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determination of loss by drying (FRX); the determination of calcination .... cells caught during division after the time of action of the algal extract. 0. 100. 200. 300.

Annals of RSCB

Vol. XVI, Issue 1

THE EVALUATION OF CERTAIN BIOLOGICAL EFFECTS OF EXTRACTS FROM MACROPHYTIC MARINE ALGAE FROM THE BLACK SEA Elena Doroftei1, Mihaela Mirela Bratu2, Daciana Sava1 1

2

FACULTY OF NATURAL AND AGRICULTURAL SCIENCES; FACULTY OF FARMACY, “OVIDIUS” UNIVERSITY, CONSTANTZA

Summary Marine macrophytic algae are a group of vegetal organisms with specific bioactive metabolites, including brominated phenols, oxygen heterocyclic compounds, sterols, terpenoids, polysaccharides etc. Even though little used in the pharmaceutical industry as such, they represent important sources for numerous raw materials and for certain extracts with therapeutic usage based on their antibacterial and antiviral properties. The present paper presents the results obtained after the chemical and biological characterization of the extracts taken from macrophytic algae collected from the Black Sea. Based on these results, we estimate that the algal extracts have important antifungal activity with the possibility of exploiting them for therapy purposes. The raw material consisted of a mixture of macrophytic algae collected from the Black Sea – Mamaia area, from a depth of 6 meters. These were stored for 48 hours in the open air for a preliminary drying. The aqueous algal extracts were obtained as cold and warm aqueous extract. The extracts obtained were characterized through general chemical and biochemical parameters, accomplishing the following analyses: the determination of loss by drying (FRX); the determination of calcination residues (FRX); the pH determination; the determination of total protein by the Kjeldahl method; the quantitative determination of proteins by the Lowry method; the quantitative determination of carotenoids; the determination of ascorbic acid from algal extracts; the determination of the phytotoxicity index by the use of the Triticum test. The physic-chemical determinations on the aqueous algal extract displayed the following values: pH 5.6; dry substance 0.065%; β-carotene 112.3 μg/dL; ascorbic acid 1.89 mg/mL and soluble protein 0.948 μg/mL for the cold extract, and pH 6.62; dry substance 0.5775%; β-carotene 68.3 μg/dL; ascorbic acid 0.862 mg/mL and soluble protein 1.048 μg/mL for the warm extract. The estimation of the mitotic activity of the obtained extracts, accomplished by the Triticum test, led to the conclusion that both extracts have a significant antifungal activity with therapeutic possibilities. Key words: macrophytic algae, biochemical parameters, Triticum test, antifungal activity.

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Introduction

(Bhakuni and Rawat, 2005; Briand, 1996; Golz-Berner and Zastrow, 2000; Khotimchenko et al., 2001; Popa et al., 1974; Smith, 2005). In what regards their role in the balance of an aquatic ecosystem, we can specify that their exaggerated multiplication – the “blooming” phenomenon – has negative consequences on the marine organisms, leading to their mass mortality. On the other hand, they have important role

Marine macrophytic algae are a group of vegetal organisms with specific bioactive metabolites, including brominated phenols, oxygen heterocyclic compounds, sterols, terpenoids, polysaccharides etc. Even though little used in the pharmaceutical industry as such, they represent important sources for numerous raw materials and for certain extracts with therapeutic usage based on their antibacterial and antiviral properties 313

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Vol. XVI, Issue 1

WARM EXTRACT:

in economy as their wide range of uses include the food and pharmaceutical industry, medicine, cosmetics, agriculture and zootechny (Hung et al., 2010). The present paper presents the results obtained after the chemical and biological characterization of the extracts taken from macrophytic algae collected from the Black Sea. Based on these results, we estimate that the algal extracts have important antifungal activity with the possibility of exploiting them for therapy purposes.

