CHITOSAN:
problems, chemistry and
application
Vladimir Tikhonov
Senior researcher, PhD, Docent
tikhon@ineos.ac.ru
A.N. Nesmeyanov Institute of
Organoelement Compounds,
Russian Academy of Sciences,
Moscow, Russia
2
Background for investigations
•
•
•
Human body is a complex
ecosystem colonized by a variety
of microorganisms;
Epidemiology of human
infections is undergoing of
significant changes;
Bacteria, fungi and yeasts strains
become more resistant towards
many commonly used antibiotics
whose misuse administration
causes severe side effects;
Therefor, there is a need for new
non-toxic biocides that would be
active against invasive and
noninvasive human pathogens
and could help to reduce
administration of classic
antibiotics
16
14
12
10
B
•
8
6
4
2
0
1983-87
1987-92
1993-97
1998-02
2003-07
2008-112012-17 march
3
Synthetic food antimicrobials
Antibacterials
Comments
Sodium benzoate
Food and cosmetic
preservative active at pH<5.
Causes aller-gy, asthma and
skin rash
Food and cosmetic
preservative active at pH<5.
Hypersensitivity reactions,
genotoxic
(E211)
Sodium (E 201)/
potassium(E 202)
sorbate
Structure
O
O
Na
O
O Na
H3C
Food preservative and
antioxidant.
Causes alpitation, allergy
Na2SO3, Na2S2O5
Food and cosmetic
preservative
C2H5COONa
Sodium nitrite (E250)
Food preservative.
Potencial carcinogenic
NaNO2
Parabens
Cosmetic preservatives
4-Hydroxybenzoic acid methyl(propyl,
butyl) esters
Sodium sulfite (E221),
sodium metabisulphite
(E223)
Sodium propionate
(E281)
4
Cosmetic antimicrobials
Antibacterials
Triclosan
Clotrimazole
Phenoxyethanol
DMDM hydantoin
(1,3-dimethylol-5,5dimethylhydantoin
Imidazolidinyl urea
{1,1'-methylenebis{3-[4(hydroxymethyl)-2,5dioxoimidazolidin-4yl]urea}
Comments
Structure
O
Disinfectant
(dioxin health hazard)
Antifungal agent for topical,
oral and vaginal administration. Specific reac-tions:
general skin irritant (erythema,
stinging, blistering, peeling,
edema, pruritus, urticaria, and
burning).
Cosmetic preservative.
Potencial skin
allergenycity
Antimicrobial form-aldehyde
(classified as carcinogenic and
allergenic) releasing cosmetic
preservative
Antimicrobial form-aldehyde
(classified as carcinogenic and
allergenic) releasing cosmetic
preservative
OH
Cl
Cl
Cl
Cl
C
N
N
O
OH
OH
N
O
O
N
OH
HO
OH
N
O
O
O
N
N
O
O
N N N N
O
N
H
H H H H
H
5
Cationic antimicrobials
Antibacterials
(ethyl-Nα
Comments
Lauric arginate
lauroyl-L-arginate
hydrochloride)
Benzalkonium chloride
Food-grade surfactant and
antimicrobial agent
Miramistin
Anticeptic
Structure
H2N
O
O
Cl
NH
NH
(CH2)10CH3
NH2
N CnH2n+1
CH3
Cl
n=8,10,12,14,16,18
O
Ph
Cationic disinfectant and
surfactant
H3C
Ph N
N
C13H27
Cl
Chlorhexidine
Cationic disinfectant
H
H
H
N
N
N
NH
NH
NH
NH
N
N
N
H
H
H
Cl
Nisin
Polyhexamethylene guanidine
ε-Polylysine
Antimicrobial specific food
preservative E234.
Low effectiveness against
Gram-negative bacteria.
