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Characterization of Compds found in Irish Moss

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A project done on the characterization of ompounds found in irish moss

A project done on the characterization of ompounds found in irish moss

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  • Have you ever had any raw? Try a cup of soaked, washed, rinsed raw Irish Moss in your smoothie, and you will see you are wrong.
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  • 1. The investigation of the chemical composition of the algal species used in the preparation of Irish Moss By Kenn Thomas
  • 2. What is Irish Moss? • Irish moss refers to a drink derived from the “Irish Moss”( Chondrus crispus,and Girgatina stellata ) species. • Local small scale producers of Irish moss rely on several species of algae (Gracilaria sp. and Euchema sp) as raw material.This was confirmed by a collector of Irish Moss at the sample site. • Gracilaria is grown commercially in the island of St.Lucia,where it is called Sea Moss. • The drink has a local folklore reputation as an aphrodisiac and energy enhancer • The Aim of this project was to identify the chemical components of Irish Moss and identify biologically active compounds by cross referencing our results with the literature • Sample consisted of Gracilaria sp.
  • 3. Gracilaria • Height of Gracilaria sp. can range from short (10cm, stiff, heavily branched) to long (60m, slender) • Phycocolloid (long chain polysaccharide) produced by Gracilaria sp. is agar or agaroid in nature • Used in food and pharmaceuticals in East Asia. • Gracilaria sp. are a good source of vitamins A, C, B2 calcium, iron and other micronutrients
  • 4. 100 known Gracilaria sp.
  • 5. G. eudelis H H H H HO H H OH HO OH poriferast-5-en-3B,7B-diol 5poriferastane-5-en-3,6a-diol
  • 6. Gracilaria verrucosa Cl O Cl O OH O 2-chlorododec-2en-1-ol OH 3-nonyloxirane-2-carboxylic methyl ester 2-chlorododec-2,11-dien-1-ol
  • 7. Sample Collection
  • 8. Location Of Sample Leith Hall St. Thomas Jamaica
  • 9. Sample Site 1(collection of sample A) Date -11th September 2006 Sample Area 1 -Rope attached to Buoy -Depth 3ft
  • 10. SAMPLE SITE 2(Collection of Sample B) Prospect St.Thomas Jamaica 18th September 2006
  • 11. SAMPLE SITE 3 (Collection of Sample B) -Date 18th September 2006 -location estuary at Leith Hall -several meters from Site 1 Sample Site 1 Sample site 3
  • 12. Bulk Sample (Algal Mat)
  • 13. Several Species of Algae in Sample
  • 14. Species Identification Gracilaria 1 Gracilaria 2 Gracilaria 3 Gracilaria 4
  • 15. Species Identification Identifed by an expert in Algal Species Gracilaria cutisaie Gracilaria gracolis Gracilaria domengensis* Gracilaria tiloanhies
  • 16. Methodology Solvent Extraction 1.Das B., Srinivas KVNS, Dihydroxysterols from the marine red Gracilaria eudelis, Phytochemistry 31,4371-4373 2.Das B. et al, 5-alpha-poriferast-9(11)-en-3-ol from the marine red Gracilaria eudelis., Phytochemistry 31, 1054-1055 •Wash Sample(20kg) H •Dry Sample •Extract with Hexane,CHCl3,MeOH(5 days per solvent) H H •Column Chromatography EtOAc-Hexane HO •1D and 2D NMR Analysis OH 5a-poriferastane-5-en-3,6a-diol H H H HO OH poriferast-5-en-3,7-diol
  • 17. Gracilaria Drying Process Colour transition, Red or green to white Air dried for 4 days
  • 18. Measurement of Dry Weight Sample A 153.6g Sample B 767.6g
  • 19. Samples and Duplicates for Solvent Extraction Sample A1 25.67g Sample B1 180.26 g Sample B2 180.33g Sample A2 25.67g Sample A3 25.66g Sample A4 25.66g
  • 20. Process Diagram Of the Chemical Investigation of Dried Gracilaria Sample Dried Sample A Boil in H2O Hexane Extraction EtOAc-H2O Extraction Hydrolyse Aqueous TLC layer Solid Phase NMR, HSQC, HMBC extraction TLC GCMS, 1HNMR
  • 21. Process Diagram Of the Chemical Investigation of Dried Gracilaria Sample Dried Sample Ca++ and Ash CH2Cl2 MeOH content Extraction Extraction determination Filter insoluble TLC Ash sample precipitate TlC MeOH Digest Ashed Silica Column Chromatography Soluble Sample Standardize Size Exclusion TlC EGTA with Chromatography CaCO3 Sample + TLC HNMR , GCMS Blank+ results 1 GCMS , H-NMR 1 C-NMR 13 Hydrolysis of MeOH insoluble HNMR, C-NMR, 13
  • 22. Solvent Extraction Samples A and B B.1 B.2 A.2 A.1
  • 23. Masses of Residues Extracted B Sample A Total Mass /g Hexane Dichloromethane Methanol A.1 25.67 0.1041 0.0271 3.4396 A.2 25.67 0.0828 0.0538 2.5477 Extraction time/hr 64:07:00 73:55:00 51:15:00 Sample B Sample Hexane Dichloromethane Methanol Total Mass B.1 180.26 0.1494 0.2324 18.2653 B.2 180.33 0.1936 0.1895 15.7436 Extraction 77:05:00 67:05:00 53:10:00 time /hr
  • 24. PRELIMINARY 1H-NMR PRELIMINARY ANALYSIS OF EXTRACTS Solvent CDCl3
  • 25. Purification of Hexane Extract
  • 26. Column Chromatography of Dichloromethane Extract
  • 27. Methanol Extract Brown Residue Insoluble White Precipitate
  • 28. GC-MS Analysis • Automated HP 6890 Gas • HP 5973 Mass Selective Detector.
