Journal of Health Science 7 (2019) 350-361 doi: 10.17265/2328-7136/2019.06.003

The Influence of Concentration of the Antioxidants in Rosemary Extract on MCF-7 Cells

Ivana Andrikj II. gimnazija Maribor, Maribor 2000, Slovenia Abstract: In my research, I studied the antioxidant effect of extracts of various rosemary samples obtained by Soxlet extraction on MCF-7 epithelial cells. I wanted to discover which of the extracts will have the highest content of total phenols determined in vitro with the Folin-Ciocalte reagent. The results show the highest content of total phenols in the rosemary sample from North Macedonia, so I assumed that it would have the biggest effective activity on MCF-7 cells. While doing the free radical inhibition test, I came to the conclusion that the plant with the highest antioxidant content does not necessarily have the best effect on the cells at different concentrations of the extracts. I suppose that it happens because antioxidants are polar and they need to transport through cell membrane conveyors whose number is limited in the membrane, that is, more antioxidants get assembled on the conveyors and cannot go through the membrane. Key words: Rosmarinus officinalis, anti-cancer, antioxidants, free radicals, MCF-7.

1. Introduction

Rosemary (Rosmarinus officinalis) is an evergreen, bushy plant that thrives in the Mediterranean. This plant is used in everyday life as a food-seasoning or as an ornamental plant. Rosemary contains 1.5%-2.5% essential oil [1]. Rosemary is also well known for its healing effects, for example, it improves concentration, it is highly recommended for mental exhaustion and depression, headache, as well as antimicrobial affection etc. [2].

Antioxidants destroy the reactive free radicals and do not convert them into destructive free radicals, but low-energy, long-lived free radicals that decompose themselves without harming the body. Many studies indicate the connection between the formation of free radicals (ROS, reactive oxygen compounds) and breast cancer. Desaulniers [3] discovered that carnosol (a phenolic diterpene found in rosemary) prevents the growth of tumors of the cancerous breast.

Given the high content of antioxidants in rosemary

Corresponding author: Eneko Jose Madorran Esteiro, asst.

prof. in pharmachology and toxicology, preclinical biomedical researcher.

and the association of antioxidants with anti-cancer properties, rosemary could be an interesting medicinal plant, as it is not difficult to be grown as well as not very demanding. I decided to research the possible anti-cancer properties of rosemary and the influence of the growing place of the plant on the antioxidant content of rosemary. I picked up and used rosemary from three different European countries (Greece, North Macedonia, Slovenia).

I asked myself the following research questions: (1) What is the relation and difference in antioxidant

content in rosemary (Rosmarinus officinalis) according to the thriving place?

Then I was interested in the antioxidative potential of rosemary within the MCF-7 cells [4] because the dangerous free radicals (ROS) are formed in those cells. So I asked another research question:

(2) What is the influence of the antioxidant content in the rosemary extract on the antioxidant potential in cancer cells of MCF-7?

The MCF-7 cancer cells, are producing many free radicals, which is exactly why these cells are very suitable for studying the effect of the antioxidants.

Hypotheses are:

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The Influence of Concentration of the Antioxidants in Rosemary Extract on MCF-7 Cells

358

I added different concentrations of extracts and DMEM colorless to the wells where I was checking the effect of rosemary extracts.

Extract concentrations: 5% of the extract contains: 100 μL of extract and

1.90 mL of DMEM 1% extract contains: 20 μL of extract and 1.98 mL of

DMEM. 0.1% of the extract contains: 5 μL of extract and 2

mL of DMEM. I needed to calculate the DCFH volume, so I did it

the following way: 50 μM DCFH-da x mL 10×10-3 M DCFH-da 10 mL

50 10 1010 10 50 µL DCFH da

I dosed 50 μL of DCFH-da in a centrifuge tube, added 9.95 mL of DMEM and mixed everything.

After 30 min, I added 100 μL of DCFH-da and DMEM solution to each well and waited again for 30 min, then measured the fluorescence of the samples, indicating the antioxidant potential in the cells.

4. Results

4.1 Antioxidant Content of Rosemary Extracts

The concentrations of the standard solutions and the absorbance gave us a proportional equation with which we could calculate the concentration of antioxidants in the extracts, which are equivalent to the corresponding concentration of GAE.

A=3.406 × (GAE) – 0.2486 In the extract obtained after 5 cycles, the GA

concentration is 0.433 g/L. In 120 mL of ethanol extract: m (GAE) = (GAE) ×

V = 0.433 mg/mL × 120 mL = 52 mg. The extract was made from 7.65 g of dried rosemary.

