OP1 Dr Ranga Rao Ambati : Biological activity of Astaxanthin from ...

Why microalgae?‣ More than 25,000 species of algae, living every where‣ Algae grown in autotrophic and heterotrophic culture conditions‣ Producing high value metabolites like pigments, fatty acids, lipids,proteins, polysaccharides, minerals and vitamins‣ Used in food, feed, pharmaceutical, nutracetuicals andpigmentation applications

Microalgae species under microscope

Microalgae-used in food, feed and medical applicationsMedical applicationsBioavailability, Toxicity,ImmunesystemNutraceutical applications,Food, Carotenoids,Vitamins,Fatty acids, Minerals,Salmon and Trout feedMicroalgaeProtection from UV lightSkin carcinogenesis,Cellular health,Cardivascular,Chronic and acute disease,Anti diabeticAge macular degeneration,Inflammatory, AntioxidantInfections, Antihypertensive,Antiulcer and gastric

Microalgae species with relevance to nutraceutical productsSpecies Product Application areaSpirulina platensisBiomass,PhycocyaninHealth food, cosmeticsChlorella vulgaris Biomass Health food, foodsupplement, feedDunaliella salina Carotenoids, -caroteneHealth food, foodsupplement, feedHaematococcuspluvialisCarotenoids,AstaxanthinHealth food, feed, colorpharmaceuticalsPorphyridium cruentum PolysaccharidesPharmaceuticals,Nutrition & cosmeticsIsochrysis galbana Fatty acids Animal nutrition

Presentation covers…………‣ Microalgae-Haematococcus‣ Cultivation‣ Isolation of carotenoids‣ Quantification of carotenoids by HPLC‣ Identification of carotenoids by LC-MS‣ Characterization of carotenoids by NMR‣ Structure of carotenoids‣ Biological activity of carotenoids Antioxidant activity Bioavailability Anticancer activity in rats Induced cell apoptosis in human cells Hepatoprotrection‣ Results and conclusion

Haematococcus Haematococcus is a green microalga It has two distinct phases in its lifecycle• Motile vegetative growth phaseImmotile encysted stageMotile cell Cyst cells accumulate astaxanthinand appears red H. pluvialis has gained commercialimportance owing to high content ofastaxanthin 2-3% (w/w)Encysted cell

Cultivation of algae

mAUIsolation of carotenoids by HPLCmAUOverlaid Spectra2004.50 min 5.10 min 8.62 min 9.02 min 22.17 min 22.75 min200150150100100505000350 400 450 500 550 600 650 700 750 800nm

Quantification of carotenoidsCarotenoids (%)Neoxanthin 0.9 ± 0.05Violaxanthin 0.3 ± 0.12Astaxanthin 3.8 ± 0.25Lutein 1.4 ± 0.20Zeaxanthin 2.2 ± 0.38β-Cryptoxanthin 2.3 ± 0.25β-carotene 1.7 ± 0.98Un identified peaks 4.5 ± 0.17Astaxathin esters(mono and di-esters)79.58 ± 3.12

Identification of carotenoids by LC-MSNo m/z Compound[M] [M+2H-FA 1] +1 MS21 536 [M+H] +1 537 α-carotene, β-carotene2 550.41 [M+H] +1 551.95 echinenone3 568 [M+H] +1 565.86 Lutein, Zeaxanthin4 578 [M] 578 4-ketoalloxanthin5 551.96 [M+H] +1 552 β-cryptoxanthin6 597.04 [M-H 2O] 579 astaxanthin7 602.04 [M+2H] +1 600 Neoxanthin, violaxanthin8 835 579 836 ME C16:09 845 579 846 ME C17:210 847 579 848 ME C17:111 849 579 850 ME C17:012 853 579 852 ME C18:513 855 579 854 ME C18:414 857 579 856 ME C18:315 859 579 858 ME C18:216 861 579 859 ME C18:117 1072 579 1071 DE C16:0/C16:018 1096 579 1095 DE C16:0/C18:219 1120 579 1119 DE C18:1/C 18:320 1122 579 1121 DE C18:1/C18:221 1124 579 1123 DE C18:1/C 18:1Xanthophyll's andother carotenoidsAstxanthinestersJohor Scientific Meeting-2013