  • 29. RESULTS
  • 30. Hexane Extract Sample A Preliminary 1H-NMR Analysis of Hexane Extract
  • 31. H-NMR Analysis of Purified Extract 1 Fraction 4 Fraction 5 Fraction 6
  • 32. Fraction 9 Fraction 10 Fraction 11
  • 33. GCMS Analysis of Hexane Extract Fraction 1+2 Compound Structure Retention Area time /min /% 12.02 14.39 heptadecane
  • 34. GCMS Analysis of Hexane Fraction 4 Abundance T IC : W G 0 1 0 .D 2800000 2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .0 0 1 8 .0 0 2 0 .0 0 T im e -->
  • 35. Retention Structure Constituent Area% Time /min O 5.30 13.03 methyl tetradecanoate O O 1.02 14.07 methyl 9-methyltetradecanoate O O 1.99 14.53 6,10,14-trimethyl -2 pentadecanone O 19.85 9-hexadecenoic acid,methyl ester 14.97 O hexadecanoic acid,methyl ester O 36.95 15.08 (methyl palmitate ) O
  • 36. DICHLOROMETHANE EXTRACT
  • 37. Preliminary 1H-NMR Analysis of Dichloromethane Extract
  • 38. HNMR Analysis of Dichloromethane Fractions Fraction 1 Fraction 2+3 Fraction 4-6 Fraction 7-10
  • 39. Fraction 11 Fraction 12 Fraction 15+16 Fraction 17-19
  • 40. Dichloromethane Fraction 7 H-NMR 1 CNMR 13
  • 41. 2D NMR ANALYSIS H-1H COSY 1 HMBC HSQC
  • 42. Dichloromethane Fraction 7 2D NMR Experiments HSQC HMBC H-1H COSY 1
  • 43. Fragment of Compound A HH O O 3.75ppm H 71.71ppm 19.27ppm 31.69ppm H H 61.80ppm 3.50ppm H O 3.55ppm H 1.59ppm 2.1.9ppm 1 H-1H COSYCorrelations ObservedHMBC Correlations COSY Position(s) H C HMBC [ 1H-13C] [ 1H-1H] 1 13 d 1.59 31.69 - 1.37 c 3.50(1H,t) 71.11 19.27,71.71 3.75 a 3.55 71.71 19.76,71.11 3.75 b 3.75 (1H,t) 71.71 - -
  • 44. Compound Fragment B O 4 3 1 O 2 COSY Position(s) H C HMBC [ 1H-13C] [ 1H-1H] 1 13 4? 1.27 22.69 - - 4? 1.27 29.14 - - 4? 1.27 29.25 - - 2 2.49 40.79 169.18,67.63 5.28 2 2.62 40.79 71.71 - 3 5.28 67.63 19.76,40.79 1.28 Infrared analysis of WGKT7-10 0.8 0.7 0.6 0.5 0.4 I.R Analysis 0.3 0.2 0.1 0 COO- 4000 3746 3237 2727 2473 1964 1709 1454 1200 3491 2982 2218 945 691 stretch cm-1
  • 45. GC-MS Dichloromethane Fraction 7-10 A b u n d a n c e T IC : W G 0 0 1 8 A .D 2 8 0 0 0 0 0 2 6 0 0 0 0 0 O 2 4 0 0 0 0 0 H 2-nonyl-Cyclopropaneundecanal 2 2 0 0 0 0 0 2 0 0 0 0 0 0 O O 1 8 0 0 0 0 0 O methyl palmitate 1 6 0 0 0 0 0 H 1 4 0 0 0 0 0 (Z)- 9-Octadecenal 1 2 0 0 0 0 0 1 0 0 0 0 0 0 8 0 0 0 0 0 6 0 0 0 0 0 4 0 0 0 0 0 2 0 0 0 0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .0 0 1 8 .0 0 2 0 .0 0 T im e -->
  • 46. Retention Constituent Structure Area% Time /min O Hexanedecanoic Acid 15.08 2.09 methyl ester O O OH Eicosanoic acid 18.15 0.21 18.30 1.88 O 11-(2-nonylcyclopropyl)undecanal O 1.88 9-Octadecenal, (Z)- 18.30
  • 47. GC-MS Dichloromethane Fraction 2-3 A b u n d a n c e T IC : W G 0 0 1 8 A .D 2 8 0 0 0 0 0 2 6 0 0 0 0 0 2 4 0 0 0 0 0 2 2 0 0 0 0 0 2 0 0 0 0 0 0 1 8 0 0 0 0 0 1 6 0 0 0 0 0 1 4 0 0 0 0 0 1 2 0 0 0 0 0 1 0 0 0 0 0 0 8 0 0 0 0 0 6 0 0 0 0 0 4 0 0 0 0 0 2 0 0 0 0 0 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .0 0 1 8 .0 0 2 0 .0 0 T im e -->
