INTRODUCTION:

Chitosan is a deacetylated chitin derivative, which is a polymer consisting of N-acetyl-2-amino-2-deoxy-D-glucopyranose, linked 1-4 glycosidic bonds (Figure 2). In obtained from natural sources chitin, as a rule, contains 5-10% of residues of 2-amino-2-deoxy-D-glucose^1,2.

Toxicity:

Current research indicates that, in general, chitosan is a relatively non-toxic, biocompatible material. However, care should be taken to ensure that it is pure, since protein, metal, or other pollutants can potentially cause many harmful effects on both derivative syntheses and
dosage forms. After derivatization (or crosslinking), unreacted reagents should be carefully removed to prevent mixed results, since many cytotoxic reagents are not related. Many enzymes appear to have activity, at least in vitro, against chitosan, but its derivatives can produce non-digestible molecules. In order for these compounds to remain clinically viable, they must remain small enough (radius <42 Å for neutral molecules) for excretion by the kidneys³.

They use chitosan as an excipient in oral dosage forms, although chitosan does not quite match the definition of excipient because it has many biological effects. Absorption of chitosan into the bloodstream is usually not investigated in oral studies. The systemic uptake and distribution of chitosan by this route of delivery may largely depend on macular mass.

It is highly likely that oligomers may exhibit some absorption, whereas chitosan which has large molecular weight are released without absorption. This effect was observed with using of chitosan with 3.8 kDa chitosan (88.4% DD), which has the highest plasma concentration after oral administration, compared with 230 kDa (84.9% DD), which were almost not absorbed. An increase in Mw was found to decrease plasma concentration³.

Chitosan does not have an irritating or allergic effect and is biocompatible with both healthy and infected human skin⁴. When chitosan was administered orally to mice, the LD50 was found to exceed 16 g / kg⁵. Oral toxicity was found in mice treated with chitosan nanoparticles 100 mg / kg (80 kDa, 80% DD)⁶.

Physical properties of chitosan:

Chitosan is not soluble in water but it is soluble in many organic acids, such as acetic acid, formic acid, and others, and in diluted mineral acids, such as hydrochloric acid.

Decomposition of chitosan was established during heating, and it was found that the rate and extent of damage to the polymer were being accelerated with increasing temperature and duration of heating:

- at a temperature of 30–110° the evaporation of residual water present in the polymer sample occurs

- decomposition of the polymer is observed in a wide temperature range, from 180 to 340°C⁷.

Table 1 : Physical properties of chitosan

The name of indicators	Characteristics and numbers	REF.
Other names	poliglusam, deacetylchitin, chicol.flonac C, flonac N	[8]
CAS No.	9012-76-4	[8]
Molecular formula		[8]
	C56H103N9O39

Formal charge	0	[8]
Molecular mass	1526.464 g/mol	[8]
pKa	6,3–6,5	[9]
		[9]
Appearance	flakes smaller than 10 mm or powders of varying degrees of grinding.	[10]
Color	from white to cream color often with a yellowish, grayish or pinkish tinge	[10]
smell	without smell	[8]
taste	electrified and astringent	[8]
Solubility	it is dissolved in weak acids ph ˂6.5	[11]
Solvents in 1%	acetic acid, L-ascorbic acid, formic, L-glutamic, Hydrochloric, lactic, Malic, phosphorous, Succinic acid	[12]
viscosity	linked with Molecular mass, temperature and deacetylation degree of chitosan\ 200-500mPa.s for 0.5% chitosan in 0.5% Acetic acid at 20deg C	[13]

Chemical properties:

The amino groups of the chitosan molecule have an ionic dissociation constant (pKa) of 6.3–6.5¹¹. When they are below of this value, the amino groups are protonated, and chitosan is a cationic, highly soluble polyelectrolyte. When they are above, amino groups are deprotonated and the polymer is insoluble. This dependence of solubility on pH allows to obtain chitosan in various forms: capsules, films, membranes, gels, fibers, etc.

The difference between chitin and chitosan is clear in C-2: when there is an amino group (-NH₂) this means it is the structure of chitosan Molecule and if there is an amide (-NH-co-CH₃) so it is the structure of chitin Molecular. to clear out this idea: in chitin polymer all the mono Molecular have the amide group, but on the other hand, the chitosan has a percentage which is called the deacetylation - it means how many amines we have in the structure of the raw material. Usually this percent is written by the factory.

Chitosan has three reactive groups: primary (C-6) and secondary (C-3). Hydroxyl groups on each repeating unit and amino (C-2) groups on each deacetylated block.

These reactive groups easily undergo chemical modification to alter the mechanical and physical properties and solubility of chitosan.