Characterization of carotenoids by NMRAX AXMEs AXDEsPosition H mult c H mult c H mult c( J in Hz)(ppm) ( J in Hz) (ppm) ( J in Hz) (ppm)1 4.32 (m, 1-OH), 4.33 (m, 1H) 68.88 5.1 71.64 5.1 (-OH, H) 71.641’ 4.35,4.36 (OH) 68.88 5.10 68.89 5.1 (-OH, H) 71.642 1.85 (m, 1H) 61.85 1.37 61.78 1.37 (m, H) 61.792` 1.83 61.85 1.36 61.78 1.36 (m, H) 61.793 -- 45.14 -- 42.41 -- 37.223` -- 45.14 -- 42.41 -- 37.174 1.23 68.76 1.26 68.81 1.26 68.814` 1.32 68.76 1.31 68.55 1.31 68.505 -- 127.77 -- 127.79 -- 127.755` -- 127.77 -- 127.79 -- 127.756 -- 200.09 -- 202.21 -- 193.496` -- 200.09 -- 196.11 -- 193.177 6.48 1H, m) 126.52 5.40 126.80 5.40(1H, m) 126.807` 6.46 (1H, m) 125.91 5.39 126.80 5.40(1H, m) 126.808 6.45( 1H, m) 142.00 5.38 131.62 5.38 131.638` 6.43 (1H, m) 141.87 5.37 131.62 5.37 131.639 -- 133.10 -- 143.32 -- 133.389` -- 133.22 -- 142.65 -- 133.3810 6.21 131.80 5.38 127.38 5.28 127.4410` 6.25 131.13 5.37 127.38 5.33 127.4411 6.70 129.00 5.35 127.44 5.40 127.5811` 6.70 (1H, m) 129.40 5.35 127.44 5.40 127.5812 6.26 (j=13Hz, 6Hz) 134.85 5.33 129.88 5.38 129.8912` 6.23 (dd, J=13Hz/6Hz) 134.25 5.33 129.67 5.37 129.8913 -- -- -- -- -- ---13` -- -- -- -- -- --14 5.31 131.80 5.38 127.28 5.40 127.2914` 5.35 131.13 5.37Johor Scientific Meeting-2013127.75 5.40 127.29

15 6.25 129.40 6.2 127.44 5.38 127.4415` 6.21 129.00 6.2 127.44 5.37 127.4416 2.02 20.01 2.00 20.22 2.00 20.2216` 2.01 20.32 2.01 20.85 2.01 20.2217 1.31 (3H, m) 25.83 1.21 26.87 1.27(3H, m) 26.3717` 1.31 (3H,m) 25.83 1.21 26.87 1.27 (3H, m) 26.3718 1.32 (3H, m) 25.83 1.20 26.70 1.29(3H, m) 26.7018` 1.32 (3H,m) 25.83 1.20 26.70 1.29 (3H, m) 26.7019 1.96 (3H, m) 13.83 2.02 13.76 2.04 (3H, m) 13.7619` 1.97 (3H,m) 13.64 2.03 13.76 2.04 (3H, m) 13.7620 1.99 (3H, m) 12.25 2.06 13.76 2.05 (3H, m) 13.7620` 1.99 (3H,m) 12.49 2.08 13.76 2.05 (3H, m) 13.7621 36.01 -- 37.2221` -- -- -- 37.2222 2.0 29.36 2.331 29.3722` -- -- 2.331 29.3723 1.39 29.01 1.384 29.0223` -- -- 1.371 29.0224 2.02 29.16 2.006 29.1724` -- -- 2.019 29.1725 1.48 28.83 1.48 28.8425` -- -- 1.48 28.8426 1.31 28.78 1.316 28.3926` -- -- 1.357 28.3927 2.00 28.38 2.006 28.8427` -- -- 2.006 28.8428 2.00 129.67 2.019 29.0228` -- -- 2.019 29.0229 2.00 129.88 2.031 129.8929` -- -- 2.031 129.8930 2.03 129.67 2.043 129.8530` -- -- 2.043 129.8531 2.00 29.36 2.071 30.1531` -- -- 2.071 30.1532 1.20 29.86 1.270 31.2032` -- -- 1.270 31.2033 1.21 30.01 1.295 31.4633` -- -- 1.295 31.4634 1.26 29.36 1.270 31.5934` -- 1.270 31.5935 1.31 29.16 1.316 29.3735` -- -- 1.316 29.3736 1.35 31.59 1.357 31.5936` --- -- 1.357 31.5937 1.61 20.22 1.625 20.2237` --- --- 1.625 20.2238 0.98 13.92 0.991 13.9238` -- -- 0.976 13.92

OCCH 3CH 3H 3CH 3CH 3CH 3C H 3OHHOOCH 3CH 3Free astaxanthinCH 3H 3C CH 3H 3COH 3C CH 3OOCRHOOH 3C CH 3CH 3CH 3astaxanthin monoesterCH 3H 3C CH 3H COCH 3CH 33OOCRRCOOOCH 3CH 3astaxanthin diesterCH 3H 3C CH 3Structure of carotenoids -Astaxanthin, mono-ester and diester- formsof Astaxanthin R= saturated or unsaturated alkylchains)

Biological activity ofcarotenoids

Mechanism of astaxanthin‣ Astaxanthin molecule isexposed both in andouside of the cellmembrane‣ β-carotene and vitaminC reside inside andoutside the lipid bilayermembrane‣ No other antioxidantposses the threecharacteristic at once

Antioxidant activityAntioxidant activity of astaxanthin on (A) -CLAMS method, and (B) Hydroxyl radicalscavenging.Anti lipid peroxidation of astaxanthin on (A)Kidney, (B) Brain, and (C) Liver.