  • 48. Retention Constituent Structure Area% Time /min 5.24 Heptadecane 12.02 O methyl 14-methylpentadecanoate 15.07 1.11 O
  • 49. Bioactivity of Compounds found • 9- octadecenal is a pheromone of two species of moths, Diatraea grandiosella(secreted by the female), Scirpophaga incertulas • .Palmitic acid as well as several other naturally occurring fatty acids and their derivatives inhibit cyclooxygenase-2(COX- 2) catalysed prostaglandin synthesis.
  • 50. METHANOL EXTRACT
  • 51. Preliminary 1H-NMR Analysis of Methanol Extract
  • 52. Purified Methanol Extract Fraction 1-3 Fraction 4 Fraction 6
  • 53. Fraction 8 Fraction 10 Fraction 13 Fraction 16
  • 54. GC-MS Results: Methanol Extract Fraction 15 A bundanc e T IC : W G 0016A .D 2800000 2600000 2400000 2200000 2000000 H 1800000 O H H O 1600000 1400000 H 1200000 H H 1000000 800000 600000 400000 200000 4 .0 0 6 .0 0 8 .0 0 1 0 .0 0 1 2 .0 0 1 4 .0 0 1 6 .0 0 1 8 .0 0 2 0 .0 0 T im e -->
  • 55. Retention Constituent Structure Area% Time /min 15.83 3.40 16.02 3.67 16.26 7.00 Cholest-5-en-3-ol (3.beta.)-, nonanoate H O H H O 15.74 to 16.87 Cholesta-3,5-diene H H H
  • 56. • No literature data regarding the bioactivity of these components could be found
  • 57. NMR Analysis of Methanol Insoluble Precipitate
  • 58. EtOAc-H2O Extraction
  • 59. Methodology Filter to remove Algae Boil in distilled H2O Extract with ethyl acetate NMR, GCMS Analysis 1hr 6 6 5 5 7 7 4 4 8 8 3 3 9 9 2 2 11 10 1 1 3 Fractions Obtained 1.)Aqueous 2.)Emulsion layer 3.)Organic layer
  • 60. Analysis EtOAc-H2O Extracts • Organic extract- analysed by 1HNMR and GCMS • Aqueous Extract - a method for the analysis of carrageenan could not be found in time hence the Phenol-Sulfuric Assay was adapted
  • 61. Phenol Sulfuric Acid Carbohydrate Assay Colourimetric Method Based on conjugation between Phenol and the hydrolysed carbohydrate 5% phenol Neutralize with NaOH Conc. H2SO4 H eO M c OA in e Et olv th ss wi Di ct tra Ex NMR Analysis
  • 62. NMR ANALYSIS OF H2O-EtOAc EXTRACTS
  • 63. H-NMR Analysis Of Organic layer 1
  • 64. The GCMS analysis of the Organic layer was inconclusive
  • 65. Aqueous Layer After Phenol Sulfuric Acid Assay H-NMR Analysis 1 C-NMRAnalysis 13
  • 66. Ethoxybenzene generated from Phenol /sulphuric assay of aqueous extract H H 8a 9a H O C C H 6a 8 9 9c H C C H 9b 6 1 8b H C C H 5a 5 2 2a C C 4 3 H H 4a 3a
  • 67. • Calcium and Ash Analysis
  • 68. Calcium and Ash Analysis 0 10 20 30 40 50 6 6 5 5 7 7 4 4 8 8 3 3 9 9 2 2 11 10 1 1 •Ash 2g Sample in duplicate O HO •.Digested with conc. Sulfuric Acid HO N O O O N OH •Neutralized NaOH O O HO •PH 11 Buffer solution(50 ml) 3,12-Bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic acid EGTA •Titrate 25ml of sample Solution with Standardized EGTA EGTA-Ca2+-EGTA 2EGTA + Ca2+ •Blank titration with distilled water
  • 69. Analysis of Ash Content of Gracilaria Sp. Sample 1 2 Mass of sample before ashing /g 2.0164 1.9982 Mass of sample after ashing /g 0.581 0.5408 Percentage Ash (%) 28.81 27.06 Error +/- 2.61%
