MARINE NATURAL PRODUCTS- Durgashree Diwakar

MARINE NATURAL PRODUCTS
BY- DURGASHREE.M.D
M.PHARM, PHARMACOGNOSY
KLE COLLEGE OF PHARMACY, BENGALURU

CONTENTS
Marine Natural Products 2
• Introduction
• Uses
• Challenges related to Marine Products
• General method of Isolation and Purification
• Study of Marine Toxins
• Recent advances in Research
• Problems faced in Research of Marine Drug and Solutions

INTRODUCTION
• Marine natural products can be defined as biologically active products as secondary metabolites as
well as enzymes, lipids, and heteropolysaccharides. [1]
• Marine Pharmacognosy is a sub-branch of pharmacognosy which is mainly concerned with the
naturally occurring substances of medicinal value from the marine. [2]
• Marine macroalgae/seaweed used as a crude drug to treat iodine deficiency-Goitre,
hypothyroidism, Example- Nori seaweed, Kombu, etc.. [3]
Nori Kombu

USES
Treatment of several Diseases
Antiviral drugs (Eudistomins from Eudistoma olliviceum)
Cytotoxic drugs (Sponges)
Antiparasitic compounds (Red Algae- Chondria Armata)
Anticoagulants (Brown algae- Fucus vesiculosus)
Antimicrobial (Cephalosporin – Cephalosporium acremonium)
Prostaglandins (Soft coral – Plexsora homomalla) [3]

CHALLENGES RELATED TO MARINE PRODUCTS
• 70% of Earth surface represents the ocean
• It is the largest habitat of the earth with high biological and chemical diversity.
• About 30,000 marine compounds are known.
• Since 2008, 1000 compounds are identified each year.
• For a long period marine natural products are used from fish and marine algae.
• Some examples are- biopolymer agar, vitamins A, D from fish liver oil, etc… [4]

CONTINUED…….
The first two Indian – trained marine medicine specialists were available to the
Submarine Arm in 1980.
They provide support for escape
training,
maintenance and
development of submarine escape
rescue equipment,
for diving operations
primary function as doctors.

CONTINUED…..
Dangers in the ocean that can affect divers include
Marine life,
Marine infections,
Polluted water,
Ocean currents,
Waves and surges and
Man-made hazards such as boats,
Fishing lines and
Underwater construction [5]

GENERAL METHOD OF ISOLATION AND PURIFICATION
Marine Natural Product 8
1. Method of Isolation
a. Conventional method of extraction
 Maceration
 Percolation
 Soxhlet Extraction
b. Modern method of extraction/ Non-conventional method
 Supercritical-fluid extraction
 Subcritical water extraction
 Ultrasound assisted extraction
 Microwave assisted extraction [6]
2. Method of Purification
a. Membrane filtration
b. Gel filtration chromatography
c. Ion exchange chromatography
d. High-performance liquid chromatography [7]

METHOD OF ISOLATION
10
• Marine macroalgae - a rich source of
bioactive compounds that can be
implemented in various food, cosmetics,
and pharmaceutical products for health
improvement.
• Bioactive compounds- polyphenols,
polysaccharides, carotenoids, and ω-3 fatty
acids.
• Marine algae, also called seaweeds, are
divided into three classes depending on
their chemical structure and pigment
distribution.
• These classes are brown algae
(Phaeophyta), red algae (Rhodophyta), and
green algae (Chlorophyta).

