Antibacterial Activity of Libyan Macroalgae

ByNada Hussein Zubi

Supervision Dr : Salmin Alshalmani

Libyan International Medical UniversityCollege of Pharmacy

Project of Graduation

Aim of the study To investigate the type and

Pharmacological activities of seaweeds originated from cost of Benghazi .

To assess the antibacterial effect of aqueous and alcoholic crude extracts of 4 marine algae from the eastern coast of Libya, against pathogenic bacteria (1 Gram-positive, 2 Gram- negative).

1. Cystoseira compressa 2. Enteromorpha intestinal 3. Ulva Lactuca4. Corallina

Techniques was used Extraction of active constituents.

Microbiological tests

Terminology

The algae are one of the most vast and diverse groups of ancient photoautotrophic pro- and eukaryotic organisms (about 30 000 known species). They are mobile and immobile, attached and free-living.

Algae are a very large and diverse group of simple, typically autotrophic organisms, ranging from unicellular to multicellular forms, such as the giant kelp, that may grow up to 50 meters in length. Algae were once considered to be plants but are now classified separately because they lack true roots, stems, leaves.

• They are thought to represent the earliest form of life on Earth, forming at a time when the Earth's atmosphere was devoid of oxygen.

• This has lead to conclude that algae are responsible for the early accumulation of oxygen in the atmosphere, paving the way for other plants and eventually animals to evolve.

Algae are Highly Adaptable, come in all sizes For example, phytoplankton, which lives in lakes and oceans, can accumulate in the thousands just on a small pinhead.

Algae are found in the sea, rivers, lakes soils, walls, in plants and animals just about anywhere there is light to photosynthesize .

Types of Algae• In 1997 some scientists introduced a classification system that

breaks all algae down in 11 different phyta classifications, • The most common are • green algae, red algae, brown algae, golden algae and

cyanobacteria (blue green algae).

• Seaweed draws an extraordinary

wealth of mineral elements from the sea

that can account for up to 36%

of its dry mass.

Algae in Benghazi coast• Benghazi coasts extend more than 18 km. at Mediterranean

Sea and very rich of marine algae but there is no recent algal list related to that point. The present work was mainly intended to collect and study the marine green algal population of Benghazi coasts to enable other researches to evaluate their chemical composition, antimicrobial activities and economic importance.

Benefits of seaweed Algae as food: Algae are used

as source of food since 6000 year ago by human beings. They are rich in carbohydrates, inorganic substances and vitamins.

Algae in industry: Diatoms (algae) are extensively used in sugar refineries and soap manufacture. It is also helpful in cement industry, in the manufacture of dynamite.

Benefits of seaweed:-• As fuel: Algae are the fastest-growing plants in the world.

Different species of algae may be better suited for different types of fuel. Algae can be grown almost anywhere, even on sewage or salt water, and does not require fertile land or food crops, and processing requires less energy than the algae provides.

Pharmacological activities of seaweeds• As Antidiabetic : Ulva rigida,

one of the green algae showed antidiabetic and antihyperlipidemic effects and improves oxidative stress in diabetic rats.

• As antihypertensive : • Six polysaccharides derived from

seaweed species were investigated, and appreciable suppression of hypertension and hypercholesterolemia .

• They also markedly enhanced the serum level of HDL

Anticancer and cytotoxic activities • The new classes of anticancer drugs that have been isolated

from marine organisms have been shown to possess cytotoxic activity against multiple tumor types.

Anti Microbial activities

• The green, brown and red algae have been extensively screened for:

antibacterialantifungalAntiprotozoal activities

ExpErimEntal SEction

Material and method Collection of Algal material Macroalgae were collected by hand picking form Benghazi coast (Alsabri) in

15 April 2013, the algae were cleaned and washed thoroughly in sterile sea water, then rinsed in distilled water to remove salt.

The taxonomic of samples was done by experts in these fields. All species tested are deposited in herbarium sheet. then dried for 48 hr in natural light in 25ºC, the dry seaweeds were crushed

in an electrical mill until a fine powder was obtained, and stored in bottles at room temp .