Hepatoprotective activityGroupsCatalase(U/mg protein)Glutathioneperoxidase(U/mgprotein)Superoxidedismutase(U/mgprotein)% Anti-lipidperoxidaseGlutathione(nmol/mgprotein)NormalToxinASX100*ASX250*ASXEs 100*ASXEs250*SASX100*457.23 ± 6.98 10.12 ± 1.42 20.81 ± 1.75 22.45 ± 2.61 11.23 ± 1.15312.51 ± 10.37 6.56 ± 1.74 10.05 ± 2.38 53.78 ± 3.80 4.25 ± 0.93428 ± 12. 61** 11.98 ± 4.63** 12.13 ± 2.66** 18.91 ± 2.13** 8.16 ± 1.84**543.01 ± 9.61** 15.98 ± 2.30** 15.02 ± 1.73** 26.46 ± 1.09** 10.47 ± 1.06**623. 84 ± 8.25*** 21.79 ± 3.06*** 26.13 ± 3.67*** 21.81 ± 2.74*** 15.03 ± 1.09***745.28 ± 6.05*** 23.65 ± 1.65*** 32.98 ± 4.01*** 10.33 ± 1.12*** 18.69 ± 2.78**561.31 ± 11.82** 11.24 ± 2.38** 16.81 ± 3.22** 27. 58 ± 2.91** 13.52 ± 2.13**

Astaxanthin(nmol/g or ml)BioavailabilityTime in(h)Catalase(U/mg protein)Superoxidedismutase(U/mgprotein)Peroxidase(U/mg protein)Plasma0 106.85 ± 3.59 a 7.57 ± 1.21 c 7.35 ± 1.55 c2 250.10 ± 12.03 b 12.12 ± 3.25 a 15.05 ± 0.92 a4 243.58 ± 8.03 b 11.14 ± 6.32 a 12.86 ± 0.61 b6 199.23 ± 5.47 c 10.01 ± 0.54 a 10.51 ± 0.61 b10009 178.55 ± 15.42 c 9.09 ± 2.51 b 9.95 ± 1.14 bLiver0 251.52 ± 2.10 c 10.34 ± 0.54 c 7.44 ± 2.64 c5002 336.67± 4.51 b 13.80 ± 4.47 b 10.11±1.47c0Plasma Liver EyeRat tissues4 383.08 ± 1.81 a 17.19 ± 3.87 a 16.28 ± 2.25 a6 374.45 ± 4.34 a 16.29 ± 1.21 a 14.63 ± 3.99 a9 366.44 ± 4.34 a 15.65 ± 0.85 a 11.32 ± 2.37 bAstaxanthin bioavailability after a single or repeated dose of H. pluvialis fed ratsas source of astaxanthin

Inhibition of cancer in rats, induced cell apoptosisTumor ItensityPhotographs of rat skin tumors and histopathologyInduced cell apoptosis byAstaxanthin

Results AX and AXEs were found to be 3% and 80% in Haematococcus followed by other carotenoids A total 13 AXEs were identified based on mass spectra data. Further, AX and AXEs werestructurally confirmed by 13 C NMR, 1 H NMR, HSQC 2D, NOESY 2D and COSY 2D NMRspectroscopy The Haematococcus extract exhibited 80-85% antioxidant activity in various methods Astaxanthin in rats showed peak levels in plasma, liver, and eyes at 2, 4, and 6 h, respectively The antioxidant enzymes levels were significantly high in plasma and liver The AXEs at the concentration of 250 µg/kg b.w showed maximum hepatoprotective activityagainst CCl 4 induced hepatic damage Further confirmed that astaxanthin & its esters showed potential anti-cancer activity in UV-DMBA induced skin cancer rats, induced cell apoptosis in human glioma cells This study implies that microalgae can be a good source of carotenoids of high biologicalactivity and nutraceutical value

Conclusion‣ Based on the current observations that astaxanthinenhances bioavailability, antioxidant activity, anticancer‣ Further theses molecules can be used for foodproperty and hepatoprotection in ratssupplements, pharmaceuticals and nutraceuticalsapplications

Research TeamAcknowledgments Prof. Dr. G. A. Ravishankar, Vice-president, DSI, R&D Life Sciences, Bangalore, India Dr. R. Sarada, Senior Scientist, CFTRI, Mysore, India Dr. Baskaran V, Senior Scientist, CFTRI, Mysore, India Dr. Shylaja M Darmesh, Senior Scientist, CFTRI, Mysore, India Dr. Udaya Sankar, Scientist & Head, Food Engineering, CFTRI, Mysore, India Prof. Dr. Phang Siew Moi, Director, IOES, University of Malaya, MalaysiaFunding Support‣ DST, DBT, ICMR, CSIR, Govt of India‣ University of Malaya Research Grant (UMRG), University of Malaya‣ Thanks to Organizers of the Symposium