1
5
2
3
4
6
1. Maceration
2. Infusion
3. Digestion
4. Decoction
5. Percolation
6. Soxhlet

1. SUPER-CRITICAL FLUID EXTRACTION
• SFE is based on the principle of extraction with fluids in their supercritical conditions, temperature, and
pressure is raised above their critical point with characteristics of both liquids and gases.
• Carbon dioxide (CO2) is the most used solvent for SFE due to its nontoxicity, safety, and low cost.
• Major advantage of supercritical fluid is increased mass transfer due to low viscosity and higher
diffusion coefficient.
• Supercritical CO2 (SC-CO2) can extract only nonpolar or compounds of low polarity since it is a
nonpolar solvent
• But the extraction of polar compounds can be enhanced by adding small amounts of polar co-solvents
such as ethanol or methanol.
• CO2 has low critical temperature and pressure, which means that bioactive compounds stay preserved
and no degradative changes can occur.
• Usually the extracts, obtained with SC-CO2, contain groups of compounds, like fatty acids, phytosterols,
tocopherols, phenolics, carotenoids, and triglycerides.

Super critical Fluid Extractions

CONT …
 Example
1.Hypnea charoides –
-Omega-3 fatty acid
-Pressure- 24.1–37.9MPa
-Temperature- 40-50C
-Time- 120min
2. Saccharina japonica, Sargassum horneri
-Fatty acids, Fucoxanthin, Polyphenols
-Pressure- 25MPa
-Temperature-45 degree celcius
-Time- 120min
-Co-solvent- Ethanol

2. SUBCRITICAL WATER EXTRACTION
• SWE operates at high temperatures (50–200 °C) and pressures (50–300 psi) for
a short period (5–10 min) with a small amount of solvent.
• The solvents are maintained near their critical region in the liquid state with the
help of applied temperature and pressure, keeping the solvents below their
boiling point.
• SWE is environmentally friendly extraction because of water, which is used as
a solvent instead of using organic solvents.
• In addition, it offers higher extraction yields because the permeability of
solvent into the material is enhanced and there is no influence on the extracted
bioactive compounds.
• But, the extraction time must be controlled because degradation of compounds
may occur.

Marine Natural Products
Subcritical Water Extraction

CONT….
• EXAMPLE-
1. Sargassum muticum
 Polyphenols, phlorotannins(Antioxidant)
 Sample- 2g
 Pressure -10.3MPa
 Temperature- 50,125,200 degree Celsius
 Time- 20min
2. Himanthali a elongata
 Polysaccharide(Antiviral)
 Sample-1g
 Pressure-10.3
Temperature-100 degree Celsius
Time- 20min

3.ULTRASOUND ASSISTED EXTRACTION
• UAE uses ultrasound waves with a frequency above 20 kHz to 100 kHz.
• These waves cause the creation of bubbles and zones of high and low pressure.
• When bubbles collapse in the strong ultrasound field, which causes the breakdown
of particles, bioactive compounds are released from the biological matrix.
• Ultrasonic bath operates at a frequency of 40–50 kHz and a power of 50–500 W,
• But an ultrasonic probe can operate only by the frequency of 20 kHz.
• Costs of the equipment are lower than the other alternative extraction techniques
and a wide variety of solvents can be used.
• UAE operates with low temperatures which enable the preservation of
thermolabile compounds and prevent complete damage to the structure.
• Low amounts of solvent are used and the working time of extraction is reduced,
which makes UAE a fast, inexpensive method as compared to traditional methods

Ultrasound-assisted extraction

CONT….
• Example
Macroalgae
Species
Ultrasound Operating Conditions
Bioactive
Compoun
ds
Bioactivity
Ultrasoun
d
Equipmen
t;
Frequenc
y [kHz];
Power
[W]
Sample
Mass [g]
Solvent
Volume
[mL]
Temp.
[°C]
Time
[min]
Hormosira
banksii
Ultrasonic
bath;
50;
150–250
1
50 (70%
ethanol)
30, 40
and 50
20, 40
and 60
Polyphen
ols
Antioxidan
t
Ascophyllum
nodosum
Ultrasoun
d probe;
20;
750
4
40
(distilled
water and
0.03 M
HCl)
- 10
Polyphen
ols,
fucose
and
uronic
acid
-