Typical reactions involving hydroxyl groups are esterifications. Selective O-substitution can be achieved by protecting the amino group during the reaction.

The presence of a nucleophilic amino group allows selective N-substitution, such as N-alkylation and N-acylation, by reacting chitosan with alkyl halides and acid chlorides, respectively. An alternative method for N-alkylation is a reductive alkylation, when the amino group is converted into an imine with various aldehydes or ketones and then reduced to an N-alkylated derivative.

Chitosan can also be modified by crosslinking or graft copolymerization^{14, 15}.

Chitosan Production Technology:

The production of chitosan is based on the hydrolysis of the acetamide group. When fungi are used to produce chitosan, alkaline treatment removes protein and deacetylates chitin simultaneously. When crustacean shells are used as a source of chitosan, two pretreatments are required: one to remove traces of organic material and the other to remove calcium carbonate. Currently, there are chemical^18,19 and enzymatic^16,17,19 methods for producing chitosan. In this section, both methods will be considered, showing the advantages and disadvantages of each of them.

The chemical extraction of chitosan it needs to be achieved with certain stages:

1- Firstly after receiving shells from different sources, they are washed, dried and reduced in size by grinding them into powder.

2- Then demineralization is carried out using acid treatment, including HCl, H₂SO₄, and HNO₃.

3- Subsequently, alkaline treatment is required to remove the protective shells, which is usually carried out with NaOH.

4- Finally, the process of deacetylation of chitin is usually carried out again using NaOH, but at a higher concentration, by heating in order to obtain chitosan. This product is dissolved in an acidic medium, such as diluted acetic acid, formic or lactic acid, and finally it is precipitated with diluted sodium hydroxide²⁰.

The weaknesses found in this method:

1. Due to the large amount of alkaline waste and organic matter, this method is unfavorable for the environment.

2. It is expensive, due to wasting of large amounts of water used for treatment after acid and alkaline reagents.

3. Continuous hydrolysis of the polymer during alkaline treatment causes a decrease in the molecular weight and, therefore, its mechanical properties.

From a chemical point of view, acids or bases can be used to deacetylate chitin. However, glycosidic bonds are very sensitive to acid; therefore, alkaline deacetylation is used more often²¹.

Enzymatic treatments offer an alternative way to extract chitosan from crustacean shells.

1- Lactic acid bacteria were used for demineralization of crustacean shells instead of acid treatment.

2- The resulting lactic acid reacts with calcium carbonate to form calcium lactate, which can be precipitated and removed²².

3- To remove the protective shells of Crustacea the bacteria’s proteases were used. Treatment consists of the fermentation of crustaceans by various types of bacteria, such as Pseudomonas aeruginosa K-187, Serratia marcescens FS-3 and Bacillus subtilis²³.

4- Commercial proteases have previously been used to produce chitosan²⁴.

5- Chitin acetyl groups are removed with chitin deacetylase²⁴. This enzyme was first found in Mucor rouxii. However, Serratia sp. and Bacillus sp. are bacteria that also produce chitin deacetylase and can be used to generate chitosan²².

The main advantages of using enzymatic methods are the following:

- acid and alkaline treatment, which can be a source of environmental problems, will be avoided.

- the molecular weight and mechanical properties of chitosan are reduced, since selective enzymes are used at each stage.

- the disadvantage of this biological method is its high cost, which limits its use only to a laboratory scale.

Pharmacological and biological effects:

Depending on their physicochemical properties, chitosan has wound-healing, anti-inflammatory, antioxidant, immunostimulating, antibacterial, anti-tumor antiviral, fungistatic, hemostatic, hepatoprotective, hypolipidemic, radioprotective, enterosorbic, anti-tumor, enteric, it is biocompatible²⁵ and biodegradable polymers.

1- Wound healing

Wound healing is a specific biological process associated with the general phenomenon of growth and tissue regeneration²⁶. The process of wound healing was described as consisting of five overlapping stages that involve complex biochemical and cellular processes. These phases are described as homeostasis, inflammation, migration, proliferation and maturation²⁷.

Several reports have described the beneficial effects of chitin and chitosan on wound healing using in vitro, animal, and clinical studies.

In patients undergoing plastic surgery, Biagini et al. treated the donor sites with soft dressings of lyophilized N-carboxybutyl chitosan to stimulate the orderly tissue regeneration²⁸. It was noted better histoarchitecture order, better vascularization and absence of inflammatory cells were observed at the germ level, while fewer proliferation was reported in the malpighian layer at the epidermal level. Accordingly, it has been suggested that N-carboxybutyl chitosan leads to the formation of regularly organized skin tissue and reduces abnormal healing.