Cationic disinfectant
Polycyclic cationic peptide
MW=3354.07 g/mol
NH
N
N
H
H
n
Specific food preservative
produced by Streptomyces
albulus
O
H
N
25–30 L-lysine residues
NH2
n
6
Chitin and chitosan
• The sources of chitin
CH2
OH
O
CH2
O
OH
NHCCH3
O
O
NH2
x
O
1-x
Chitosan market was valued at USD 476.6 Million in 2016 and is
projected to reach USD 1,088.0 Million by 2022
7
Chitin deacetylation→ Chitosan
• Degree of deacetylation >60% -product
becomes soluble in diluted acids (HCl,
AcOH);
• Conditions of chitin deacetylation determine
its molecular weight, degree of acetylation,
polydispersity, acetyl-groups distribution
along polymer chains, and end-subunit
structure, i.e. molecular heterogeneity of
chitosan
8
What is oligochitosan?
• Chitooligosacharides (COS) – lowest
oligomers of N-acetylglucosamine and/or
glucosamine - MW< ~ 2 kDa
• Oligochitosan – highly depolymerized chitosanMW ~ 2 ÷~16 kDa
• Low molecular weight chitosan – product of
partial depolymerization of chitosan, MW ~ 16
÷~100 kDa
• High molecular weight chitosan- MW> 300 kD
9
Oligochitosan: preference pattern over
LMW/HMW chitosan
–
–
–
–
–
–
–
Higher solubility and lower solution viscosity
Increased biocidal and antioxidant activity
Low hemostatic effect
Enhanced absorption in GIT system
Prebiotic effect
Higher compatibility with cationic and neutral
surfactants, stabilizers and emulsifiers, sugars,
ethanol, salt, glycerin, organic acids and colorants;
Application of oligochitosan in pharmaceutical,
cosmetic, house-hold goods, and food products does
not interfere with current technologies
10
Aim
• The current work is aimed at finding
out whether oligochitosan can be
used as an alternative or additive to
classic antibiotics and other biocides,
i.e. whether there is a possibility to
use oligochitosan in medical,
cosmetic, house-hold goods, and
food/fodder compositions
Oligochitosan preparation
methods
• Oxidative method
• Enzymatic method
• Acidic hydrolysis
Vladimir Tikhonov, Oligochitosan preparation methods
comparative approach
DOI: 10.13140/RG.2.1.1425.4803
11
12
Enzymatic depolymerization
• Specific enzymes (chitinases, chitosanases)
• Unspecific enzymes( lysozyme,
hemicellulases, pectinases, lipases, and
pepsin, chimotripsin, plant proteases)
Merits: high yield, moderate shelf-life
Drawbacks: low hydrolysis rate, high enzyme
consumption, extraction of residual enzyme is
required, enzyme-dependent solubility and
biocidal activity
13
Oxidative decomposition
а) sodium nitrite
Merits: very fast N-selective
reaction
Drawbacks: multiple by-products
of deamination and condensation,
formation 2,5-anhydro-Dmannose at reducing end, low
shelf-life
b) hydrogen peroxide
Merits: moderate reactivity;
Drawbacks: oxidative side
modification, partial deamination,
low shelf-life
OH
NaBH4 or NaCNBH3
CHI
CHI O
O
CH2NH
HO
CHI
branched
oligochitosan
HNO2
OH
CHI O
HO
OH
NH 2OH
O
CHI O
CHO
HO
O
CH NOH
linear
oligochitosan
E.A. Bezrodnykh, I.V. Blagodatskikh, S.N. Kulikov, Pavel V. Zelenikhin, I.A.