4. MICROWAVE-ASSISTED EXTRACTION
• MAE is based on ionic conduction and dipole rotation which act directly on the
molecules and occur simultaneously.
• Microwave heating causes absorption of energy by molecules where no heat is lost
into the environment.
• Due to absorption of energy by polar molecules, disruption of cells is inevitable.
• Destructed cells facilitate faster mass transfer (constituents).
• MAE can be performed in open or closed vessels.
• Open vessels operate at atmospheric pressure, while closed vessels operate at a
pressure that is higher than atmospheric.
• Due to operation at atmospheric pressure, open vessels can be more effective,
safer, and it is possible to process larger samples.
• Also, process conditions are suitable for thermolabile compounds.
• An advantage of MAE is that it is an economical and environmentally friendly
process because of the reduced process time and solvent amount. [6]

Microwave-assisted extraction

CONT….
• Example
Macroalgae
Species
Ultrasound Operating Conditions
Bioactive
Compounds
Power [w];
Frequency
[MHz]
Solvent Temp. [°C] Time [min]
Enteromorpha
prolifera
300–700;
-
10–60%
ethanol
-
5–40 (1–4
cycles)
Polyphenols
Carpophyllum
flexuosum
-
H2O, acetone,
ethanol,
propan-1-ol,
ethyl acetate
135, 160 and
185
1, 3, 5, 10, 15
and 20
Phloroglucinol

METHOD OF PURIFICATION
1. Membrane filtration
 Membrane filtration can be used at different
levels.
 Ultrafiltration with a high molecular weight
cut-off (MWCO) can be used for the
separation of macropeptides and
nonhydrolyzed proteins.
 Membrane filtration can operate at normal
temperature, and there are no chemical
reactions during the process.
 Membrane filtration can provide a large
number of separations compared to other
chromatographic separations.

2. Gel Filtration Chromatography
Gel filtration chromatography (GFC), also called size exclusion chromatography,
For the separation, desalting, and molecular weight estimation of peptides and
proteins.
GFC is the simplest and mildest of all of the chromatography techniques
Separates molecules on the basis of differences in size.

CONT….
Some smaller molecules enter the pores of the gel and travel a longer distance,
While larger molecules show much shorter retention times.
Advantage of GFC is that elution conditions can be varied to suit the type of
sample
Some limitations, such as the loading amount being rarely compared to the
membrane filtration and collecting sample costing a lot of time.

3. Ion Exchange Chromatography
IEX media have charged functional groups that bind molecules with an
opposite charge.
Bound molecules are eluted from the medium by displacement, via the
application of an increasing concentration of a similarly charged molecule.
Proteins have numerous functional groups that can have either positive or
negative charges.
By adjusting the pH or the ionic concentration of the mobile phase, proteins
can be separated.
IEX is used for capturing the target protein or bulk impurities from large
volumes.
As an intermediate purification step or as a final step for high-resolution
purification to remove impurities.

Marine Natural Product 29
CONT…

4. High-Performance Liquid Chromatography
HPLC is the most widely used technique for the separation, identification, and
purification of bioactive peptides.
In addition, RP-HPLC can be used to fractionate peptides based on their
hydrophobic properties.
The main advantages as it always uses a short time to get the elution spectra
compared to the GFC and IEX.
In recent years, there are many researchers that have used HPLC to purify marine
organisms, like Enteromorpha, Cyanobacterium, Thornback Ray, Sponge, Tuna,
Marine Snail, and so forth.
HPLC also has some limitations, like chromatographic columns being expensive,
Elution composition containing an organic solvent and being environmentally
unfriendly, and so forth. [7]

CONT…..
High-Performance Liquid Chromatography

MARINE TOXINS

MARINE TOXINS
• Microorganisms from the sea which are
autotrophic, also act as a source of toxins.
• These toxins are both ectocrine or external
metabolites and endotoxins.
• Such endotoxin are some of the most potent
materials.
• The best-known marine biotoxins and
tetradotoxin, paralytic shellfish toxin, and
ciguatera toxin.