2- Anti-inflammatory activity:

Davydova and his colleagues tested the anti-inflammatory activity of chitosan with high (MW: 115 kDa) and low molecular weight (MW: 5.2 kDa), and both samples of chitosan demonstrated enhanced induction of the anti-inflammatory cytokine IL-10 in the blood of animals and suppression of colitis²⁹. The authors concluded that the main contribution to the anti-inflammatory activity of chitosan was made by the structural elements that make from its molecule, but do not depend on MV. Friedman et al. reported the inhibitory ability of chitosan-alginate nanoparticles against inflammatory cytokines and chemokines induced by P. acnes, and the results showed that chitosan-alginate nanoparticles effectively inhibit P. acnes-induced cytokine production in human monocytes and keratinocytes in a dose-dependent manner³⁰.

3- Antitumor:

Many scientists are inspired to search for more effective and harmless drugs for cancer patients. Chitosan and its derivatives are considered as one of the potential anti-cancer drugs obtained naturally. Many efforts to obtain an effective anticancer agent from natural products are causing a growing interest in natural polysaccharides. Zongetal published a review article on the anti-cancer activity of polysaccharides of fungi, plants, algae, animals and bacteria³¹.

Azuma and his colleagues examined in detail the antitumor activity of oligosaccharide chitosan (cos) using in vivo and in vitro cell models, and demonstrated effectiveness in growing tumors, reducing the number of metastatic colonies, suppressing the growth of cancer cells and enhancing acquired immunity³².

4- Immunostimulating:

In mice, chitosan strongly enhanced the local and systemic immune response (production of IgA and IgG antibodies) against influenza viruses A (Texas H1N1) and B (Panama) together with antigen (purified hemagglutinin and neuraminidase)³³. Thus, chitosan can affect the induction phase of immune responses in animals and many effectors mechanisms of the immune system.

5- Hemostatic agents:

The specific mechanism of action of chitosan remains unknown, but the data indicate three possible ways to control bleeding:

- plasma sorption: Chitosan can absorb from 50 to 300% of the liquid from its basis weight, which leads to the concentration of red blood cells and platelets in the damaged area.

- coagulation of red blood cells: Coagulation of red blood cells is directly related to hemostatic properties. Agglutination of Erythrocytes was enhanced in the presence of chitosan due to red blood cell crosslinking

- adhesion, aggregation and activation of platelets: the main cause of the hemostatic effect of chitosan is associated with adhesion, aggregation and activation of platelets. It was demonstrated that films of chitosan can cause adhesion of platelets, aggregation and activation of their own blood coagulation [Wang XH, 2003]³⁴.

6- Hepatoprotective effect:

The study investigated the hepatoprotective effect of β-chitosan, obtained from gladiard squid Sepioteuthis lessoniana, on oxidative stress caused by carbon tetrachloride (CCl4) and liver damage in rats.

Treatment with β-chitosan alone markedly increased the levels of hepatic and circulating superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx), as well as decreased levels of glutathione (GSH) and reduced levels of malonic dialdehyde.

Histopathological observations manifested a pronounced hepatoprotective effect of β-chitosan. CCl4-induced changes in the blood circulation and liver antioxidant protection system were normalized by β-chitosan, and it can be concluded that the hepatoprotective effect of chitosan may be due to its antioxidant and anti-lipidemic properties. Therefore, β-chitosan can be considered as an antihepatotoxic agent³⁵.

In another study, the results showed that chitosan has a protective effect on APAP-induced liver damage in rats³⁶.

7- Anti-lipidemic properties of chitosan:

It was shown for the first time that chitosan reduces the level of serum cholesterol in humans in 1993, when adult men were eating chitosan-containing cookies for two weeks (3 g / day for a week, 1.6 g / day for a week), there was a significant decrease in total cholesterol by 6%. Subjects also demonstrated an increase in HDL cholesterol by 10%.

The study reported a decrease in serum cholesterol levels with chitosan treatment. Obese women, consuming 1.2 g of microcrystalline chitosan for 8 weeks, showed a significant decrease in LDL, but not total serum cholesterol. Most recently, women with mild to moderate hypercholesterolemia who received 1.2 g of chitosan per day experienced a significant decrease in total serum cholesterol³⁷.

8- Antioxidant activity of chitosan:

The antioxidant activity of chitosan attracts a great attention of many scientists. Chitosan has shown marked cleansing activity on various types of radicals, which has great potential for its widespread use. The absorption activity of chitosan derivatives against free radicals is due to the donation of a hydrogen atom.