Yamskov, V. E. Tikhonov, Consequences of chitosan decomposition by nitrous
acid: approach to non-branched oligochitosan oxime, Carbohydrate Polymers,
2018, v. 195, 551-557
14
Acidic hydrolysis
Complete or partial hydrolysis →
(oligo)chitosan hydrochloride
(1÷12 M hydrochloric acid, 50÷100oC)
A-A> > A-D > D-D
Advantages: high yield, low degree of
acetylation, stability at storage
Drawback: requires excessed HCl utilization
and plastic-made (non-stainless steel)
equipment
15
Anti-MRSA activity of oligochitosan:
MW and pH dependencies
pH
5.75
6
6.25
6.5
6.75
7
7.25
7.5
7.75
0
100
MIC
800
600
400
200
05
7.7 .5
7 5
7.2 7
pH
5
6.7 .5
6
5
6.2
6
5
5.7
20
18
16
14
12
10
6
8
,k
4
Da
2
Minimal inhibition
concentration
MIC(μg/ml)
of oligochitosan
towards methicillinresistant S. aureus
(MRSA)
vs pH and Mw
Mw
Antifungal activity of oligochitosans (short chain chitosans) against some Candida species
and clinical isolates of C. albicans: molecular weight – activity relationship, Sergey N.
Kulikov, Svetlana A. Lisovskaya, Pavel V. Zelenikhin, Evgeniya A. Bezrodnykh, Diana R.
Shakirova, I.V. Balgodatskikh, Vladimir E. Tikhonov,
Eur. J. Med. Chem., 2014, 74, 169-178
15
16
Oligochitosan (10 kDa) demonstrates antifungal
activity towards C. albicans strains cross-resistant to
classic antifungals: (1) nystatin, (2) ketoconazole, (3)
fluconazole, (4) terbinafine, (5) itraconazole, (6)
clotrimazole, (7) pimafucin
Human
body
side
oral
cavity
genital
tract
nails
MIC, μg/ml (resistance to antifungals)
2.0 kDa
6 kDa
12 kDa
256 (1234567)
64 (23567)
512 (123567)
32 (1234567)
8 (23567)
256 (123567)
32 (1234567)
8 (23567)
128 (123567)
64 (1234567)
512 (24)
256 (1234567)
8 (1234567)
32 (24)
64 (1234567)
8 (1234567)
32 (24)
64 (1234567)
64 (23456)
8 (23456)
8 (23456)
17
Oligochitosan demontrates fungicidal and antihyphal
activities
Flow cytometry analysis of C. albicans cells treated with
oligochitosan 10 kDa (1 MIC)
Cells status
Intact cells, %
Treated cells, %
Living cells
98.6
4.8
Membrane-damaged living cells
1.0
3.1
Membrane-damaged dead cells
0.4
80.9
Membrane-intact dead cells
0.0
11.2
Oligochitosan supresses hyphae formation (C. albicans culture)
untreated
hyphae
treated
(1/4 MIC of
oligochitosan 10 kDa)
19
Approaches to encreased oligochitosan solubility
and bioactivity at physiological pH values:
Application of biocompatible D-glucosamine suppresses
intermolecular binding
5.5
6.0
6.5
7.0
7.5
2.5
800
1.5
-5
Scattered light intensity x10 , Hz
two-phase
system
Oligochitosan:
single-phase
system
600
400
1.0
0.5
200
0.0
0
4
800
3
Oligochitosan+ glucosamine:
single phase system
2
two-phase
system
600
400
1
200
0
5.5
6.0
6.5
pH
0
7.0
7.5
Minimal ingibition concentratyion, g/ml
2.0
D-glucosamine
shifts the critical
pH of phase
separation in
oligochitosan
solution and
enhances its
antibacterial
activity in neutral
and alkaline
media
Оligochitosan Mw= 10 kDa, bacterium: E. coli
19
20
Betaineted oligochitosan
CH2OH
CH2OH
H
O
H
OH
H
H
H
H
O
...
O
...
R
m
O
H
H
O
H
H
NH
C O
O
H
OH
CH2OH
CH2OH
...
NH
C O
O
H
OH
O
H
O
H
H
H
...
C O
CH3
O
H
O
H
H
NH
n
O
H
OH
p
NH2
...
r
CH2
N+ Cl
CH3
CH3
CH3
DegreMw: 5.7—42 kDa (-NR3 + = 12%)
e of substitution: -NR3 + = 4—23 % (Mw  10 kDa)
I. V. Blagodatskikh, O. V. Vyshivannaya, A. V. Alexandrova, E. A. Bezrodnykh,
P. V. Zelenikhin, S. N. Kulikov, and V. E. Tikhonov, Antibacterial Activity and
Cytotoxicity of Betainated Oligochitosane Derivatives, Microbiology, 2018, Vol.
