Marine bio-prospecting

1. Dr. T. Citarasu
Associate Professor
Tele-fax: + 91-4652-253078
Mobile: +91-9994273822
Email: citarasu@gmail.com
citarasu@msuniv.ac.in
Website: www.msuniv.ac.in
MANONMANIAM SUNDARANAR UNIVERSITY, TIRUNELVELI

2. BIO-PROSPECTING OF MARINE
NATURAL PRODUCTS AND THEIR
HEALTH BENEFITS

3. Importance of Marine Biotechnology
Contribute to oxygen cycle & help to regulate the
Earths’ climate
Explore medicines, cosmetics, nutraceuticals,
enzymes, industrial products, bio fuels, foods,
aquaculture etc.
Defined as “ Application of scientific & engineering principles to the
processing of materials by marine biological agents to prove goods and
services”.

4. 7516.6 km
News Courtesy :
Daily Thanthi 23.02.2014
Strengthen our marine biotechnology research…….
Indian Council of Agricultural Research (ICAR)
Department of Biotechnology (DBT)
Ministry of Earth Sciences (MOES)
Ministry of Environment and Forest (MOEFS)
Council of Scientific & Industrial Research (CSIR)

5. Need for Marine Bio-prospecting
Repeated search in terrestrials leading to known compounds
Marine ecosystems are unique and rich reservoir of biodiversity with an
enormous potential towards improving the quality of human life.
Marine organisms have highly developed defense system in order to
survive in the hostile conditions such as extreme temperatures, varied
pressures (low or high), low energy and lack of sunlight

6. Multi-OMICS methodologies: Genomics, Transcriptomics, Proteomics,
Metabolomics, Metagenomics and Meta transcriptomics
Production and analysis of massive biological data
Once, the compound of interest is identified, bioactivity screening,
heterologous expression, genetic engineering and/or chemical synthesis
can follow
Characterize the activity and produce the desired bioproducts with
pharmaceutical, nutraceutical, cosmeceutical and biofuel production
applications.

7. Defined as “Searching for bio molecules from marine sources and the
search for new and unique bioactive components with potential
commercial applications”
Technical details & drug development includes…
Identification
Screening
Efficacy
Safety testing
Clinical trials
Large scale commercial
production

8. Untapped reservoir of biologically active compounds, which leads to the
development of new functional foods with vast diverse of novel
chemicals.

9. The soft bodied and unmoved organisms
are under threat with predators &
environmental factors. They escape from
the predators they have to synthesize
molecules with diverse of structures with
potent biological activities.
Even though marine bio prospecting
started the past 50 years unfortunately
only few products available in the market
and most of them under clinical trials.
Most of them treated cancers,
inflammations and antimicrobials.
Difficulties in synthesis. Structures of
some compounds are extremely
complex, the synthesis, in many case, is
not feasible.

10. Secondary metabolites
Produced after active growth has taken place & which perform no
vital function for the producing organisms
Interesting array of diverse and novel chemical structures with High
bioactivity potential
Heart of commercial venture & turn over - more than billion 100 $ / yr
Non Ribosomal Peptide Synthetase (NRPS) & Polyketides (PKS)
Peptide secondary metabolites produced by bacteria & Fungi
NRPS / PKS system involved diverse of bimolecular secretion
Independent of mRNA & Broad biological activities

11. MNP RESOURCES

12. MICROBIAL SOURCES
Microbial Metabolites
Microbes from marine sources have a
rich potential of antimicrobial active
principles for protecting themselves
from their predators
Due to the rich potential bioactive
metabolites in the marine microbes, it
may be used as drugs directly or
used as lead structures for drug
discovery
Nowadays the antibiotic resistance
and problems increased and
alternative discovery needed

14. Metagenomic Approach
Less than 1% of the microbes have been cultivated and characterized.
Offering access to the genetic information present in uncultured
bacteria by screening of libraries constructed from DNA isolated from
different habitats.
Marine microbes form highly specific and symbiotic relationships with
filter-feeding organisms like sponges, alcyonarians, ascidians and
marine plants.
The host organism synthesizes these compounds as non-primary or
secondary metabolites to protect themselves and to maintain
homeostasis in their environment
A diverse array of bioactive compounds can be isolated from the
extracts of marine organisms.
Many of them have novel chemical structures which may lead to
development of novel drugs.