FRESH ALGAE ↓ SUSPENSION IN DISTILLED WATER (1: 5) ↓ WARMING (48°C, 4 h) ↓ DECANTING ↓ FILTRATION Fig. 1. The scheme for the obtaining of algal extracts

Material and methods The raw material consisted of a mixture of macrophytic algae collected from the Black Sea – Mamaia area, from a depth of 6 meters. These were stored for 48 hours in the open air for a preliminary drying. The aqueous algal extracts were obtained as follows (Bratu and Crâsmaru, 2002; Ciulei and Istudor, 1995): a) Cold aqueous extract: The algae were suspended in distilled water in a 1:5 (m : m) ratio and were maintained in cold water (9°C) for 84 h. Afterwards, the liquid part was decanted and clarified by filtering. b) Warm aqueous extract: The algae were suspended in distilled water in a 1:5 (m : m) ratio and were maintained at 48°C for 4 hours. After cooling, the liquid part was decanted and clarified by filtering. The scheme for the way in which the

The extracts obtained were characterized through general chemical and biochemical parameters, accomplishing the following analyses: the determination of loss by drying (FRX); the determination of calcination residues (FRX); the pH determination; the determination of total protein by the Kjeldahl method; the quantitative determination of proteins by the Lowry method; the quantitative determination of carotenoids; the determination of ascorbic acid from algal extracts; the determination of the phytotoxicity index by the use of the Triticum test (Raicu et al., 1983).

Results and discussions Double samples were used both for the raw material (fresh algae) and the algal extracts obtained. The determinations of the dry substance on the raw material reveal a percentage of 85.439% water and 14.561% dry substance for sample 1, and a percentage of 84.312% water and 15.687% dry substance for sample 2. The physicchemical determinations for the raw material reveal a percentage of 15.124% dry substance and 0.899 ‰ ash, while the value of the total Kjeldahl protein was 1.966. The determinations of dry substance on algal extracts displayed a percentage of 99.935% water and 0.065% dry substance for the cold extract and a percentage of 99.4225% water and 0.5775% dry substance for the warm extract. The physic-

two extracts were obtained is presented in figure 1: COLD EXTRACT: FRESH ALGAE ↓ SUSPENSION IN DISTILLED WATER (1: 5) ↓ DECANTING ↓ FILTRATION 314

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chemical determinations on the aqueous algal extract displayed the following values: pH 5.6; dry substance 0.065%; β-carotene 112.3 μg/dL; ascorbic acid 1.89 mg/mL and soluble protein 0.948 μg/mL for the cold

extract, and pH 6.62; dry substance 0.5775%; β-carotene 68.3 μg/dL; ascorbic acid 0.862 mg/mL and soluble protein 1.048 μg/mL for the warm extract.

Table I. Determinations of the dry substance on raw material Det. no.

Dry substance mass

Water mass

% dry substance

% water

Sample 1

0,2921

1,7139

14,561

85,439

Sample 2

0,3142

1,6887

15,687

84,312

Table II. Physic-chemical determinations on raw material Sample

Dry substance (%)

Ash (‰)

Fresh algae

15.124

0.899

Total Kjeldahl protein 1.966

Table III. Determinations of the dry substance on algal extracts Type of extract

Dry substance mass

Water mass

% dry substance

% water

Cold extract

0.0112

1.9887

0.560

99.935

Warm extract

0.0115

1.9884

0.5775

99.4225

Table IV. Physic-chemical determinations on aqueous algal extracts Sample

pH

Dry (%)

Cold extract Warm extract

5,6 6,62

substance β-carotene μg/dL 0,560 112,3 0,5775 68,3

Ascorbic acid mg/mL 1,89 0,862

Solubile protein μg/mL 0,948 1,048

Etalon curve for Lowry Method 2.7 2.4

y = 0.2098x + 0.2231

2.1

ΔA660

1.8 1.5 1.2 0.9 0.6 0.3 0 1

2

3

4

5

6

7

8

9

10

Albumine concentration (μg/mL)

Fig. 2. Standardization curve for the Lowry method for the determination of soluble protein

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Table V. Results obtained for the calibration curve for the Lowry method Tub. No.

