1. Presented by Dr. Bharti Singh, Director VitaeGen Biotech, Varanasi
Biomedical Application of Natural Molecule Chitosan and its Chitooligosaccharides
Presented by
Dr. Bharti Singh, Director
VitaeGen Biotech, Varanasi

2. INTRODUCTION
Chitosan is a natural polycationic biopolymer derived by deacetylation of chitin and can be obtain from crustacean shells, cell wall of fungi and algae (Tesson et al., 2008).
Chitosan has attracted tremendous attention as a potentially important resource for its biological properties including nontoxicity, biodegradability, and biocompatibility absorption properties but due to poor solubility long chain polymer makes them difficult to use as biomedicine (Rinaudo et al., 2006).
The chitooligosaccharides (COS) is a highly potent molecules used as biomedicine because COS is water-soluble due to their shorter chain and free amino groups in D glucosamine units and easily absorbed through the intestine, can quickly get into the blood flow and have systemic biological effects in the organism.

3. Chitin: a brief history
1811 Chitin was first discovered by Professor Henri Braconnot, who isolated it from mushrooms and name it “Fungine”
1823 Antoine Odier found chitin while studying beetle cuticles and named “chitin” after Greek word “chiton” (tunic, envelope)
1838 Cellulose was discovered and noted
1859 Rought discovered chitosan, a derivative of chitin..
1920s Production of chitin fibers from different solvent systems
1930s Exploration of synthetic fibers
1950s The structure of chitin and chitosan was identified by X-ray diffraction, infrared spectra, and enzymatic analysis
1970s “Re-discovery” of the interest in chitin and chitosan
1977 1st international conference on chitin/chitosan
Henri Braconnot ( )

4. Chitin: a promising material
Unique characteristics of chitin and chitosan:
Biocompatible
Biodegradable
Non-toxic
Remarkable affinity to proteins
Ability to be functionalized
Renewable
Abundant
Muzzarelli R. et al, Chitin in Nature and Technology. Plenum Press NY, 1985

5. Chitin is a Co-polymer Pure chitin does not exist in reality
Chitin and chitosan tend to form co- polymer
# of N-acetyl-glucosamine units > 50% => Chitin
# of N-glucosamine units > 50% => Chitosan
Degree of N-acetylation, DA = acetamido / (acetamido+amino)
Degree of N-deacetylation, DD = amino / (acetamido+amino)
In nature, chitin is commonly 70~90%
Structure of Chitin-Chitosan co-polymer
Kohr E. Chitin: fulfilling a biomaterials promise. Elsevier Science, 2001

6. Crystalline structure
Chitin has 3 polymorphic forms: α-chitin, β-chitin, γ-chitin
α-chitin: the most abundant form anti-parallel configuration highly ordered crystalline structure strong H-bonding (N-H····O=C) rigid, intractable, insoluble
β-chitin:
- found in diatom spines and squid pens - parallel configuration weak H-bonding unstable, soluble in water
γ-chitin:
- mixture of α and β-chitin intermediate properties
[1]
H-bonding in α-chitin
H-bonding in β-chitin
[2]
[1] Muzzarelli R. Chitin. Pergamon Press, 1977
[2] Kohr E. Chitin: fulfilling a biomaterials promise. Elsevier Science, 2001

7. Diabetes Mellitus- Outline
Diabetes mellitus (DM) is a chronic disease & a heterogeneous group of metabolic disorders characterized by insulin activity and clinically by hyperglycemia and other manifestable disorders (WHO, 1994).
Chronic hyperglycemia is associated with long-term damage, dysfunction and eventually the failure of organs (Susheela et al., 2008). Diabetics exhibit high oxidative stress markers and ROS in pancreatic islets due to persistent and chronic hyperglycemia (Kanter, 2003).
COS can be attract a greater interest as antidiabetic agents because COS are readily absorbed through the intestine, can quickly get into the blood flow and have systemic biological effects in the organism (Katiyar et al., 2011).