15. Associated microbes
Secondary metabolites produced with the association between Sponges and
bacteria
Obligatory mutualism (symbionts play an essential role in the metabolism of their
host), facultative mutualism (beneficial effect on their host, but the host will
survive without the symbiont) or commensalism (they are present without
providing obvious beneficial effects to their host).
More than 30 different phyla of Bacteria and Archaea as being associated with
sponges.
However, less than 1% of these microorganisms can be cultivated in laboratory
conditions.
Bioactive metabolites originally isolated from sponges, were in fact synthesized
or transformed by bacterial strains.
More than 5,300 different products are known from sponges and their associated
microorganisms, and more than 200 new metabolites from sponges are reported
each year.

16. Table. Sponge associated bacterial compounds and its bioactivities
Compounds Activity Bacterial Sources Association
2-undecyl-4-
quinolone
Antimalarial
& anti-HIV
Pseudomonas sp. 1537-E7. Homophymia sp
3,6-
diisopropylpiperazin
-2,5-dione
Antimicrobial Pseudomonas sp. NJ6-3-1 Hymeniacidon perlevis
Andrimid Antibacterial Vibrio sp. M22-1 Hyatella sp.
2,4,4’-trichloro-2’-
hydroxy-
diphenylether
Antibacterial Micrococcus luteus
R-1588-10
Xestospongia sp.
Cyclo-(l-pro-l-phe) Antibacterial Alcaligenes faecalis A72 Stelletta tenuis
Iturin Antifungal Bacillus subtilis A202 Aplysina aerophoba
Manzamine A Antimalarial Micromonospora sp. Acanthostrongylophora
sp.
Surfactin, Iturin and
Fengycin
Antibacterial
antifungal
Bacillus subtilis A184 Aplysina aerophoba
Trisindoline Antibacterial Vibrio sp Hyrtios altum

17. Nonanoic acid

18. 9-Octadecenamide -Fatty acid

19. Mannosamine
Polymeric BS

20. Actinomycetes Origin
Actinomycetes are the intermediate group between bacteria and fungi having
high G+C (>55%) content in their DNA and abundantly present in the marine
sediment
Top most producers of antibiotics @ 70%
The important genera are Streptomyces sp, Nocardiopsis sp, Marinispora sp,
Salinispora sp and the culture- independent Dietzia sp and Rhodococcus sp
Major producers of antibacterial, antifungal, insecticidal, antitumor, anti-
inflammatory, anti-parasitic, antiviral, antifouling and anti-infective compounds

22. Important Secondary Metabolites
Surfactin A Synthetase C (SRFA-C)

25. Fungal Origin
Widely distributed in deep sea to polar ice
covers, occur in sediments, dead organic
matter, driftwood, algae, corals and
sponges etc.
Huge potential for new natural products
and an increased number of new
metabolites have become known over the
past years.
In hydrothermal sea ecosystem,
unsuspected high diversity of fungal
species identified with novel secondary
metabolites recently
Vast source of diverse antimicrobial
compounds mostly inhibit Gram negative
bacterial pathogens, HIV virus and fungus

26. Figure. Diversity of fungal genera obtained from the orange puffball
sponge,Tethya aurantium from the Mediterranean Sea with 29 identified genera
among 160 isolates

27. Table. Screening bioactive compounds from marine fungal strains (Mi-Hee et al., 2008).

28. Table. Antibacterial compounds from marine derived fungi (Punyasloke et al., 2006).

29. Table. Antifungal compounds from marine derived fungi (Punyasloke et al., 2006).

30. Antiviral compounds
Asperxanthone
Asperbiphenyl
Aspergillus sp MF-93
TMV
Anti-protozoan compounds
VarixanthoneAscosalipyrrolidinone AAigialomycin D

31. Invertebrate Resources
Group of animals have no backbone
In halobiotic environment, they have
special adaptations, metabolic
activities, secretions helps to produce
diverse of bioactive substances,
protecting from harsh environment.
Among the invertebrates poriferans,
cnidarians, annelids, arthropods,
molluscs and echinoderms have
attracted attention due to their
antiviral, antimicrobial, antiprotozoal,
antifungal, antihelminthic and
anticancer activities

