) and tested on osteoblast (MC3T3-E1) and chondrocyte (C28/I2) cultures. Both EPSs, HE800 and GY785, exhibited a significant improvement of the mechanical properties of Si-HPMC hydrogel and stimulated the attachment of MC3T3-E1 and C28/I2 cells on the top of scaffolds. Moreover, Si-HPMC hydrogel containing 0.34% HE800 or 0.67% GY785 showed the best compressive modulus (9.5–11 Kpa) while also supporting the proliferation of chondrocytes, and has the most promising features for cartilage engineering application [128]. A previous paper already described the bone regeneration property of the HE800 evaluated by using the Critical Size Defect (CSD) technique. A 5 mm-diameter hole was made on each parietal bone of male rats. The left hole was used as control (without any treatment), while the right hole was fixed with either HE800 and with collagen (negative control). After 15 days, in sample treated with HE800, bone healing was almost complete, in which a layer of osteoblasts on bone surface and an increase of osteocyte inclusion were observed. The collagen-treated rats did not exhibit any significant bone healing [7].

Ruiz-Velasco et al. also investigated the effects of over-sulfated exopolysaccharides, in comparison to the native EPS, produced by Alteromonas infernus on the bone biology. They noted that the long-term administration of OS-EPS produced cancellous bone loss in mice due to an increase of osteoclast number binding the trabecular bone surface. Native EPS did not show any significant activity, pointing up the importance of sulfation in trabecular bone loss. To explain the mechanism of action of OS-EPS, the authors investigated the effect of OS-EPS on osteogenesis. Results showed that OS-EPS inhibited osteoclastogenesis in two cell models, CD14+ (human monocytes) and RAW 264.7 (murine monocyte/macrophages). OS-EPS formed a heteromolecular complex OS-EPS/receptor activator of NF-kB ligand (RANKL)/RANK and pre-incubating with OS-EPS RANK had a higher affinity for RANKL than for RANKL alone, which means an increase of the bone resorption. In vitro, OS-EPS inhibited the cell fusion step by means of an inhibition of initial steps of osteoclast precursor adhesion. Moreover, OS-EPS decreased the proliferation and aided the differentiation of osteoblasts, causing an inhibition of nodule formation and an increase in bone resorption. These results showed a proresorptive effect of EPSs by means of the regulation of different levels of bone resorption [129].

Recently, because the capacity of EPSs to establish polymeric matrices enables their in-vitro manipulation to create novel structures in which bioactive compounds are encapsulated, new applications are being developed in drug delivery systems [130,131].

Halomonas smyrniensis strain AAD6 (JCM 15723) strain was described as a producer of high levels of levan exopolysaccharide in the presence of sucrose in defined media. Studies on biocompatibility were performed and the results showed that this EPS did not affect cellular viability and proliferation of osteoblasts and murine macrophages. Moreover, the toxicity test carried out in a brine shrimp test revealed a protective effect of levan against a toxic agent [132]. Because of the amphiphilic nature of levan, it is able to form nanoparticles by self-assembly in water [133]. The levan EPS isolated from strain AAD6 was tested for its potential use as a biopolymer in a nanoparticle drug-delivery system. Levan nanoparticles encapsulated with bovine serum albumin (BSA) were prepared and used as a model to investigate their different properties. The size of nanoparticles varied between 200 nm and 537 nm and their encapsulation capacity also varied (ranged between 49.3% and 71.3%), depending on the levan concentration used. Moreover, the increasing in-vitro release of BSA from the nanoparticles was shown to be a controlled release of proteins [8].

Natural polysaccharides have attracted growing interest as drug carriers because they are commercially available at low cost, they show a wide range of physicochemical properties and they can be easily modified by simple chemical reactions for definite applications. For example, chitosan, obtained from chitin, is the second most abundant natural polysaccharide. Chitosan and its derivatives have exhibited excellent biocompatibility, biodegradability, low immunogenicity, and biological activities [134,135]. These unique physiochemical properties of chitosan have stimulated its study in the development of drug-delivery systems for a wide range of biological agents [136–139].

A further interesting biotechnological application of natural polysaccharides is in the development and design of new biomaterial. Natural biopolymers possess the capacity to establish chemical and physical intramolecular interactions, resulting in a cohesive polymeric matrix able to form biofilms. These biofilms show specific properties (mechanical and barrier properties, transparency, biodegradability and biocompatibility) that make them suitable for applications in edible coating for food products and packaging purpose [140–143]. The biobased economy is expected to grow substantially in Europe within the coming 20 years. An important part of the bioeconomy is biorefineries in which biomass is processed in a sustainable manner to various exploitable products and energy. Bioeconomy can be seen as an expansion of the biorefinery concept as it also includes the exploitation of biotechnology in processing of non-biological raw materials or production of non-bio products exploiting certain biological principles.

Waste management is a significant affair for the food industry. The new biotechnologies could allow the reuse of wastes as a source of bioproducts with high added value [144]. From solid tomato (Lycopersicon esculentum variety Hybrid Rome) processing industry wastes a high grade polysaccharide was extracted and characterized. Specific analysis revealed the presence of glucose and xylose as main carbohydrates and a low level of uronic acids. The yield of biopolymers represented 7.5–10.0% of lyophilized biomass. By addition of glycerol, the polysaccharide was able to form a solid, clear, elastic and biodegradable film [145]. Moreover this polysaccharide was used as the sole carbon source in a media for the growth of a thermohalophilic strain, Samu-Sa1, isolated from the hot springs at Mount Grillo (Baia, Naples, Italy), obtaining a yield of 3.5 g l–1 [146]. In accordance with the obtained results, the polysaccharide isolated from a renewable source could be employed to produce biodegradable film and get cheaper bacterial biomasses.

1.7 Conclusions and Future Perspectives

Polysaccharides are used in all sectors of human activities like materials science, nutrition, health care and energy. They are polymers with exceptional properties, far from being fully recognized, able to open routes for completely novel applications in pharmaceutical products, medical engineering and bioplastics. Polysaccharides and polysaccharide-based polymers offer credible answers to the challenges faced by the world in terms of global sustainability. They offer numerous product development opportunities that are increasingly attractive in the field of renewable energy and biodegradation issues. The use of renewable raw materials such as polysaccharides is one of the targets of the European Union policies, with objectives to increase the share of renewable energy and to promote biodegradation. In recent years, there has been a significant increase in the research on added-value products and new technologies based on the use of polysaccharides, showing a wide range of applications of these natural polymers and their great potential.

Acknowledgments

This article is part of the project PON01_01966, Filiere agro-industriali integrate ad elevate efficienza energetica per la messa a punto di processi di Produzione Ecocompatibili di Energia e Bio-chemicals da fonte rinnovabile e per la valorizzazione del territorio (EnerbioChem), 28/10/2011 prot. n. 881/Ric – Programma Operativo Nazionale (PON) Ricerca e Competitività 2007 – 2013 Regioni Convergenza.

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