1. Ciguatoxin
It is present in red tide dinoflagellate Gam bier discuss – toxins.
The term “Ciguatoxin” was first used to describe an ailment associated with the ingestion
of marine snails.
Ciguatoxin is the factor isolated from pacific red snapper and considered to exert toxicity
i.e., ciguatera poisoning.
The toxin symptoms are associated with neurological, cardiovascular, and gastrointestinal
systems
Like respiratory depression and bradycardia, the release of nor-epinephrine from the
presynaptic sides in the neuromuscular junction of guinea pig, etc.

2. Palytoxin
It is a long chain polyhydroxy macromolecule present in Palythoa species
and is the most potent coronary vasoconstrictor.
Palytoxin can exert a full effect on coronary arteries in an isolated guinea
pig heart even at very low concentrations.
Besides such effect it also has actions on nervous tissues and muscles.
It elevates T wave and S-T segment in ECG of Rat, Dog, and Guinea Pig.

3. Red Tide Toxins (Paralytic Shell Fish Toxins)
These include brevetoxins, saxitoxin, gonyautoxin, etc.
These causes a change in color of the water surface, like yellow, brown, green,
and not always red.
They are dinoflagellates which bloom periodically and contain peridinin, a red-
colored pigment.
The most deleterious among them are :
- Gonyaulax catenella giving Saxitoxin.
- G.Tamerenin which gives Gonyautoxin
- Ptychodiscus breuis gives Brevetoxin

Adverse Conditions like
-O2 deficiency,
-an environmental imbalance of nutrients,
-accumulation of H2,
-Sulphide in the surrounding,
-Red tides exert drastic effects due to their toxins
and may turn a rich fauna into a biological dessert.

• Marine toxins have not yet been introduced in Clinical medicine.
• They might prove to be useful as a research tool in investigating the process of
biolectogenesis, chemical transmission, etc.
• Research work- Biochemistry from Ottawa in Canada
- found a protein in sea tortoise which is present in blood cells.
- because of this protein (not named) tortoise can lower the temperature of
its body fluid below freezing point.
- such protein may be employed to facilitate certain surgical operations
like cardiac surgery where it is essentials to lower the body temperature
to an appreciable level. [8]

RECENT ADVANCES IN RESEARCH IN MARINE DRUGS
Currently, natural product-derived therapeutics are mostly developed from
compounds of terrestrial origins.
Recently, the research focus for natural products has been shifted from terrestrial
to marine sources. (because- superior chemical and biological novelties).
In general, natural molecules isolated from the marine environment show higher
and more significant bioactivity than those from the terrestrial environment.

CONT…
In cytotoxicity screenings, approximately 1% of the tested marine samples
showed anti-tumor potentials versus 0.1% of the tested terrestrial samples.
Marine natural product research faced several challenges, ranging from
limited access to the marine environment to chemical and biological
characterizations of promising natural products.
Advancement in sampling techniques and structural determination
strategies have played crucial roles in the recent progress of marine drug
discovery.

1. Sampling Techniques
• Sampling in the ocean requires specific techniques
and sophisticated equipment.
• This hurdle has been the main reason for the
limited exploration of natural products in the
oceanic environment.
• This submersible has produced several impressive
achievements in research
• The origin of deep submersible was motivated by
biologist William Beebe, who observed fish and
other diverse forms of deep-sea life with the naked
eye in 1934.

CONT…
• After several decades, a human-occupied deep submergence
vehicle, SHINKAI 6500, was launched in 1991, and was capable of diving to the
maximum depth of 6500 m.
• This submersible has produced several impressive achievements in research on the
topography and geology of the ocean floor, as well as life inhabiting the deep seas
• Because of the cost and risk, human-occupied vehicles cannot be used in research
at depths greater than 6500 m.

