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.

CHLORELLA SP.

SYNECHOCYSTIS SALINA

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.