Albumin solution

Distilled water

μg/mL albumin

ΔA

1

0

1,0

0

0

2

0,1

0,9

10

0,466

3

0,2

0,8

20

0,480

4

0,3

0,7

30

0,819

5

0,4

0,6

40

1,067

6

0,5

0,5

50

1,363

7

0,6

0,4

60

1,537

8

0,7

0,3

70

1,735

9

0,8

0,2

80

1,937

10

0,9

0,1

90

2,151

11

1,0

0

100

2,241

Etalon curve for determination of concentration in β-carotene 0.7 0.6

ΔA

0.5 0.4 0.3 0.2 0.1 0 0

0.5

1

1.5

2

2.5

3

3.5

β-carotene concentration μg/mL

Fig. 3. Standardization curve for the determination of β-carotene concentration Table VI. Results obtained for the calibration curve in the determination of β-carotene Crt. no. 1

β-carotene solution 2.5 mg% 0,7 mL

2

Benzene la 10 mL

β-carotene concentration μg/mL 1,75

0,33

0,8 mL

la 10 mL

2

0,37

3

0,9 mL

la 10 mL

2,25

0,42

4

1,0 mL

la 10 mL

2,5

0,47

5

1,1 mL

la 10 mL

2,75

0,51

6

1,2 mL

la 10 mL

3

0,56

7

1,3 ml

la 10 mL

3,25

0,61

316

ΔA

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Vol. XVI, Issue 1

The Triticum test is used to determine the phytotoxicity index. Thus, seeds of Triticum aestivum (wheat) are chosen and set to germinate in Petri dishes, on watermoistened filter paper. Each Petri dish contains 10-12 seeds, while the filter paper is water-moistened every day until the development of 1-1.2 cm radices. At this point, the extracts that need testing are added (cold algal extract and warm algal extract) in two Petri dishes, while the third is considered witness. The filter paper

continues to be moistened. After 6, 12 and 24 hours respectively from the application of the mentioned extracts, the seeds with radices are collected in 2-3 ml of fixing solution and are kept in the fridge for 16-24 hours. After this interval, the best developed radices are detached and colored by the Feulgen technique. Afterwards, microscopic preparations can be made by the squash method and analyzed under the microscope.

Table VII. The variation of the number of cells caught during division, depending on the action time period of the algal extract Analyzed version

Martor Alge 6h Alge 12h Alge 24h

Total number of counted cells 500 500 500 500

Number of cells at interphase

Number of cells at prophase

Number of cells at metaphase

Number of cells at anaphase

Number of cells at telophase

Number of cells at cytokinesis

385 430 476 488

65 32 14 7

3 1 -

2 2 1 -

20 15 5 3

25 20 4 2

The formula used to calculate the mitotic index is as follows: MI = number of cells at mitosis / number of analyzed cells x 100

For the witness: MI =

385 ⋅100 = 77, so on. 500

The variation of the number cells caught during division after the time of action of the algal extract 600

Cells number

500 Proba martor Extract algal la rece 6h Extract algal la rece 12h Extract algal la rece 24h

400 300 200 100 0 1

2

3

4

5

6

Cell division phases (1- intephase, 2- prophase, 3- metaphase, 4 - anaphase, 5 - telophase, 6 cytokinesis)

Fig. 4. The variation of the number of cells caught during division after treatment with algal extract at 6, 12 and 24 hours respectively, compared to the witness.

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number of prophases, telophases and cytokinesis being reduced compared to the previous determination. The microscopic analysis of the samples, 6 hours from the treatment with the algal extract, emphasized: - Triticum aestivum meristematic cells in which prophase is noted ( fig. 5); - Triticum aestivum meristematic cells in which prophase is noted and the two nucleoli are also visible (fig. 6); - Triticum aestivum meristematic cells in which anaphase is noted (fig. 7); - Triticum aestivum meristematic cells in which telophase is noted (fig. 8); - Triticum aestivum meristematic cells in which plasmolyzed cells are noted (fig. 9); - Triticum aestivum meristematic cells in which there are cells with the parietal nucleus at cytokinesis (fig. 10);

By following the results obtained in terms of the number of cells during division and of the mitotic index, it can be appreciated that there is a decrease of these two values. The decrease is more accentuated in the samples treated for 12 and 24 hours, respectively. In the case of the samples treated for 6 hours, there is a marked decrease of the number of cells at prophase, telophase and cytokinesis. The number of metaphases and anaphases remains very low. The next determination, at 12 hours, emphasized the absence of cells at metaphase and the marked decrease of the other values (cells at prophase, telophase and cytokinesis are in much reduced number). The absence of cells at metaphase and anaphase is a particularity of the determination realized at 24 hours, the

Fig. 5 (200X)

Fig. 6 (200X)

Fig. 7 (200X)

Fig. 8 (200X)