32. PORIFERA
Sponges
Most primitive metazoans (700-800 mill. yrs)
which appeared in the Cambrian period
Widely found in coastal platform to deep
waters
Sessile filter feeders with efficient defense
mechanisms against foreign attackers such
as viruses, bacteria or eukaryotic organisms.
Considered as gold mine during the past 50
years (> 10000 compounds)
Occasionally develop symbiotic relationship
with algae and microbes

33. Sponge derived Compounds
sesterpene
Fascaplysinopsis sp
Antibacterial
Topsentin
Spongosporites ruetzleri
Antibacterial
Anti-inflammatory
Colon cancer
2-(2’,4’-dibromophenoxy)-4,6-
dibromophenol
Dysidea granulosa
MRSA
Cyclodepsipeptides
Mirabamides A-D
Siliquariaspongia mirabilis
HIV

34. cyclodepsipeptide
Antifungal, anthelmintic & antimalarial
Japis sp.
Pachypelliina sp
Anti-infective

35. Bengamides & Derivatives
Fijian Sponge Jaspidae
Antibiotic , anticancer &
Antihelmintic
Contigna sterol
Petrosia contianata
Curing Asthma
DBH (Debromohymenialdisine)
Stylotella aurantium
Antibiotic , anticancer

36. COELENTERATES
Cnidarians are the richest natural sources of
prostaglandins
Jelly fish, sea nettle and sea anemones mostly
secreted toxins with mixture of enzymes and
polypeptides

37. Hydra, Jelly fish, coral & sea anemones
1. Pseudo pterosins
Pseudopterogorgia elisabethae
Anti-inflamatory
2. Eleutherobin
Octocoral Eleutherobia sp
Anti cancer
Ascidians derived Compounds
1. Apilidine
Mediterranian tunicate Aplidium albicans
anticancer
4. Ectein ascidin 743
Ecteinascidia turbinate
Anti cancer

38. Table. Most promising compounds from soft corals, Anthozoa

39. ANNELIDS
Of the approximately 9000 species
of annelids, more than 8000 are
polychaetes including lugworms, clam
worms, bristleworms, fire worms, palolo
worms, sea mice, featherduster worms, etc
They are found in most marine
environments from tidal zones to
hydrothermal vents
They has been found to treat several
pathophysiological conditions such as
arthritis, osteoporosis, bone cancer etc.

40. Arthritis
Oesteoporesis
Bone cancer
Antimicrobial
Antibacterial
Antifungal
Arenecins Hedistins
PeptidesAMP
Arenicola marina Perinereis aibuhitensis
Perinereis cultrifera

41. ARTHROPODS
They having an exoskeleton (external skeleton), a segmented body, and
jointed appendages (paired appendages).
Several bioactive compounds have also been isolated from marine
arthropods including horseshoe crab, crab, krill and shrimps

42. Krill Oil and Enzyme (Euphausia superba)
Tiny shrimp @ Whale food
Omega 3s, especially EPA and DHA
Heart disease, reduce high cholesterol, high
blood pressure, stroke, cancer,
osteoarthritis, depression

43. Chitin, Chitoson & Oilgomers
Shell waste with biomedical
applications
Chitin a high molecular wt polymer
N-acetyl-D-glucosamine
Chitoson-heteropolymer
Reducing LDL
Antioxidants & anticancer
Immunostimulants
DNA Vaccine delivery
Biopesticide – Agricultural
Shell waste
Wash
deprotenize- 3 % NaoH
Alkali removal by water wash
Demineralization-1.25 N Hcl
Wash
Dry and package

44. Application Bioactivity
Antimicrobial Antibacterial, Fungal & measuring
mold contamination
Nutritional quality Dietary fibre, hypocholesterolemic
effect, live stock & feed additive,
reduction of lipid absorption,
antigastric effects & infant feed
ingredients
Others Encapsulation of nutraceuticals &
vaccine delivery
OTHER HEALTH BENEFITS

47. Lectin
Triglycerides Limulin
Limulin
Carcinoscorpin
Thiol ester protein
Fatty acids
Triglycerides
Carotenoids
Lipids
Antibacterial
Anti cancer
Antioxidant
Antiproliferative
Antimutagenic
Antiinflamatory
immune response

48. MOLLUSCA
Largest marine phylum, comprising about 23% of all the named
marine organisms.
Snails, octopuses, squid, clams, scallops, oysters, and chitons.
These creatures have been important to humans throughout history as a
source of food, jewelry, tools, and even pets.

