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ISOLATION OF ANTIOXIDANT AND ANTI-INFLAMMATORYNATURAL COMPOUNDS FROM SRI LANKAN MARINE ALGA: Pdina commersonii

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Abstract
This study was collaborative research project with Faculty of Science, University of Colombo, Sri Lanka and Marine Bio-resource Technology Laboratory, Marine Life Sciences, Jeju National University, Republic of Korea. Sri Lankan University was kindly gave us the seaweed sample or crude extract to evaluate the biological activity and isolate the active compounds. Among those samples Padina commersonii, (PC) brown algae selected for this study. PC was extracted to the 80 % methanol and get the crude extract. Crude extract was fractionate using organic solvent such as hexane, chloroform, ethyl acetate and remaining one considered as water fraction and they were named as PCH, PCC, PCE and PCW in respectively. Total phenolic content was estimated for these four fractions. Anti oxidant activity was evaluated using three types of radicals, DPPH, hydroxyl and alkyl by ESR technique. According to the results PCE was shown high amount of total poly phenol content and low IC50 values for the all three radical scavenging activities. They were 7.44 ± 0.29 GAE mg/G for the total phenolic content and 0.71±0.03 mg/mL for the DPPH radical scavenging, 0.017±0.001 mg/mL for alkyl radical scavenging and 0.25±0.06 mg/mL for the hydroxyl radical scavenging activity. Furthermore all four fractions were screen for the anti-inflammatory activity by checking the inhibition of the NO production in sample pre treated LPS induced RAW 264.7 macrophages and cell viabilities were measured after 24 h. PCE was recorded 26.14±0.9 μg/mL as low IC50 value without toxicity. PCE was exhibited better activities compared to the other three fractions and it was selected for further purification. TLC experiment was carried out to find out the best mobile phase condition for the RP-open column fractionation and 70 %, 80 %, 90% and 100 % methanol selected as suitable mobile phase condition for RP-open column fractionation and four fractions were obtained and named as PCEF1, PCEF2, PCEF3 and PCEF4 and checked antioxidant and anti-inflammatory activities. PCEF1 and PCEF2 were shown better results for the both radical scavenging and anti-inflammatory activities with 0.014±0.00 mg/mL DPPH radicalscavenging , 0.01±0.001 mg/mL for alkyl radical scavenging, 0.100±0.003 mg/mL for hydroxyl radical scavenging and 44.11±2.3 μg/mL for inhibition of NO production as IC50 values. After that all four column eluted fraction were analyzed by RP-HPLC and according to the results PCEF1 and PCEF2 were shown same chromatogram with different concentration. Therefore PCEF1 and PCEF2 were combined together and named as PCEF-A. Partition coefficients values between top and bottom phase of the CPC different solvents were calculated by RP-HPLC and 1:11:2:8 of hexane:ethyl acetate:methanol: water ratio was shown better separation in K values and it was selected for the suitable solvent system for the CPC preparative separation. After elution from the CPC 120 fractions were obtained and each other fractions were analyzed by RP-HPLC and identified the same fractions and pooled them together. Depending on the results there we three partially pure compounds and they were subjected to the further activity checking. That three compounds were named as PCEF-A-1, PCEF-A-2 and PCEF-A-3. They were tested for the sample toxicity by treating with different concentration on Vero cells and checking by cell viability. According to the results all three compounds were not observed sample toxicity for the tested concentration. All three compounds were tested for the intracellular ROS scavenging activity by treating on Vero cells and after 1 h treated with H2O2 and scavenging ability of the H2O2 was evaluated by DCFDA assay and PCEF-A-1 was recorded better ROS scavenging activity compared to other two compound. In addition cell viabilities were calculated under H2O2 oxidative stress and to that pre sample treated Vero cell were treated with H2O2 and incubated for 24 h and checked the cell viabilities and PCEF-A-1 was shown protective effect under H2O2 oxidative stress. In addition compounds were tested for the apoptotic body formation in Vero cells. All these compounds were shown low number of apoptotic body with high amount cell population at high concentration and at low concentration all three compounds wee shown low cell population and high number of apoptotic body formation. Furthermore all threecompounds tested for antioxidant activities in zbrafish embryos and PCEF-A-1 was shown protective effect in zebrafish.