CONT…
Marine Natural Produts 43
• So far, KAIKO is one of the deepest diving devices which is capable of diving to a
maximum depth of 10,000 m.
• Since 2009, more than 4400 novel marine natural compounds have been
identified.

2. Structural Determination
• Modern research for marine natural products has relied heavily on fractionation based
on the combination of a valid fractionation by a high-resolution separation technique
and detection systems.
• Combining different instrumentations as hyphenated devices, such as
-CE-MS (capillary electrophoresis-mass spectrometry),
-HPLC-MS (high-performance liquid chromatography-mass spectrometry),
or HPLC-NMR (high-performance liquid chromatography-nuclear magnetic
resonance)
for separation and detection, is one of the most active research fields in modern
analytical chemistry.
• These techniques play a predominant role in natural product discovery-related fields
such as phytochemistry or pharmacognosy. [9]

CE-
MS
HPLC-MS
HPLC-NMR

3. Tyrosinase Inhibitors from marine algae (Enteromorpha
species) [Lima Rodriguesa, Supriya Tilvi b, Michelle S. Fernandes, etc.]
• Marine green macroalgae Enteromorpha sp. belonging to family Ulvaceae.
• It houses numerous bioactive secondary metabolites such as sterols, chlorophyll-
related compounds, phenolic polyketides, α-pyridones, polysaccharides, etc.
• Tyrosinase enzyme, also known as polyphenol oxidase is a complex copper-holding
enzyme, found in prokaryotes as well as in eukaryotes.
• It bio-catalyzes the biosynthesis of melanin from tyrosine through a chain of oxidative
reactions.

CONT…..
• Melanin plays an essential role in protecting the skin from getting damaged upon
exposure to harmful UV radiations.
• Skin whitening is a classical issue in many human cultures.
• There are several synthetic, semi-synthetic, and natural tyrosinase inhibitors
derived from countless natural sources.
• Health safety is the primary motto, it has drawn the attention of scientific
researchers towards exploiting naturally occurring sources for tyrosinase
inhibitors.
• Crude methanol extract of Enteromorpha sp. mildly inhibited the mushroom
tyrosinase.
• It is also reported -major metabolites 4- hydroxycoumarin, ergosta-5,7,22-trien-
3β-ol, ergosterol peroxide, from the anti-tyrosinase active crude methanol extract
of Enteromorpha sp. [10]

4. Synthesis of Varitriol: an Anti-tumour Agent from Marine-
Derived Fungus Emericella variecolor. [Mahesh S. Majik, Supriya Tilvi,
Prakash T. Parvatkar]
• Marine fungi are one of the important sources of natural products, exhibiting a
wide range of biological activities.
• (+)-Varitriol, has shown 100-fold increased potency over the mean toxicity
towards a variety of cancer cell lines.
• Varitriol (+)-5 is such a marine natural product isolated by Barrero and co-
workers.
• From a marine strain (M75-2) of the fungus Emericella variecolor along with
other metabolites-
-varioxirane 6 and
-dihydroterrein 7 (in the year 2002)

• The fungus was isolated from a sponge collected in
Venezuelan waters of the Caribbean Sea.
• Varitriol shows more than 100-fold increased potency
toward
-RXF 393 (renal cancer),
-T-47D (breast cancer), and
-SNB-75 (CNS cancer) cell lines
and lower potency against prostate cancer, leukemia,
ovarian cancer, and colon cancer cell lines.
• Since 2006, about 15 total syntheses of varitriol have
been reported in the literature. [11]
CONT…

5. Bryozoa
• Bryozoans such as moss animals and lace corals are abundant in marine
environments.
• Shallow water bryozoan species have produced such medicinally important
compounds as the anti-cancer and Anti-Alzheimer lead (lead Bryostatin 1 isolated
from Bugula neritina.)
• Deep water bryozoan metabolites, have been isolated from Amathia tortuosa. [12]

PROBLEM FACED IN RESEARCH AND SOLUTION
1. Supply
Problem:
It is a critical point in the process of drug development from marine organisms.
Permanent availability of sufficient amounts of organisms and compounds without
harming the marine environment if a difficult process.
Only if supply can be addressed in an economically and ecologically feasible
fashion,
This marine drug will get a chance on the market.