49. Molluscan derived Compounds
1. Dolastin (Sea slug)
Dolastin 10 &15
Dollabella auricularia
Anticancer activity
2. Kahalaide F
Sea slug Elysia rufescens
Induce apoptosis
3. Spisulosine
Arctic surf clam Spisula polynyma
Anti proliferative & Anti cancer
4. Cadalmin Green mussel extract
Perna viridis –leukotrenes
Arthritis; Anti-inflamatory
Known wt of tissue + water or
methanol
Chop & homoginize/ sonicate
Centrifuge
Collect supernatant
Further purification need
Assay

50. commercially important marine
“archeogastropod” mollusk
Rich nutritional value, superior
taste, and various other benefits to
human health
Ginseng in the ocean- 25 vitamins
and minerals and is particularly
rich in selenium, magnesium and
B12
Abalone is favoured for its high
nutrient content that has
traditionally been used to support
eye health
Abalone

51. ECHINODERMATA
The phylum contains about 7000 living species including sea stars, sea
urchins, sand dollars, and sea cucumbers, as well as the sea lilies
Found at every ocean depth, from the intertidal zone to the abyssal zone
The bioactive compounds mainly are saponins

52. Saponins
Sterol derivatives
Arpenoids
Glycoproteins
Cerebrosides
pyrimidine nucleaosides
Thymine deoxyriboside
Uracil deoxyribose
Polysaccharides
β- carotene
Hemolytic
Antibacterial
Antifungal
Antineoplastic
Antitumor
Antiviral
Anti HIV
Antiinflamatory
anti cancer
anti allergic
Holothuria fuscocinereaToxopneustes pileolus Lytechinus variegates Strongylocentrotus droebachiensis

53. FISH AND OTHER VERTEBRATES
Metabolites extracted from fish, sea
snakes and aquatic mammals are
scanty.
Fish oil, rich in omega-3 fatty acids,
useful for various remedies including
arthritis etc.
500 species of fish are considered
toxic (Tetradotoxin (TTX))
Squalamines, a water soluble
antibiotics from dogfish shark,
Squalus acanthias
An anticancerous drug, namely “Fu-
anntai”, from sea snake had anti-
cancerous effects

54. Omega-3 fatty acids eicosapentaenoic
acid (EPA) and docosahexaenoic
acid (DHA)
Safety and potential nutritional and
therapeutic effects.
Heart health, Liver tonic, antioxidant &
cure cancers
Good for health, Good for Eye
Anti stress
Rich Vitamins
Fish Oil (Cod Liver oil)
Fresh Livers without Gall bladder
and veins
Wash & Chop
Steam cooking (70° C – 20 min)
Remove the floating oil
Filter & Centrifuge
Keep overnight with NaSo4
Filter & store
Thermal rupture to release oils

55. Steroids for fast growing !!!!! XXX

56. Shark Fin Cartilages and Rays
Caudal, Dorsal & pectoral fins
Known wt of dried fins
Wash
cooking with acidified water
until hydrolyze to gelatin
Remove adhering muscle
Dry in artificial dryer
Grade and pack
Shark Fin soup
Boost sexual potency
Enhance skin quality
Prevent heart disease, lower
cholesterol
Beneficial to lung & kidney
Shark Bone cartilage
Prevent variety of illness
Angiogenesis
Prevent Cancer

57. Shark cartilage has long been valued by traditional herbalists in China,
where it has been consumed for centuries as shark fin soup.
Shark cartilage contains mucopolysaccharides, a family of complex
carbohydrates including the now well-known chondroitin sulfate.
It is an excellent source of calcium and phosphorus.
Some Shark cartilage based products available in market

