Phenolic composition of some Tunisian medicinal plants associated with anti-proliferative effect on human breast cancer MCF-7 cells

The incidence of cancer in Mediterranean countries is generally lower than in the rest of European countries and in the United States (1); the major reason of this, in addition to genetic factors, could be attributed to their eating habits. The traditional Mediterranean diet is characterized by a high consumption of plant-based foods, rich in compounds with nutraceutical properties (2). With this respect, it is well known that plants (fruits, vegetables, medicinal herbs, etc.) usually contain a important variety of antiradical compounds, such as phenolic (e.g. phenolic acids, flavonoids, coumarins, lignans, stilbenes, tannins), nitrogen (alkaloids, amines, betalains) and terpenoids (including carotenoids) molecules which are recognized for their efficient antioxidant activities (3). Moreover, it has been proposed that there is an inverse relationship between dietary intake of antioxidants-rich food and the incidence of many human diseases, such cancer (4).

Accordingly, the National Cancer Institute (USA) began to screen plant extracts with antitumor potency (5) and many researchers pay attention to ethnomedicinal plants as rich sources of novel anticancer drugs. With this respect, the ethnobotanical survey and documentation of traditional knowledge, mainly on the medicinal uses of plants, has provided important clues for the discovery of new drugs (6).

In Tunisia, thanks to the diversity of its climate, the traditional pharmacopoeia comprises a large arsenal of medicinal plants (7). Although several of these species are nearly facing extinction (8), only a small proportion of them have been scientifically studied. It is also important to highlight that Tunisian medicinal and aromatic plant; especially those growing in arid and desertic lands are equipped with powerful antioxidant systems and were labeled as valuables to treat several human diseases (7,6,9). For that reason, five traditional Tunisian medicinal plants (TMP): Cymbopogon schoenanthus (L.) Spreng, Crithmum maritimum (L.) Spreng, Hammada scoparia (Pomel) Iljin, Retama raetam (Forssk.) and Zizyphus lotus (L.) Desf., were selected to be tested in this study (Fig. 1). The main aims of this paper were: 1- to determine the bioactive compounds (especially phenolic ones) present in aqueous and ethanolic extracts of the five selected TMP; 2- to assay their antioxidants capacities (AC); 3- to explore their antiproliferative on a breast cancer cell lines (MCF-7) using the MTT tetrazolium salt colorimetric assay.

Figure 1

Since most of the Tunisian medicinal plants are generally used for consumption of herbal tea or soup or medicinal bolus to treat/prevent different pathologies, the aqueous extraction performed in this study resulted necessary. In order to test the efficacy of the extraction using only water, a comparison with the alcoholic solvent extraction, largely used in chemical industry, was carried on. Aqueous ethanol mixtures are generally used for the extraction of several phenolic compounds from aromatic and medicinal plants. This is mainly due to the diverse range of polyphenols that the aqueous ethanol mixtures can successfully dissolve. Besides, ethanolic mixtures, up to certain percentage, have an high degree of acceptability for human consumption models (26).

Several published studies showed that different solvents, due to their polarity, have significantly various extraction capacities for phenolic molecules in plants (27, 28). Plant materials may contain different percentages of phenolic acids, phenylpropanoids, anthocyanins, and tannins, among others, which can interact with other plant molecules such as carbohydrates and proteins that may lead to the formation of complexes, often insoluble. Furthermore, the phenolics solubility is affected by the polarity of solvent(s) used. For these reasons, it is very difficult to set up an extraction method suitable for all phenolics present in plant kingdom (29). Thus, there is no uniform or completely satisfactory procedure for the extraction of all phenolics or, at least, a specific class of phenolic compounds in plant materials (27,28). For this reason, it is important to properly analyse the composition of phenolic compounds in plants so that their health-promoting properties can be adequately studied.

In addition, these wild TMP significantly showed to contain more phenolics compared to many other (not only Tunisian) medicinal and common dietary plants (30, 31). Among TMP extracts, Z. lotus, a spontaneous edible plant commonly used in the Mediterranean regions for its beneficial therapeutic impact in nutrition and health care, represents an important source of phenolics such as flavonoids and tannins (9). Phenolic compounds are ubiquitous in plants and have been associated with the healthcare derived from consuming high levels of fruits and vegetable (30). In particular, flavonoids are associated with a broad spectrum of health benefits and are an essential component in a variety nutraceutical, medicinal, pharmaceutical and cosmetic applications. This is due to their antioxidative, anti-inflammatory, anti-mutagenic and anti-carcinogenic activity associated with their ability to modulate key cellular enzyme functions (32).