CONT…
Solution: Genetic Engineering
The principle of this method is the transfer of genetic information of the desired
compound into host cells.
This will be easier to cultivate and and get sustainable production of the compound in
the host cell.
Such techniques would permit the isolation and expression of genes of the organisms,
which can not be cultivated.
Till now, this approach is realized on a research-level but not on an industrial scale for
marketable marine drugs.

2. Taxonomical Identification
Problem:
Lack of taxonomic knowledge for marine species,
And the still large number of unidentified species and
strains,
So this is a major blockage faced by marine natural products
programs.
The selection for- Pharmacological Purpose
- Macro and Micro-organism
either from terrestrial or marine, must be grounded on
correct taxonomic identification and classification.
Incorrect classification of species may compromise an entire
drug discovery project.

CONT….
Solution:
Scientific community should be encouraged to publish
research journals that describe new Natural Products.
This should be done to improve their standards
concerning the Taxonomic identification and Geographic
information on the origin of screened biological material.
Such accurate information will ultimately maximize the
success of future screening efforts.
This also allows meta-analysis using cross-disciplinary
approaches that may lead to a significant breakthrough in
research.

3. Chemical Screening
Problem:
The growing recognition that marine
microorganisms associated with invertebrates hosts
are involved in the biosynthesis of secondary
metabolites.
This offers new alternatives for the discovery and
development of marine natural products.
However, the discovery of microorganisms
producing secondary metabolites,
Which is, previously attributed to an invertebrate
host poses the significant challenge of efficient
chemical screening.

CONT…
Solution:
Conventional activity-based screening, though today is driven by new
methodologies. (Such as – TLC, bioluminescence assay, Dilution Method, etc…)
Mining orphan secondary metabolic pathways.
Engineers know antibiotic pathways to make new molecules. [12]

57
REFRENCES
1. Marine natural product – an overview by science
direct topics.
2. Marine pharmacognosy – Wikipedia
3. Pharmacognosy- Trease and Evans (page-115)
4. Marine-Derived Pharmaceuticals – Challenges and
Opportunities- Biomolecules and Therapeutics-NCBI
5. Impact and Challenges of Marine Medicine to Man
and its Environment – Poultry, Fisheries and Wildlife
Sciences.
6. Overview on the Application of Modern Methods for
the Extraction of Bioactive Compounds from Marine
Macroalgae(https://www.ncbi.nlm.nih.gov/pmc/articl
es/PMC6213729/)
7. Characterization, Preparation, and Purification of
Marine Bioactive
Peptides.(https://www.hindawi.com/journals/bmri/201
7/9746720/)
8. Pharmacognosy- C.K.Kokate (17.7)

REFERENCES
9. Recent Advances and Applications of Experimental
Technologies in Marine Natural Product Research.
(https://www.mdpi.com/1660-3397/13/5/2694/htm)
10. Isolation and Identification of Tyrosinase Inhibitors
from Marine Algae Enteromorpha sp[Lima Rodriguesa,
Supriya Tilvib, Michelle S. Fernandes,
etc.](https://drs.nio.org/drs/handle/2264/8604)
11. Recent developments towards the synthesis of Varitriol: An
antitumor agent from marine-derived fungus Emericella
variecolor.[Mahesh S. Majik, Supriya Tilvi, Prakash T.
Parvatkar](https://drs.nio.org/drs/handle/2264/4541)
12. Recent Advances in Marine Drugs.
(https://www.slideshare.net/VarshaSrivastav/recent-advances-
in-marine-drugs)

MARINE NATURAL PRODUCTS- Durgashree Diwakar

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