Let us assume that we extract all the polyphenols, then we get: 52 mg (GAE) / 7.65 g rosemary = 6.8 mg/g.

The results clearly show that the more cycles are carried out in a Soxhlet apparatus, the higher the content of antioxidants is in the extract. The highest

concentration of antioxidants is contained in rosemary extract from North Macedonia, probably because the sun is the highest in the area, which is very good for the growth of the rosemary.

4.2 Antioxidant Potential inside the Cells

From these results we can see that in 5% and 1% of the extract from Macedonia, the fluorescence is the highest, which means that it is expected to have the greatest effect on MCF-7 cells or maximum antioxidant potential and consequently less ROS (Fig. 13 Comparison of antioxidant content of rosemary (Rosmarinus oficinalis) from different countries after the fifth Soxhlet extraction cycle).

I measured low fluorescences at 0.1% of the extracts, the differences between them are small, not necessarily due to the influence of the extracts, but also to other substances in the mixture. Table 2 Antioxidant concentration in the rosemary extract from North Macedonia.

Cycle A γ (GAE) g/L

1 0.587 0.245 2 0.709 0.281 3 0.810 0.311 4 1.110 0.399 5 1.225 0.433

Table 3 Antioxidant concentration of the rosemary extract from Greece.

Cycle A c (GAE) mg/g

1 0.337 0.172 2 0.460 0.208 3 0.490 0.217 4 0.570 0.240 5 0.670 0.270

m (GAE) = (GAE) × V = 0.270 mg/mL × 120 mL = 32.4 mg; 32.4 mg (GAE) / 7.65 g rosemary = 4.2 mg/g.

Table 4 Antioxidant concentration of the rosemary extract from Slovenia.

Cycle A c (GAE) mg/g

5 0.660 0.267

m (GAE) = (GAE) × V = 0.267 mg/mL × 120 mL = 32.04 mg; 32.04 mg (GAE) / 7.65 g rosemary = 4.2 mg/g.

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The Influence of Concentration of the Antioxidants in Rosemary Extract on MCF-7 Cells

360

(2) In preparing the ethanol extract of rosemary in the Soxhlet apparatus, 5 cycles will be sufficient.

The hypothesis was correct. The concentration of antioxidants in extracts (GAE)

is expected to increase from the first to the fifth cycle, but there is no longer a significant difference between the fourth and fifth cycles. Further cycles would be uneconomical, especially from an energy point of view.

The third hypothesis was: (3) The extract with the highest content of phenols

and polyphenols has the highest antioxidant potential in MCF-7 cells.

If we compare the results, we see that the hypothesis is correct. The highest antioxidant potential was measured with a 5% extract from Macedonia which also has the highest concentration of phenols and polyphenols (GAE).

At lower concentrations of extracts in cells, however, there is no such clear correlation between the antioxidant potential of extracts and the concentration of phenols and polyphenols in them.

6. Conclusion

The aim of the research was to demonstrate the anti-cancer effect of rosemary extracts. Using spectrophotometry method, I have found that most of the total phenols contain the rosemary extract from Macedonia.

I analyzed the antioxidant potential using the DCFH method (Fig. 14).

The analysis of the results also showed that the plant with the most antioxidants had the best effect on the cells, that is, most ROS react.

The field of study of the medicinal effects of rosemary is still largely unexplored. Nevertheless, many studies provide us with encouraging results that indicate the positive effects of phenolic compounds of rosemary in the medical field.

The research mission opens a new perspective on the already confirmed practice rosemary health benefits

that rosemary certainly helps us and that we should definitely continue exploring these fields.

7. Social Awareness

In preparing my research assignment, I followed the basic principles of social responsibility. With it I wanted to point out the solutions that nature offers us. The common rosemary plant, Rosmarinus officinalis, has many healing effects from improving digestion, reducing stress, increasing memory and concentration. Recently, its anti-cancer properties, its ability to protect against macular degeneration, and the rest (etc.) have also been discovered in this modern world when the incidence rate of cancer is more and more increasing. While being interested in the problem of increasing the number of cancers in the modern world, it seemed important to me to add a bit of knowledge in developing successful treatment methods based on helpful products already present in the plant world.

Acknowledgement

I sincerely thank all the people who helped me do this research. I also thank the doctor who gave me access to a laboratory at the University of Maribor Faculty of Medicine in which we were growing MCF-7 cells and then checked the effect of extracts on them.

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