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Fig. 9 (400X)

Fig. 10 (400X)

The microscopic analysis of the samples, 12 hours from the treatment with algal extract, emphasized: - Triticum aestivum meristematic cells in which prophase is noted with vacuolized nucleus (fig. 11); - Triticum aestivum meristematic cells in which anaphase is noted (fig. 12); - Triticum aestivum meristematic cells in which the precocious formation of large numbers of chloroplasts is noted (fig. 13);

- Triticum aestivum meristematic cells in which cytokinesis and the presence of many chloroplasts are noted (fig. 14); - Triticum aestivum meristematic cells in which prophase, telophase and cytokinesis are noted, with the formation of the longitudinal (and not transversal) separating wall (fig. 15); - the formation of nucleated absorbing hairs from Triticum aestivum meristematic tissue developed as defense mechanism (fig. 16).

Fig. 11 (200X)

Fig. 12 (200X)

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Fig. 13 (600X)

Fig. 14 (600X)

Fig. 15 (400X)

Fig. 16 (600X)

- Triticum aestivum meristematic cells at cytokinesis, slightly plasmolyzed and with wavy separating wall (fig. 19); - Triticum aestivum meristematic cells, slightly plasmolyzed, with numerous chloroplasts (fig. 20); - Triticum aestivum meristematic cells, slightly plasmolyzed, with numerous chloroplasts (fig. 21); - Triticum aestivum meristematic

24 hours after the treatment with algal extract, the following were emphasized: - Triticum aestivum meristematic cells at prophase, with the development of numerous chloroplasts (fig. 17); - Triticum aestivum meristematic cells at prophase, with the development of numerous chloroplasts (fig. 18);

cells with numerous chloroplasts (fig. 22).

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Fig. 17

(400X)

Fig. 18

Fig. 19 (400X)

(400X)

Fig. 20 (400X)

Fig. 21 (400X)

Fig.22 (400X)

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Bratu, M.M., Crâsmaru M.: Îndrumar de lucrări practice de biochimie, Ovidius University Press, Constanta, pag. 50, 87, 2002. Briand, X.: Utilization of algae extract for the preparation of pharmaceutical, cosmetic, food or agricultural compositions, United States Patent 5508033, 1996. Ciulei, L., Istudor, V.: Analiza farmacognostică si fitochimică a produselor vegetale, vol. 2, 1995. Golz-Berner, K., Zastrow, L.: Cosmetic cleansing and skin care preparation containing plant and algae extracts, United States Patent 6221372, 2000. Hung L. V., Phong N. T. D., Tlusty M. F.: Effect of astaxanthin and cholesterol on growth, survival, and pigmentation of adult spini lobster Panulirus ornatus (Decapoda, Palinuridae). AACL Bioflux 3, 261-268, 2010. Khotimchenko, Yu.S., Kovalev, V.V., Savchenko, O.V., Ziganshina, O.A.: Physical-chemical properties, physiological activity, and usage of alginates, the polysaccharides of brown algae, Russian Journal of Marine Biology, 53-64, 2001. Popa, V., Rusan, V., Simionescu C.: Chimia Algelor Marine, Editura Academiei R.S.R., Bucuresti, pp. 29-31, 1974. Raicu, P., Anghel, I., Stoian, V., Duma, D., Taisescu, E., Gregorian, L.: Genetica – Metode de laborator, Ed. Academiei, Bucureşti, 47-53, 1983. Smith, J.A.: Medicinal and pharmaceutical uses of seaweed natural products: A Review, Journal of Applied Phycology, 245-262, 2005.

Conclusions •







Mixtures of thallophytic algae collected from the Black Sea were processed and characterized by means of general chemical parameters; Warm and cold macerates were accomplished from the collected algae by means of simple and duplicable methods; The extracts obtained were characterized by means of general chemical and biochemical parameters; The estimation of the mitotic activity of the obtained extracts, accomplished by the Triticum test, led to the conclusion that both extracts have a significant antifungal activity with therapeutic possibilities.

References Bhakuni, D.S., Rawat, D.S.: Bioactive Marine Natural Products, Anamaya Publishers, New Delhi, India, pp. 2-12, 18-19, 65-67, 69, 73-74, 81-84, 94-95, 105-112, 2005.

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