Author(s)
H.H. CHAMINDA LAKMAL
Issued Date
2014
Awarded Date
2015. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000007165
Department
대학원 해양생명과학과
Table Of Contents
1. INTRODUCTION 1
1.1.Medicinal natural products 3
1.2.Marine natural products 4
1.3.Availability of seaweed and studies in Sri Lanka 5
1.4.Brown seaweed Padina commersonii 8
1.5.Reactive Oxygen species 9
1.6.Importance of natural antioxidant 12
1.7.Inflammation and importance of anti-inflammatory natural products 13
2. METERIALS AND METHODS 14
2.1. Chemicals and reagents 14
2.2. Sample collection 14
2.3. Extraction of 80 % crude methanol extract 15
2.4. Fractionation of the 80 % methanol crude extract 15
2.5. Sample preparation 16
2.6. Determination of total phenolic content 16
2.7. DPPH radical scavenging assay 16
2.8. Hydroxyl radical scavenging assay 17
2.9. Alkyl radical scavenging assay 19
2.10. Cell cultures 19
2.11. Determination of sample toxicity on Vero Cells 20
2.12. Determination of anti-inflammatory activity by measurement of NO concentration on RAW 264.7 cells 20
2.13. Thin Layer Chromatography (TLC) analysis of PCE 21
2.14. Open column fractionation of the PCE 22
2.15. Radical scavenging activity assessment of the open column eluted fractions of PCE 22
2.16. Anti-inflammatory activity assessment of the open column eluted fraction of PCE 22
2.17. HPLC analysis of the open column eluted fractions 23
2.18. Investigation of antioxidant activity by the ABTS+ Online HPLC 23
2.19. High Performance Centrifugal Partition Chromatographic Solvent System 25
2.20. Centrifugal Partition Chromatographic Separation 26
2.21. NMR spectroscopy of the Compounds 28
2.22. LC-MS/MS analysis 28
2.23. Sample toxicity on Vero cells 28
2.24. Antioxidant activity on Vero cells 29
2.25. Assessment of intracellular H2O2 scavenging ability of compounds by DCF-DA assay 29
2.26. Evaluation of apoptotic body formation by nuclear staining with Hoechest 33342 30
2.27. Origin and maintenance of the zebrafish 30
2.28. Waterborne exposure of compounds and hydrogen peroxide to the embryos 31
2.29. Measurement of heart rate and survival rate 31
2.30. Intracellular ROS and cell death estimation in zebrafish embros 31
2.31. Statistical analysis 32
3. RESULTS AND DISCUSSION 33
3.1. Total phenolic content 33
3.2. DPPH radical scavenging activity 34
3.3. Hydroxyl radical scavenging activity 34
3.4. Alkyl radical scavenging activity 35
3.5. Anti-inflammatory activity by measurement of concentration on RAW 264.7 macrophages 38
3.6. Thin Layer Chromatographic analysis of PCE 41
3.7. Reverse phase open column fractionation of the PCE 43
3.8. Radical scavenging activity of the open column eluted fractions of PCE 45
3.9. Anti-inflammatory activity of the open column eluted fractions of PCE 45
3.10. HPLC investigation of PCE and Column fractions 49
3.11. Investigation of antioxidant activity by the ABTS+ onlie HPLC 49
3.12. Determination of High performance Cenrifugal Partitiom chromatographic solvent system and preparative isolation of the compounds 52
3.13. NMR spectroscopy of the compounds 56
3.14. LC/MS/MS analysis of the compounds 56
3.15. Sample toxicity of compounds on Vero cells. 62
3.16. Antioxidant activity assay on Vero cells 62
3.17. Evaluation of apoptotic body formation by nuclear staining with Hoechest 33342 65
3.18. Heart rate and survival rate of zebrafish under oxidative stress 67
3.19. ROS generation and cell death estimation in zebrafish under oxidative stress 70
4. CONCLUSION 73
REFERENCES 74
ACKNOWLEADGEMENT 77
Degree
Master
Publisher
제주대학교 대학원
Citation
H.H. CHAMINDA LAKMAL. (2014). ISOLATION OF ANTIOXIDANT AND ANTI-INFLAMMATORYNATURAL COMPOUNDS FROM SRI LANKAN MARINE ALGA: Pdina commersonii
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General Graduate School > Marine Life Sciences
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