Herbarium sheet of (Cystoseira compressa , Ulva lactuca ,

Enteromorpha intestinal)

Microscopically examination for the powder

• 1- Corallina

2- Cystoseira

3- Enteromorpha 4 . Ulva Lactuca

Extraction:1. The powder (5 g) of dried algae

extracted in shaker apparatus using 100ml of solvent for 24h at 25ºC.

2. The extraction done by several solvent (water - ethanol( 99%) – methanol (99%) – methylated spirit (95%).

3. then the extract filtrated to remove the insoluble material.

4. Then concentrated by evaporated the solvent to dryness and the solid residue then dissolved in 2 ml by the same solvent and stored at 4ºC .

Antibacterial activity test:

• The tested microorganisms selected were spoiled microorganisms or common human pathogen, and were comprised gram- positive bacteria staphylococcus aureus SH100, of the two gram- negative bacteria pseudomonas aeruginosa ATCC 17933 and Escherichia coli ATCC 25922. The organisms were obtained from the department of microbiology, Benghazi medical center ,all cultures were kept on nutrient agar and stored at 4ºC.

Antibacterial Assay:

The test was performed by the Agar Diffusion Method .

by a smear of microorganism applied to( Mueller-Hinton) plates using a cotton swap .

the crud extracts were placed in 6 mm diameter wells made on the plates with sterilized borer , the extract algae compound (100µl) was introduced into the well with electronic pipit.

solvent was also used as negative control .

the plates were incubated at 37ºC, for 24hr

Antibacterial Assay: the diameter of the inhibition halo is considered

to be indicative of the bioactivity of the seaweeds extract .and was measured

( including the well diameter) with caliper

standardized values for diameters of inhibition halo , expressed in mm.

diameter less than 7mm was interpreted as inactive (-) .

diameter less than 9mm was interpreted as trace active (+) .

diameter between 10 and 14 was interpreted as moderately active (++) .

diameter more than 14 was interpreted as highly active (+++).

Results and Discussion Antimicrobial activity of liquid algae extract according to

solvent• Methanolic extract had the highest antimicrobial activity and the broadest

spectrum. • Water extract had the lowest activity with low bioactivity and narrow

spectrum of action • Methanol, methylated spirit, ethanol and water extract showing the

antimicrobial activity respectively.

• The results showed the effect of the methylated spirit extract of Ulva lactuca. significantly inhibited the growth of pseudomonas aeruginosa. And this is clear since the discoloration of the plate.

Antimicrobial activity of methanolic extract of the 4 algae

1. The results indicated that Green alga ulva lactuca is the most active algae as antimicrobial activity with highest and the broad spectrum activity .

2. S. aureus was more sensitive than all the stocks, with the largest inhibition diameter.

3 . E.coli was more resistance against all the solvents .

Effects of highly concentration of methanolic extract of 4 algae

• when we study the highly concentration of methanolic extract (10% , 15% , 20%, 25%) of 4 algae with the same bacteria, significant result obtained from S. aureus, at 10% conc there is no growth of S. aureus in the all plats ,the algae kills all bacteria.

• With Sp.sp was obtained discoloration for most bacteria in the plate

• no activity on E.coli even in the high concentration.

Effect of algae extract after one year of storage to the dried powder

• in April 2012 sergassium algae was collected and stored in bottle in room temperature for one year , in April 2013 was extracted and

• there are no deference in the antibacterial activity between the stored algal powder collected in 2012

• the active constituent are still stable.

Conclusion:

1. algae are used by human beings for food, from ancient times. researches are being done in phycology (study of algae) to find out the food value of algae.

2. their importance in industries and importance in agriculture.

3. Macroalgae from Libyan coast are potential bioactive compounds and should investigated for natural antibiotics this study has shown that the production of antibacterial substances is regular occurrence among those found on the coast of Libya especially Green algae,.

RECOMENDATION

1 . Continuous researches on the other possible activities on these 4 algae.

2. Consider the economic importance of these type of studies, since the long Libyan coast and high availability of different types and species.

3. Continuous education and awareness about the Marine products and their use daily life as food, cosmetics, in agriculture and more importantly as a fuel.

4. More study on methods of isolation and separation of their active constituents .

THANK YOU