10. Table 1: Composition of f/2 Medium culture medium for Diatom growth
Isolation of Diatom
Table 1: Composition of f/2 Medium culture medium for Diatom growth
Component
Stock Solution
Quantity
Molar Conc.
NaNO3
75 g/L dH2O
1 mL
8.82 x 10-4 M
NaH2PO4 H2O
5 g/L dH2O
3.62 x 10-5 M
Na2SiO3 9H2O
30 g/L dH2O
1.06 x 10-4 M
trace metal solution
(see Table 2)
---
vitamin solution
(see Table 3)
0.5 mL
Sample site
Table 2: Composition of f/2 Trace Metal Solution culture medium for Diatom
Component
Primary Stock
Quantity
Final M Conc.
FeCl3 6H2O
---
3.15 g
1.17 x 10-5 M
Na2EDTA2H2O
4.36 g
CuSO4 5H2O
9.8 g/L dH2O
1.0 mL
3.93 x 10-8 M
Na2MoO42H2O
6.3 g/L dH2O
2.60 x 10-8 M
ZnSO4 7H2O
22.0 g/L dH2O
7.65 x 10-8 M
CoCl2 6H2O
10.0 g/L dH2O
4.20 x 10-8 M
MnCl2 4H2O
180.0 g/LdH2O
9.10 x 10-7 M
Substratum having diatom

11. Left part further centrifuged 2000 × g for 20 min.
Culture
Centrifuge at 1500 × g for 20 min.
Supernatant decanted
Left part further centrifuged 2000 × g for 20 min.
Supernatant was collected and centrifuged at × g for 30 min.
5 % KOH is added in the precipitate
Kept for 12 hrs. at room temp. & Filtered
Chitin was collected & dispersed in methanol
Maintained 600C for 2 hrs. & again filter
The filterate was dispersed in 0.1M/L aq. Hydrofluoric acid
Boiled for 1 hr to dissolve component & filtered

12. Dispersed in 0.34% aq. NaClO2 and maintained at 800C for 2 hrs
Filtration
Pellet was dispersed in 1% aq. Hydrofluoric acid
Allow to stand at room temp. for 12 hr.
Insoluble matter collect by filtration, dried & purified Chitin was obtained
Chitin hydrate was dried at 1050C for 2 hr. to obtain anhydrous chitin.
For the preparation of Chitosan , Chitin was treated with NaOH at a concentration of 30-50% w/v at high temperature (800C) for 1 hr.
Chitosan was treated with papain to obtain Chitoligosaccharides (COS)
Characterization by FTIR chitin, Chitosan Chitoligosaccharides (COS)
The extraction of chitin from diatoms followed by method of Edwin and Werner, (1995) with some slight modification.

13. Research Findings Isolation of Diatoms Yield of Chitin
4 centric diatoms-Cyclotella, Thalassiosira and Skeletonema & Melosira.
Chitin of 120 mg-180 mg / gm ( %) of the diatoms biomass weight.
Fig1:The growth of diatom genera Cyclotella, Thalassiosira, Skeltonema & Melosira (mean± SE).

14. a b
Fig 3: General morphological structure of Diatoms (SEM Pictures) 1. Cyclotella meneghiniana, 2.Thallassiosira 3. Melosira 4.Skeletonema
Fig2: Photomicrographs of centric diatoms a. Cyclotella b. Melosira

15. Characterization by FTIR
FTIR spectra are usually recorded in the middle infrared (4000 cm-1 to 400 cm-1)
OBSERVATION
STANDARD
EXTRACTED
O-H Stretching
3450 cm-1
cm-1
C-H stretching
cm-1
cm-1
Amide I (N-H)
1661 cm-1
cm-1
Amide II
1560 cm-1
cm-1
CH2 bending
1420 cm-1
cm-1
C-O-C stretch
1070 cm-1
cm-1
C-O-C bridge
897.0 cm-1
cm-1

16. Characterization of Chitosan
Fig. 4: FTIR spectra of chitosan
In the IR spectra of chitosan cm-1 (O-H stretching overlapping the N-H stretching), 2930and 2366 cm-1 (C-H stretching), cm-1 (amide band, N-H stretching) 1463–1390 cm-1 (asymmetrical C-H bending of the CH2 group) and cm-1 (O bridge stretching) of the glucosamine residue (Domszy and Roberts, 1985).

17. Characterization of COS
Fig. 5: FTIR spectra of chitooligosaccharides
A slight shift in the peak around cm-1 (chitosan) was indicated at cm-1In this figure (OH stretching) of orderliness of the product probably due to increase in DA. The region of amide I shifts to (1626–1633 cm-1) cm-1 in this figure. The region between , is considered conformation sensitive for polysaccharide which depend on the orientation of OH group and its shift in the chitooligosaccharides compared with chitosan.