58. Fish Gelatin
Epinephelus sp, Rastrelliger sp etc
Elasticity, consistency & stability of
the products
Acid extraction
Washed skin soaked with 0.2 %
NaOH
Wash
0.2 % H2So4 + 1% citric acid
Coldwater wash
DH2o incubation @ 45° C-18hr
Filter
Dry

59. Fish Bone
60-70 % of inorganic substances & rich in
calcium
Essential elements for human health
Solve the problems related to bone
Collagen
Structural protein in connective tissue
Cosmetic, biomedical, pharmaceutical,
leather and film industries
Alkaline and acid extraction
Fish Bone and Collagen

63. ALGAL NATURAL PRODUCTS
Algae have a tremendous impact on the
sustainability of the marine ecosystem
as being the primary producers and,
therefore, a food source for other marine
organisms.
microalgae synthesize bioactive
molecules such as pigments and lipids
that exhibit health properties.
Types of Algae
Seaweed: Macro; benthic & muticellular
algae
Microalgae: Diverse group of unicellular;
occupy fresh, marine & extreme
environments

64. MICRO ALGAL PRODUCTS

65. Carotenoids- Powerful antioxidants
Beta carotene
Algotene
Axtaxanthin
Dunaliella salina

66. Carotenoids
Solvent Extraction
Astaxanthin
Isolated from Micro algae
Powerful antioxidants in aquaculture
Fucoxanthin
Brown carotenoids in certain sea weeds
Promote DHA & omega-3 fatty acids
Beta carotenoid
Found in many algae
Colored carotenoids
Antioxidants & anticancer
Immunostimulants

67. ANTIBACTERIAL COMPOUNDS
C-lactone malyngolide
(Lyngbya majuscula)
ANTIFUNGAL COMPOUNDS
Majuscuiamide (cyclic depsipeptide)
Gambieric acid
(Gambierdiscus toxicus –Dinoflagellate)
IMMUNOSUPPRESSIVE COMPOUNDS
Lipoproteins, microcolins A and B
ANTICANCER COMPOUNDS
Curacin A – anticancer (antimitotic
agent)
Cytotoxic pentapeptides:
majusculamide D
Deoxymajusuculamide D
Amphidinolide B
(dinoflagellate, Amphidinum sp.)
ANTIMICROBIAL & ANTICANCER COMPOUNDS

68. FATTY ACIDS AND HIGH VALUE OILS
Very Long chain PUFA (vlc PUFA)
Eicosapentaenoic (EPA)
Docosahexaenoic acid (DHA)
Arachidonic acid (AA)
Flexibility, fluidity and selective permeability
to cellular membranes
Brian development
Beneficial for the cardiovascular system
Nutraceutical in human and animal health

69. ALGAL SMALL MOLECULES
Group of secondary metabolites synthesized by biosynthetic gene
clusters (Enzyme systems producing products such as antibiotics)
Cyclic peptide (microcystin), depsipeptide ( majusculamide)
Lyngbyatoxin-A ( Lyngbya majuscule) – antifungal activity
Cryptophycin from Nostoc sp. shows a fungicidal activity
&microtubule depolymerizing agent for Cancer cells.
Heterocyclic compounds, polycyclic ethers, oxygenated polyketides
and macrolides from dinoflagellates
Dinoflagellate toxins like saxitoxin , neosaxitoxin and gonyautoxins
are sodium channel blockers

70. ALGAL BIOSURFACTANTS
Extracellular Amphiphilic compounds
Glycolipids, lipopeptides, fatty acids, polymeric surfactants &
particulate natures
Broad range of applications
Antibacterial, antiviral, antifungal, anti-adhesive, anti-cancer,
antioxidants, adjuvant etc
Arthrospira sp., Synechococcus nidulans, Chlorella minutissima, C.
vulgaris and C. homosphaera used for various industrial and
pharmaceutical applications

71. ALGAL OIL PRODUCTION
Storing the Sun’s Energy
(Photosynthesis)
What is needed Storage of Energy
Sunlight Lipids and oils
CO2 Carbohydrates
Nutrients