87, No. 5, pp. 725–731
21
Betainated oligochitosan Mw(Q 12%)/solubility relationship
I, Гц
2x10
Rh,нм
5
QХЗ Mw = 5700
2000
QХЗ Mw = 5700
QХЗ Mw = 24500
1600
QХЗ Mw = 24500
QХЗ Mw = 42000
1x10
QХЗ Mw = 42000
1200
5
800
400
0
0
2
4
6
8
10
12
14
2
pH
]
N, %
OEt,
%
Mw
pHc
(AcOH)
pHc
(NaCl)
Q2-296
12
17
5700
нет
нет
Q2-287
12
18
9500
нет
нет
Q2-277
13
14
24500
7.8
7.7
Q2-334-8
12
16
42000
7.4
7.2
4
6
8
pH
10
12
14
The higher Mw is, the
lower pHc is.
Solubility in alkaline
media at Mw 25 kDa
22
From crystalline chitin to chitosan:
α-chitin(crustacean): antiparallel chains; β-chitin (squid, sepia):
parallel chains; γ-chitin (fungi): two parallel/one antiparallel chains
23
Reacetylated oligochitosan (10kDa)
50
1
2
3
4
40
30
I/I0
20
10
4
3
2
1
Dependence of scattered
light intensity of parent
and RA-oligoCHIs
varying in DA:
1 - DA=1%
2 - DA=9%
3 - DA=16%
4 - DA=20%
in solution on pH
2 3 4 5 6 7 8 9 10 11 12 13 14
pH
I.V. Blagodatskikh, S.N. Kulikov, O.V. Vyshivannaya, E. A. Bezrodnykh, V. E. Tikhonov, NReacetylated Oligochitosan: pH Dependence of Self-assembly Properties and Antibacterial Activity,
2017, Biomacromolecules, 2017, 18(5), 1491-1498
24
25
USA and EU official documents
• “Chitosan”, The United State
Pharmacopoeia, NF31, 2013, p.1959;
• “Chitosan hydrochloride”, European
Pharmacopoeia 9.0, 2016, p. 2028-2029
26
Heavy metals and trace elements in commercial
chitosan
Element Limit, Heppe
ppm Medical
Chitosan
85/200/A1,
Chitosan T8S Chitosan LMW
Marine
Kitomer Chitosan,
BioProducts, Marinard ALDRICH,
Germany
Germany
Biotech,
Canada
USA
Chitosan
Sinochem
Qingdao
Co., China
Fe
Cr
Ni
Pb
Cd
10.0
1.0
1.0
0.50
0.20
112.0
7.92
0.91
0.70
0.020
2.3
0.72
1.75
0.05
0.005
83.4
33.51
7.90
0.18
0.003
207.3
26.5
6.31
0.46
0.015
382.5
30.31
3.82
0.18
0.006
Hg
As
0.20
0.5
0.0005
0.205
0.0150
0.005
0.0091
0.103
0.0004
0.067
0.0405
0.306
27
Oligochitosan hydrochloride preparation process
28
In vitro relative (towards control) Cow embryonic lung
(LEC) cells viability vs reacetylated chitosan molecular
weight and concentration
2.5 mg/ml
1 mg/ml
0.2 mg/ml
Relative cells viability,%
100
80
60
40
20
0
6.5
10.6
15.0
Mw, kDa
24.5
45.6
29
Unsolved problems of oligochitosan application
1.
Interaction with GIT enzymes and
microorganisms
2.
Interaction with food and cosmetic
components
3.
