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BIOCHEMICAL PROFILE OF MACRO AND MICRO ALGAE
AND THEIR IMPLICATION FOR FURTHER COMMERCIAL APPLICATIONS
PhD. RALUCA MIHAI
ANTIOXIDANTS
• Excessive amounts of ROS (Oxygen related free radicals: superoxide and hydroxyl radicals and reactive species: hydrogen peroxide, nitric oxide, peroxynitrile and hypochlorous acid) may be harmful because they can initiate bimolecular oxidations which lead to cell injury and death, and create oxidative stress which results in numerous diseases and disorders.
Free radical formation is controlled naturally by various beneficial compounds known as antioxidants. They protect the key cell components by neutralizing the damaging effects of free radicals, which are natural products of cell metabolism
Marine organisms comprise over a half million
species.
Because of the immense biological diversity in the
sea as a whole, it is increasingly recognized that a
huge number of natural products and novel
chemical entities exist in the ocean, with biological
activities .
exceeds 10,000, with hundreds of new
compounds still being discovered every year
From 1969-1999 approximately 300 patents
on bioactive marine natural products
MACROALGAE
• Macro-algae or “seaweeds” are multicellular plants growing in salt or fresh water. They are often fast growing and can reach sizes of up to 60 m in length.
• They are classified into three broad groups based on their pigmentation: • brown seaweed (Phaeophyceae)
• red seaweed (Rhodophyceae)
• green seaweed (Chlorophyceae)
Field work was performed at several coastal sites
around Greece, by free diving in rocky midlittoral
and upper sublittoral levels (0–3 m depth) and by
scuba diving to 42 m depth. 42 m depth.
Taonia atomaria
Gelidium pulchellum
Ulva rigida
Enteromorpha linza
Halimeda tuna
Caulerpa prolifera
Organism Type
Ulva rigida Green
Halimeda tuna Green
Udotea petiolata Green
Codium vermilara Green
Dassycladus clavaeformis Green
Caulerpa prolifera Green
Enteromorpha rinza Green
Coralina mediterranea Red
Gelidium sp Red
Laurencia obtusa Red
Sargasum vulgare Brown
Dictyopteris membranaceae Brown
Taonia atomaria Brown
Posidonia oceanica seaweed
Halopteris (filicina) Brown
Cladostephus spongiosus Brown
Laurencia papillosa Red
• For each sample, on this experimental step three solvents of different polarities (CH3OH 40%, CH2Cl2, MeOH) were used for the exhaustive extraction of compounds from the solid algal tissue into the solution.
• AOA evaluation of polar extracts using:
luminol chemiluminescence
DPPH method
Results: Posidonia oceanica > Taonia atomaria > Caulerpa prolifera > Laurencia papillosa
> Udotea petiolata > Cladostephus spongiosus > Dassycladus clavaeformis > Halimeda
tuna > Corallina mediteraneea > Sargassum vulgare > Halopteris filicina > Enteromorpha
linza > Dictyoperis membranaceea > Codium vermilara > Ulva rigida > Laurencia obtusa >
Gelidium sp
• AOA evaluation of nonpolar extracts
Peroxyoxalate Chemiluminescence Assay
Hydrogen donating method with EtOAc
• Fractionation of Posidonia oceanica extract by flash chromatography
• Two different eluents (CH2Cl2 and MeOH) were used in different proportions for the separation of the Posidonia extract. The presence of compounds in the fractions obtained during flash chromatography was monitored by TLC using the mobile phase CH2Cl2 and MeOH (3:2).
• Evaluation of antioxidant activities of the fractions obtained by flash chromatography.
1H NMR spectrum of Fraction 6 showed several resonances in chemical shifts that were characteristic of oxygenated methines.
The complex spin systems in the region of
3.0 ppm – 4.1 ppm is indicative of
carbohydrates.
The deshielded protons at 5.4 ppm,
5.6 ppm and 6.1 ppm are the
anomeric protons of these carbohydrates.
IDENTIFICATION OF THE MAJOR METABOLITES BY GC-MS
• Since the polarity of the oligo / polysaccharides is prohibitive for their analyses by GC-MS, it was decided to derivative those in order to reduce their polarity and allow better separation on the Gas chromatograph.