Xie et al. They offered several theories³⁸:

i Hydroxyl groups in the polysaccharide unit can react with hydroxyl radicals through a typical reaction of H-abstraction.

ii OH can react with residual NH amino groups to form stable macromolecular radicals.

iii NH2 groups can form ammonium NH3 + groups, absorbing H + from solution, and then they react with hydroxyl radicals through addition reactions.

9- Antiviral:

The findings suggest that chitosan and its derivatives suppress viral infections in various biological systems. However, the mechanism of this antiviral activity is not understood quite well. Most likely, the effects of anionic derivatives of chitosan, which inhibit retroviral infections in animal cells, are mediated by other mechanisms than the effects of polycationic chitosan molecules that are active against plant viral infections and phage infections.

Chitosan-initiated processes that prevent the development of viral infection in plants and microbial cultures may not have many similarities. However, it cannot be ruled out that the effects of chitosan interaction with cell surfaces may be somewhat similar to those observed in the interaction of chitosan with the plasma membranes of plant cells.

This interaction can lead to an increase in membrane permeability and its destruction caused by the unusual binding of polycationic chitosan molecules³⁹.

10- Antibacterial effect:

Chitosan, a biopolymer of marine origin, has recently attracted attention due to its significant antimicrobial properties and benefits, since it is non-toxic, biodegradable and biocompatible⁴⁰.

Mechanisms of Antibacterial effect:

Chitosan and its derivatives show differences in their activity against gram-positive and gram-negative bacteria, which is evident from most studies. This difference in activity may be due to the difference of the composition of the cell wall.

In gram-positive bacteria, the cell wall consists of a thick layer of peptidoglycan, where negatively charged teichoic acids are covalently bound with N-acetylmuramic acid, while lipopolitichoic acids form covalent bonds to the cytoplasmic membrane. These teichoic acids perform functions such as ensuring the strength of the cell wall and placing uniform charges of high density in the cell wall, thereby affecting the passage of ions through the outer surface layers⁴¹.

In the case of gram-negative bacteria, a thin layer of peptidoglycan over the cytoplasmic membrane is additionally covered with an additional outer membrane, called the outer membrane (OM). Lipoprotein and lipopolysaccharide (LPS) are the main components of OM, and therefore the hydrophilic O-specific side chains present in LPS help in the identification of bacteria⁴². Hydrophobic compounds and macromolecules are usually not active against gram-negative bacteria, and therefore it is important to overcome the barrier of the outer membrane to interact with gram-negative bacteria⁴³.

The method of antibacterial action of chitosan, apparently, is due to the interaction with the surface of bacteria (either a wall or OM), and four models have been proposed to explain this mechanism.

· The first and most common interaction model involves the electrostatic attraction of cationic groups of chitosan to negatively charged components present on the surface of bacteria⁴⁴.

· “The second mechanism proposed is that, in the case of gram-negative bacterial species, chitosan can cause changes in the permeability of the outer shell, forming the ionic type of bond^45-47. These changes prevent the transport of nutrients into the cell and create internal osmotic pressure. Ultimately, the cell dies due to nutrient deficiencies⁴⁸.

· In the third proposed method of action, chitosan is supposed to be able to cope with microorganisms intracellular. According to this theory, chitosan is able to penetrate a microbial cell in the case of bacteria and fungi and interact with DNA^49,50.

· The fourth mechanism of action is proposed when metal ions present on the surface of bacteria are chelated by the amino groups of chitosan. This chelating effect was found to suppress the electrostatic effect when the pH of the medium is higher than the chitosan’s pKa⁵¹.

CONCLUSION:

Since all these good properties of chitosan have been discovered by scientists, people have started to use it as food additives or as exipients in the pharmaceutical industry or in cosmetics.

One of the many uses of chitosan today as a dietary supplement internally is slimming down due to its anti-fat properties.

The use of chitosan as an accelerator for wound healing has increased in recent days and it has been increasingly used in oral and dental medicine as a disinfectant. Chitosan has aroused a great interest in the cosmetic field as a moisturizer or as a protective agent for skin or in hair care. It also increases interest in nanotechnology for the delivery of the medical drugs.

Now that we have this polymer, it is more important to direct it in the pharmaceutical industry. For example it is possible to use it in oral medicine or in bacterial or fungal infections of the digestive system because the polymer is not absorbed by the relatively high or medium molecular weight and not only as an exipients, but also as an effective substance in itself.

The research of chitosan should be carried out thoroughly. On the one hand to avoid its toxic effect, on the other to get the most out of this bio-polymer and to achieve substitutability with other toxic and less effective drugs.

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Received on 22.06.2019 Modified on 28.08.2019

Research J. Pharm. and Tech 2020; 13(2):1043-1049.

DOI: 10.5958/0974-360X.2020.00192.4