Digestibility of proteins, polysaccharides etc
in the presence of oligochitosan
30
In collaboration and assistance:
I.V. Blagodatskikh, O.V. Vyshivannaya,
(INEOS RAS, Moscow),
S.A. Kulikov (Institute of Epidemiology
and Microbiology, Kazan),
S.G. Markushin, (Mechnikov Institute
of vaccine and sera, Moscow ),
V.P. Kurchenko (Belorussian State
University, Minsk, Belorussia)
31
Main results are shown in:
•
•
•
•
•
•
•
•
•
•
•
B.B. Berezin, E.A. Bezrodnykh, I.V. Blagodatskikh, I. A. Yamskov, V.E. Tikhonov, EXTRACTION OF RESIDUAL HEAVY
METALS FROM COMMERCIAL CHITOSAN AND APPROACH TO PREPARATION OF OLIGOCHITOSAN (SHORT
CHAIN CHITOSAN) HYDROCLORIDE, submitted to Carbohydrate Polymers on 10.01.2019;
E.A. Bezrodnikh, I.V. Blagodatskikh, S.N. Kulikov, Pavel V. Zelenikhin, I.A. Yamskov, V. E. Tikhonov, Consequences of
chitosan decomposition by nitrous acid: approach to non-branched oligochitosan oxime, Carbohydrate Polymers, 2018, v.
195, 551-557
S.G. Markushin, I.I. Akopova, I.V. Blagodatskikh, S.N. Kulikov, Е.А. Bezrodnykh, A.V. Muranov, I.A. Yamskov, V.E.
Tikhonov, Biochemical properties of chitosan derivatives as adjuvants for vaccines, Appl. Biochem. Microbiol., 2018,
54(5), 512-517
I. V. Blagodatskikh, O. V. Vyshivannaya, A. V. Alexandrova, E. A. Bezrodnykh, P. V. Zelenikhin, S. N. Kulikov, and V. E.
Tikhonov, Antibacterial Activity and Cytotoxicity of Betainated Oligochitosane Derivatives, Microbiology, 2018, Vol. 87,
No. 5, pp. 725–731
V.P. Kurchenko, T.V. Radzevich (Butkevich), S.V. Rizevsky, V.P. Varlamov, I.V. Yakovleva, V.E. Tikhonov, L.R. Alieva,
Influence of molecular weight of chitosan on interaction with casein, Appl. Biochem.Microbiol., 2018, 54(5), 501-504
I.V. Blagodatskikh, S.N. Kulikov, O.V. Vyshivannaya, E. A. Bezrodnykh, V. E. Tikhonov, N-Reacetylated Oligochitosan:
pH Dependence of Self-assembly Properties and Antibacterial Activity, 2017, Biomacromolecules, 2017, 18(5), 14911498;
S. Kulikov, P. Zelenikhin, R. Murtazina, R. Khayrullin, E. Bezrodnikh, V. Tikhonov, Induction of apoptosis of tumor cells by
oligochitosan (short chain chitosan), BioNanoScience, 2016, DOI: 10.1007/s12668-016-0243-8;
S.N. Kulikov, L.B. Bayazitova, O.F. Tyupkina, P.V. Zelenikhina, M.M. Salnikova, E.A. Bezrodnikh, V.E. Tikhonov,
Evaluation of a method for determination of antibacterial activity of chitosan, Appl. Biochem. Microbiol. 2016, 52(5), 502507;
Kulikov S.N., Tikhonov V.E., Bezrodnykh Е.А., Lopatin S.А., Varlamov V.P., Comparative evaluation of antimicrobial
activity of oligochitosans against Klebsiella pneumoniae // Russian Journal of Bioorganic Chemistry, 2015, v.41(1), 57-62;
Antifungal activity of oligochitosans (short chain chitosans) against some Candida species and clinical isolates of C.
albicans: molecular weight – activity relationship, Sergey N. Kulikov, Svetlana A. Lisovskaya, Pavel V. Zelenikhin,
Evgeniya A. Bezrodnykh, Diana R. Shakirova, I.V. Balgodatskikh, Vladimir E. Tikhonov, Eur. J. Med. Chem., 2014, 74,
169-178
Total publications number for 2014-2018: 17 publication + 1 patent
31
Thank you for attention!
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