GS-MS spectrum of the acetylated Fraction
• The GC-MS spectrum showed the presence of 2 major constituents which, when compared with the library spectra were identified with a good confidence as α-D-Glucose pentaacetate and Sucrose octaacetate.
GS-MS spectrum of the acetylated Fraction
• The 1H NMR spectrum of the acetylated Fraction showed the presence of a number of Acetyl groups (1.9 – 2.2 ppm), supporting the assumption of the oligosaccharide presence.
1H- NMR spectrum of acetylated Fraction
In recent years, algal polysaccharides have been demonstrated not
only to play an important role as free- radical scavengers and
antioxidants for the prevention of oxidative damage in living
organisms, but also to have diverse physiological activities including
anticoagulant, antiviral and antitumor activities.
MICROALGAE • Microalgae are microscopic organisms that grow in salt or fresh water. The three
most important classes of micro-algae in terms of abundance are the diatoms (Bacillariophyceae), the green algae (Chlorophyceae), and the golden algae (Chrysophyceae).
• Their photosynthetic mechanism is similar to land based
plants, but due to a simple cellular structure, and
submerged in an aqueous environment where they have efficient
access to water, CO2 and other nutrients, they are generally more efficient in
converting solar energy into biomass.
APPLICATIONS OF MICRO-ALGAE
• Therapeutic supplements from micro-algae comprise an important market in
which compounds such as β-carotene, astaxanthin, polyunsaturated fatty
acid (PUFA) such as DHA and EPA and polysaccharides such as β-glucan
dominate.
ANTIOXIDAT ACTIVITY (DPPH METHOD)
139,5
140
140,5
141
141,5
142
WATER MEOH ETHYL ACETATE
130
135
140
145
150
155
160
WATER MEOH ETHYL ACETATE
SYNECHOCYSTIS SALINA CHLORELLA Sp.
PHENOLIC CONTENT EXPRESSED IN TERMS OF GALLIC ACID
EQUIVALENT (MG OF GA/G OF EXTRACT)
0
20
40
60
80
100
120
W AT E R M E O H
E T H Y L
AC E T A T E H E X AN E
0
50
100
150
200
250
W AT E R M E O H
E T H Y L
AC E T A T E H E X AN E
CHLORELLA SP. SYNECHOCYSTIS SALINA
CONTENT OF CHLOROPHYLL AND CAROTENOIDS (MG/ML)
0
10
20
30
40
50
60
70
SYNECOCYSTIS ME OH
24,17
67,03
14,78
Chl a Chl b β-C
0
5
10
15
20
25
30
35
40
45
SYNECOCYSTIS ETOH
40,05
17
6,18
Chl a Chl b β-C
Chlorella vulgaris MeOH
27,71
67,17
14,84
Chl a Chl b β-C
Chlorella vulgaris EtOH
26,32
76,38
14,93
Chl a Chl b β-C
CHLOROPHYLL
• Chlorophyll is one of the valuable bioactive compounds that can be extracted from microalgal biomass. It is used as a natural food colouring agent and has antioxidant as well as antimutagenic properties.
• Chlorophyll has been found to accelerate wound healing by more than 25% in some studies. Since chlorophyll stimulates tissue growth, it prevents the advancement of bacteria and speeds up the wound healing process. Chlorophyll is similar in chemical structure to haemoglobin and, as such, is predicted to stimulate tissue growth in a similar fashion through the facilitation of a rapid carbon dioxide and oxygen interchange. Because of this property, chlorophyll is used not only in the treatment of ulcers and oral sepsis but also in proctology.
• Pay a key role in cancer prevention due to their high displays of antioxidant and antimutagenic activities
CONCLUSIONS
• Microalgae are a diverse group of microscopic plants with the wide range of physiological and
biochemical characteristics and contain up to 50-70% protein (up to 50% in meat, and 15-17% in wheat),
30% lipids, over 40% glycerol, up to 8-14% carotene and a fairly high concentration of vitamins B1, B2,
B3, B6, B12, E, K, D, etc., compared with other plants or animals .
• Microalgae are meant to be an important raw material for amino acids, vitamins and productions of
other pharmateuticals. The cultivation of microalgae is known to be the most profitable business in the
biotechnological industry. It is a wasteless, ecologically pure, energy and resource saving process.