72. How to produce more oils ?
The lipid content of an algae species can be increased by imposing a stress
factor, such as starving of nitrogen or silicon.
In this manner, lipid fractions up to 70-85% have been reported for nitrogen
starvation and 60% for silicon starvation

73. Health aspect of algal oils
The oil extracted from
Crypthecodinium cohnii contains 40-
50% DHA useful for infant formula
Schizochytrium produce algal oil
known as DHA Gold for use as an
adult dietary supplement in food and
beverages as well as animal feeds
(OmegaTech (USA)).
Other applications include food for
pregnant and nursing women and for
cardiovascular health

74. SEAWEED PRODUCTS
Bioactivity Seaweeds Compounds
Antioxidant Cymopolia barbata Cymopol
Avrainvillea longicaulis Avrainvilleol
Halimeda opuntia Polyphenols
Undaria pinnatifida Fucoxanthin
Anti-inflammatory Laureafincia claviformis Pacifenol
Stypopodium flabelliforme Styfotriol
Undaria pinnatifida Fucoidon
Anticancer Gracilaria corticata Polysaccharides
Gracilaria tenuistipitata
Plocamium telfairiae
Capsosiphon fulvescens
Sargassum oligocystum

76. Alginate
polyuronic saccharaide
Laminaria and Fucus
D- mannuronic and L-galuronic acid
Emulsifier in food industries
Sargassum
Fresh seaweed washed & Dried
Pulverize to get powder
Treat with 1N HCl (pH 2-3)-ON
Wash with water & add Na2Co3
Filter & bleach with 2.5 % NaClo
Evaporate @ 60° C
Pure sodium alginate

77. Carrageenan
Isolated from sea weeds
Sulfated polysaccharides
Powerful antioxidants
Broad range of applications in
food and cosmetics

78. Fucoidon (Foo–Koy–Den)
Sulfonated polysaccharides
Antioxidants & immunostimulants
Brown sea weeds
Blood purifier
Reduced Cholesterol
Fight off viral infections
Scavenge free radicals
Liver tonic
Anticancer

79. Ulvan
Composed of rhamnose,
glucuronic acid, iduronic acid,
xylose and sulfate
Green seaweed, Ulva
Good gelling agent
Prevent biofilm formation
Good Nutraceuticals
Antioxidants
Anti-thrombotic activities
Treatment of gastric ulcers
Anticancer
Immunostimulants

80. Laminarian
Water-soluble polysaccharide
(1,3)-β-D-glucan with β (1,6) branching
and contains 20–25 glucose units
Regulator of intestinal metabolism
Tumor-inhibiting agent
Antibacterial agent
Protection against severe irradiation
Boosting the immune system
Reducing cholesterol levels in serum

81. Sea Lettuces
Genus Ulva, a group of edible green algae that is widely distributed along the
coasts of the world's oceans.
Food source for humans in Scandinavia, Great Britain, Ireland, China,
and Japan
Used as salads and cooked in soups.
High in protein, soluble dietary fiber, vitamins and minerals, especially iron.

82. Ulva reticulata

84. Cadalmin Green Algal extract
Cadalmin Green Algal extract contains a
unique blend of 100% natural marine
bioactive anti-inflammatory ingredients
extracted from selected seaweeds or marine
macro algae with a patented ecofriendly
“green” technology .
The product is effective to combat arthritic
pain and inflammatory diseases in human
beings
Patients suffering with chronic joint pain and
arthritis clinical trial-based studies revealed
that more than 98% of the respondents were
satisfied with the product with about 70-85%
relief in joint pain and arthritis

85. CONCLUSIONS & FUTURE DIRECTIONS

86. Current medications have severe drawbacks, and to the scientific
appraisal of marine-based remedies that might be safer, cheaper,
and less toxic for self-medication than existing prescription
medicines.
Marine derived pharmaceuticals provide a novel and rich source of
chemical diversity that can contribute to design and development
of new and potentially useful pharmaceutical agents.
Metagenomic approach help to find novel lead structure for drug
development. By utilizing the bioprocess technology we can
produce the compounds at large scale levels.
A proactive interaction between researchers, the pharmaceutical
marine biotech sector and government regulating agencies is
crucial to the incorporation of this challenging new tool in clinical
medicine.

87. THANK YOU