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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
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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.
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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 ( )
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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
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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
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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
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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).
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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
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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
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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.
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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).
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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
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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
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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).
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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.
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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.
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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 %
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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.
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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.
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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.
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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*
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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.
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a* b* b* c c Fig 4.17 : Effect of COS treatment on Lipid profile in normal and diabetic mice.
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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
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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.
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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.
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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
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HISTOPATHOLOGICAL FINDINGS
Fig: Photomicrographs of mice section pancreas (Hematoxylin- Eosin stain)
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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.
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