18. Experimental Design
Group1: Normal control mice received only distilled water during the experimental
Group 2: Diabetic control- freshly prepared alloxan (a single dose of 150 mg/kg b.w. through ip)
Group 3: Diabetic mice were daily administered with COS (LD) at a dose of 5 mg/kg b.w. (125µg/25 µl) ip.
Group 4: Diabetic mice were daily administered with COS (HD) at a dose of 10 mg/kg b.w.(250µg/25 µl) ip.
Group 5: Normal mice were daily administered with COS (LD) at a dose of 5 mg/kg b.w. ip for 21st days.
Group 6: Normal mice were daily administered with COS (HD) at a dose of 10mg/kg b.w. (250µg/25 µl) ip.

20. Antidiabetic Effect of COS 21 Days after administration
Body weight
Table 1: Changes in Body Weight of control, diabetic and COS treated mice. (mean ± SE, n=6, p<0.05)
Group
Treatments
(Dose/Kg b.w.)
Body weight (gm)
Initial Day
7 Days after Alloxan administered
21 Days after administration I
Control
24.3 ± .88
29.1 ± .40
29.6 ± .21 II
Diabetic
27.0 ± 1.63
22.1 ± .94
21.1 ± .90
III
D + COS (LD)
25.8 ± .83
23.0 ± .57
25.8 ± 1.22 IV
D + COS (HD)
27.0 ± .88
20.8 ± .94
24.8 ± .30 V
COS(LD)
25.6 ± .83
27.6 ± .91
29.5 ± .56 VI
COS(HD)
26.1 ± .41
28.5 ± .50
29.1 ± .60
C.D at 1 %
C.D at 5 %

21. Fig 6 : Effect COS on body weight changes in diabetic and Normal mice.
Data are expressed as mean ± S.E of 6 mice in each group. Values are statistically significant at p < Significance determined by ANOVA was compared within the group as follow: a alloxan induced diabetic group vs control; b D + COS (LD) and D + COS (HD) group vs alloxan induced diabetic group; c COS (LD) and COS (HD) vs control group. In graph figures bearing *is significantly different at p < 0.05 between the group.

22. Fig 8 : Effect of COS on glycogen levels in diabetic and normal mice.
Groups
Treatments
(Dose mg/Kg b.wt.)
Glycogen I
Control
50.0 ± .73 II
Diabetic
9.8 ± .69
III
D + COS(LD)
37.1 ± 1.85 IV
D + COS(HD)
44.9 ± .58 V
COS(LD)
51.3 ± 1.57 VI
COS(HD)
43.9 ± .81 c b* c b* a*
Fig 8 : Effect of COS on glycogen levels in diabetic and normal mice.

23. SGPT and SGOT Groups Treatments (Dose mg/Kg b.wt.) SGPT (IU/L)
SGOT (IU/L) I
Control
38.4 ± .78
56.0 ± 1.54 II
Diabetic
96.4 ± 4.73
143.8 ± 5.65
III
D + COS (LD)
66.2 ± 1.70
108.4 ± 3.48 IV
D + COS (HD)
53.7 ± 2.17
83.5 ± 2.76 V
COS (LD)
39.9 ± 2.75
60.5 ± 2.92 VI
COS (HD)
44.2 ± 3.20
59.2 ± 3.80 a* a* b* b* b* b* c c c c
Fig 9 : Effect of COS treatment on SGPT in normal and diabetic mice.

24. HYPOLIPIDEMIC ACTIVITY OF COS
Groups
Treatments
(Dose mg/Kg b.wt.)
Total cholesterol
Triglycerides I
Control
144.5 ± 4.64
120.5 ± 4.46 II
Diabetic
375.8 ± 13.3
239.8 ± 3.31
III
D + COS(LD)
182.3 ± 4.30
173.1 ± 4.00 IV
D + COS(HD)
153.6 ± 3.26
168.5 ±10.58 V
COS(LD)
142.3 ± 4.02
133.5 ± 5.91 VI
COS(HD)
148.3± 3.21
129.3 ± 6.00
TC- CD at 1% = CD at 5% = TG - CD at 1% = CD at 5% = 17.85 a* a* b* b* b* b*

25. a* a* b* b* b* b* b* b* a*
Fig : Effect of COS treatment on LDL-cholesterol , VLDL-cholesterol HDL-cholesterol levels in normal and diabetic mice.