The concentration and nature of phenolic compounds in plants are very sensitive to the environmental and climatic conditions (33) and a geographical localization may contribute to increase genetic differentiation between different taxonomic entities (34). The studied saharian plants are very locally distributed in in the ecoregions of Tunisia. Their significant phenolics contents could be an adaptive trait to face the oxidative stress caused by the severe climatic conditions (warm temperatures, dryness, high solar exposure, short growing season) of their habitat. In plants, polyphenol synthesis and accumulation are generally stimulated in response to biotic/abiotic stresses, leading one to think that secondary metabolites may play a role in the adaptation of extremophile species to this constraint (30). Flavonoids, acting as UV filters, protect plants from different biotic and abiotic stresses; moreover they include signal molecules, allopathic compounds, phytoalexins, detoxifying agents and antimicrobial defensive compounds.

Flavonoids have roles against frost hardiness, drought resistance and may play a functional role in plant heat acclimatisation and freezing tolerance (32). Moreover, data showed that the H. scoparia ethanol extract and the C. schoenanthus aqueous extracts depicted an important reducing power compared to other halophytes plants with medicinal properties such as the previously cited Tamarix gallica (205 μg mL^-1) and Salsola kali (L.) (165 μg mL^-1) (28).The significant chelating effect showed by Z. lotus ethanol extract (0.11 mg mL^-1) can be attributed to the high content in tannins. Tannins have several controversial effects on biological systems since they are biological antioxidants and potential metal ion chelators, but they are also responsible of the protein precipitation (30).

The high total phenolic content found in H. scoparia, R. raetam and Z. lotus ethanolic extracts and in C. schoenanthus aqueous extracts seems, therefore, to be related to their important antioxidant capacities. In accordance with this statement, previous published studies confirmed the direct relationship between antioxidant activity and total phenolic content in selected herbs (30,3). In the human body, these phytochemicals are thought to provide health benefits by different mechanisms, including: (1) free-radical scavenging; (2) protection and regeneration of other dietary antioxidants (i.e. vitamin E); and (3) chelating of pro-oxidant metal ions (29). Flavonoids are oxidised by radicals, resulting in a more stable, less-reactive radical. In other words, flavonoids stabilise the reactive oxygen species by reacting with the reactive compound of the radical (32).

However, in C. shoenanthus and C. maritimum, no relationship between their antioxidant activities and total phenolics was found. It is known that different structures or levels of glycosylation of phenolic compounds determinate different functional properties (29); hence, the total phenolics content in plants is not very informative indicator of their possible antioxidant capacities (31). These results agreed with those ones observed by Shahidi and Naczk (37) who reported that differences in antioxidant activities of plants extracts could be attibuted to different chemical structures of the phenolic compounds as well as on their derivatives. In general, phenolics free radical scavenging and antioxidant potency depends on the number and position of hydrogen-donating hydroxyl groups on the aromatic ring of the phenolic molecules, and is too affected by other factors, such as glycosylation level, or the presence of H-donating groups (-NH, -SH), to name a few.

Oxidative stress is amongst the principal causes of cancer-related death, and antioxidants such as flavonoids have emerged as potential chemoprevention candidates for cancer treatment (44). With this respect, the highest accumulation of phenolic compounds in H. scoparia, R. raetam, Z. lotus and C. schoenanthus extracts (ethanolic or aqueous) and their important antioxidant activity confer to these species a strong antiproliferative activity. These results are in good agreement with previously studies confirming that phenolic compounds possess antioxidant and anticarcinogenic activities, particularly by inhibiting cancer cell proliferation (32,44). For instance, Zhang and colalborators (45) reported that the polyphenols isolated in strawberry (Fragaria sp.) including kaempferol, quercetin, anthocyanins, coumaric and ellagic acids, were demonstrated to inhibit the growth of human breast (MCF-7), colon (HT-29), and prostate (DU-145) tumour cell lines. A case-control study involving women from Klang Valley and Selangor, Malaysia, shown that good antioxidant consumption can reduce oxidative stress and consequently prevent breast cancer risk. On the other hand, it should be noticed that some cancer deseases develop in a highly oxidative state due to the decrease in enzymatic antioxidant defence. Ultimately, phenolic compounds, as potent antioxidant agents, are proven to prevent cancer disease (44).

Based on their anti-proliferative activities against breast cancer cell lines, TMP such as H. scoparia, R. raetam, Z. lotus and C. schoenanthus have constituted a rich source of natural secondary metabolites, useful in preventing the onset of different cancer deseases. In conclusion, the present work showed variations in the antioxidant and anti-proliferative capacities and phenolic composition among the five investigated Tunisian ethnomedicinal plants.These plants demonstrated important biological activities and contained much more phenolic compounds than many common vegetables and fruits. These findings may confirm that traditional TMP might represent an interesting natural source of powerful antioxidants and chemo-preventive agents. The use of these TMP can emphasizes disease prevention and whole-body homeostasis instead of disease-oriented therapy.