  • 70. Masses of Reagents used Volume Concentration Error +/- Mass/g RMM # of moles /dm Moldm moles 3 -3 CaCO3 5.0033 100 0.05 1 0.05 2.00E-06 NaOH 4.0091 40 0.1 1 0.1002 8.04E-06 EGTA 9.506 380.35 0.025 1 0.025 5.00E-07 Titration of Sample 1 with EGTA run rough 1 2 initial volume /ml 0.3 0.22 1.69 final volume /ml 27.5 28.1 29.52 titre Volume /ml 27.2 27.88 27.83 Average Volume/ml 27.855
  • 71. O HO HO N O O O N OH O O EGTA-Ca2+-EGTA HO 2EGTA + Ca2+ 3 -B ,12 is(ca xym th rbo e yl)-6,9-dio -3 -dia te d can ioic a xa ,12 za tra e ed cid ETGA # Of moles Ca++= 0.025dm3 x 0.05M = 1.25E-03 moles # Of moles of EGTA= (# of moles Ca++) x 2 =2.50E-3 moles
  • 72. •Titration of Sample 1 with EGTA run rough 1 2 3 blank initial volume 0.65 5.36 7.952 10.513 5.991 /ml final volume /ml 5.36 7.952 10.513 13.11 6.23 titre Volume /ml 4.71 2.592 2.561 2.597 0.239 Blank subtraction 4.471 2.353 2.322 2.358 0 Average volume 2.344 /ml Tolerance+/- 0.03 ml, error 4.243E-03 ml Calculation of Ca ++ Concentration # of moles of EGTA transferred=2.344x 10-4 dm3 x 2.50E-03 moles =5.86E-07 moles # Of moles Ca ++ = 5.86E-07 moles/2 =2.93E-07 moles Mass of Ca++ present = 2.93E-07 moles x 40.078g/mol =1.1743E-05 g =1.1743E-02 mg Mass of sample =0.581g = 5.81E-4 Kg Concentration of Ca++ by mass= 1.174E-2mg/5.84E-4 Kg= 20.10ppm
  • 73. • Titration of Sample 2 with EGTA run rough 1 2 3 blank initial volume /ml 0.95 0.45 0.861 3.432 5.991 final volume /ml 5.49 3.76 3.432 5.991 6.23 titre Volume /ml 4.54 3.31 2.571 2.559 0.239 Blank subtraction 4.301 3.071 2.332 2.32 0 Average volume /ml 2.326 •# of moles of EGTA transferred=2.326x 10-4 dm3 x 2.50E-03 moles • = 5.82E-07 moles •# Of moles Ca ++ = 5.82E-07 moles/2 • =2.91E-07 moles •Mass of Ca++ present = 2.91E-07 moles x 40.078g/mol • = 1.1663E-05 g =1.1663 E-02 mg •Mass of sample =0.581g = 5.81E-4 Kg •Concentration of Ca++ by mass= 1.1663 E-2mg/5.81E-4 Kg • • = 20.07ppm •
  • 74. • The sample collected does not compare favourably with G. changgi (65.5ppm) • This is based on a wet weight value and flame atomic absorption analysis, and G. changii was not the specie of Gracilaria collected
  • 75. Conclusion • The Gracilaria species sampled contained fatty acid methyl esters (hexadecanoic acid methyl ester), two sterols (Cholesta-3,5-diene , • Cholest-5-en-3-ol (3b.)-nonanoate ) , 2-nonyl- cyclopropaneundecanal, 9-Octadecenal, and an ash content of 27.06% and a calcium content of 20.10ppm. • The bulk of the sample consisted of a polysaccharide which was not characterized. The methanol extract contained an insoluble white precipitate which also was not analysed. • Several cholesterols were found and sterols as a group are used in the biosynthesis of certain hormones such as testosterone,but the human body also produces cholesterols hence • Irish Moss does contain at least one compound proven to be an aphrodisiac,however this relates to moths