26. a* b* b* c c
Fig 4.17 : Effect of COS treatment on Lipid profile in normal and diabetic mice.

27. ACTION OF COS ON HYPERLIPIDEMIA
MOBILIZATION OF FATTY ACID FROM ADIPOSE TISSUE
Alloxan
Destroys B cells
HYPERGLYCEMIA
COS
Positively charged
Binds to FREE FATTY ACIDS
EXCESS ACCULULATION OF FREE FATTY ACID IN LIVER
Binds to BILE ACIDS
Increase rate of LDL LOSS as bile formed from Cholesterol of LDL
CONVERTED TO TRIGLYCERIDES
IMPROVES LDL & HDL RATIOS
ELEVATED LEVEL OF VLDL,LDL,IDL
LCAT activated , Thus HDL Cholesterol decreases

28. ANTIOXIDATIVE ACTIVITIES OF COS
Groups
Treatments
(Dose mg/Kg b.w.)
Superoxide Dismutase
Catalase
IU/mg Protein
IU/g tissue wt.
(nM of H2O2
/min./mg Protein) I
Control
12.2 ± 0.79
321.0 ± 14.3
53.7 ± 6.2 II
Diabetic
0.38 ± 0.01
4.60 ± 1.3
13.7 ± 2.07
III
D + COS(LD)
8.46 ± 0.82
227.9 ± 8.07
48.28 ± 1.53 IV
D + COS(HD)
11.43 ± 0.92
320.4 ± 9.69
51.25 ± 2.10 V
COS(LD)
12.63 ± 0.50
± 3.84
56.5 ± 4.65 VI
COS(HD)
13.1 ± 1.04
235.8 ± 18.7
60.6 ± 4.04 b* b* b* b* a* a*
Fig : Effect of COS treatment on liver super oxide dismutase level in normal and diabetic mice.

29. Groups Treatments (Dose mg/Kg b.wt.) MDA nM of MDA/mg Protein
nM of MDA/g tissue wt. I
Control
15.2 ± 0.8
44.5 ± 2.4 II
Diabetic
105.5 ± 5.3
181.5 ± 9.1
III
D + COS(LD)
21.7 ± 0.95
38.1 ± 1.7 IV
D + COS(HD)
25.2 ± 2.3
49.9 ± 4.5 V
COS(LD)
13.4 ± 0.57
38.7 ± 1.66 VI
COS(HD)
13.3 ± 0.56
41.8 ± 1.8
CD at 1% = 9.56
CD at 5% = 7.10 a* b* b*
Fig : Effect of COS treatment on liver Malonaldehyde levels in normal and diabetic mice.

30. PARAMETERS
10 mg/Kg b.w. COS
5 mg/Kg b.w. COS
BLOOD GLUCOSE
74.10
40.5
GLYCOGEN
64.9
59.3
SGPT
50.2
44.2
SGOT
41.9
37.1
CREATININE
67.69
57.69
UREA
55.0
45.8
TOTAL CHOLESTEROL
59.12
53.8
TRIGLYCERIDE
63.4
61.3
LDL
71.7
67.3
SOD
70.3
62.1
CATALASE
66.6
59.2
MDA
79.4
76.11

31. HISTOPATHOLOGICAL FINDINGS
Fig: Photomicrographs of mice section pancreas (Hematoxylin- Eosin stain)

32. The four different genera of Diatoms were isolated i. e
The four different genera of Diatoms were isolated i.e., Cyclotella, Thalassiosira, Skeletonema and Melosira. The Diatom has a good amount of chitin in their frustules. The isolated Chitin from Diatom was confirmed by FTIR spectroscopy.
The COS was hypoglycemic agent and highly effective in managing the complication associated with diabetes mellitus such as hypercholesterimia, hypertriglyceridimia and impaired renal function. The 10 mg/kg b.w of COS was showing significant dose compared 5 mg/kg b.w. The COS also act as antioxidant agent.
This present research findings proved that this study is helpful for developing new drugs from micro algae diatom for managing diabetes and associated complications. The COS seems promising for the development of a new medicine for diabetes mellitus.