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):
The all plant material used in the present study were obtained from wild plants which were collected from the Southeast of Tunisia. This area is arid to semi-arid with a typical Mediterranean climate, characterized by irregular rainfall events and a harsh dry summer period. Annual precipitation is around 186 mm and annual mean temperature is 19.4°C with a minimum temperature 3.9°C in January and 35.9°C maximum in August correspond to climatic data where original population of the plants occurred. These plants species, belonging to five different plant families from South East of Tunisia (arid land), were collected (200–250 g per species) during the vegetative phase. The harvested plants were authenticated by botanist Dr. Mohammed Neffati according to the “Flora of Tunisia” catalogue (10). Voucher specimens were deposited at the herbarium of the IRA. The scientific names and the parts used in folk medicine are detailed (
Medicinal properties of the plant species investigated
Family and scientific | Medicinal | Use(s) | Major active compounds | Reference(s) |
---|---|---|---|---|
Arial parts | Indicated for the treatment | Essential oil: limonene (10.5–27.3%), | (11) | |
Leaves, | Appetizers and condiments. | vitamin C, carotenoids, flavonoids, | (12) | |
Shoots | Indicated for the treatment | Alkaloids, flavonol triglycoside, | (14) | |
Leaves | Used for the treatment | Alkaloids and flavonoids (daidzein, | (16) | |
Fruits, | Treatment of digestive | Cyclopeptide alkaloids, (lotusines B, | (18) |
For the aqueous extraction, an amount of 5g of the powdered sample was extracted with 100 mL Milli Q water at 105°C for 15 minutes. After cooling, each extract was centrifuged at 800 x g for 10 minutes and filtered using a 0.45μm nylon membrane (Millipore). The filtrated extracts were finally stored in shady condition until use.
For the alcoholic extraction, the same amount (5 g) of powedered sample was extracted using 100 mL of 70% ethanol at room temperature for 24 hours in a shaking condition. After centrifugation and filtration, as well as for the aqueous extract, the plant extracts were stored at 4°C before assays.
Total phenolic (TPC), flavonoid (TFC) and Total Condensed Tannins Content (TCTC) contents in the extracts were determined as gallic acid and catechin equivalents, respectively, by spectrophotometry, while Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) was used for their qualitative analyses. The analysis was performed by Agilent Technologies 121100 series HPLC system. The separation was carried out using a reverse phase ODSC18 (250mm × 4.6mm ID, 5 μm particle size Hypersil) column in gradient elution as mobile phase using acetonitrile (Solvent A) and (0.2%) water sulphuric acid (Solvent B). Experimental conditions are given in the supplementary material in detail.
Total Antioxidant Capacity (TAC) of plant extracts was determined monitoring the capacity of the extract to reduce Mo (VI) to Mo (V) with the subsequent formation of a green phosphate / Mo (V) complex at acidic pH (22). The ability of plant extracts to scavenging the DPPH radical was measured according to Hanato and colleagues (23). Iron reducing power, defined as the capacity of plant extracts to reduce Fe3+, was assessed by the method of Oyaizu (24). The ferrous ion chelating activity of the extracts was assessed as described by Falleh and colleagues (22). Experimental conditions are given in the supplementary material in detail.
In order to investigate the antiproliferative effect of the examined plant extracts towards the MCF-7. The cell viability and proliferation were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide or MTTassay. Data are presented as percentage of cell proliferation against a control (100% of cell proliferation) using different concentration of the plant extracts. Experimental conditions are given in the supplementary material in detail.
For all the studies, samples were analyzed in triplicates. Data are shown as mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) with Duncan’s test was carried out to test any significant differences between species at
In this study, both aqueous and ethanolic extracts of the five TMPs were compared. Total Polyphenolic Content (TPC), Total Flavonoids Content (TFC) and Total Condensed Tannins Content (TCTC) indicated large differences among all the plant extracts (
Total polyphenolics, flavonoid and condensed tannin content in the five investigated plant extracts. Data are expressed in mg g-1 of dry matter
Extracts | Cymbopogon | Crithmum | Hammada | Retama | Zizyphus | |
---|---|---|---|---|---|---|
Total phenolic | Ethanolic | 63.86±0.01d | 62.47±0.05e | 93.87±0.03c | 112.12±0.11b | 139.08±0.17a |
Aqueous | 128.63±0.12a | 116.13±0.2b | 41.90±0.15d | 97.27±0.03c | 35.17±0.21e | |
Total flavonoid | Ethanolic | 7.09±0.07d | 6.05±0.13e | 70.00±0.10b | 15.93±0.23c | 70.71±0.09a |
Aqueous | 16.00±0.09c | 21.26±0.08b | 14.11±0.20d | 14.50±0.12d | 29.17±0.03a | |
Total condensed | Ethanolic | 22.84±0.09d | 16.62±0.07e | 82.44±0.20b | 28.15±0.16c | 129.17±0.12a |
Aqueous | 59.97±0.16a | 58.31±0.19b | 35.22±0.11d | 40.83±0.08c | 22.22±0.06e |
Data are the mean of three replicates±SD. Different letters represent values that are significantly different between control and treatments at 5% according to the Duncan’s multiple range test.
TPC ranged from 62.5 to 139.1 mg GAE DW-1 for the ethanolic extracts and from 35.2 to 128.6 mg GAE DW-1 for the aqueous ones. TFC ranged from 6.1 to 70.7 mg CE DW-1 for the ethanolic extracts and it resulted lower than 30 mg CE DW-1 for the aqueous ones in all the plants. Finally, TCTC reached 129.2 mg CE DW-1 in alcoholic extracts, while it was limited to 60 mg CE DW-1 in the aqueous ones. Interestingly, among the ethanolic extracts, the one obtained from
Antioxidant activities of fruits and vegetables are measured by using different assays. Some of these methods were applied to quench and scavenge Reactive Oxygen Species, (ROS) which include both free (O2•−, OH•, HO2•, and RO•) and non-radical forms (35). Several analyses such as total antioxidant activity, DPPH and ABTS assays, ROS quenching assay, metal chelating, reductive potential, β-carotene linoleate system and linoleic acid method are commonly used for the determination of antioxidant activities of plant extracts (36,7). In this study, according to the results of the total polyphenols, results showed that antioxidant activity (TAA, antiradical activity, chelating effect and iron reducing power) measured on each plant extract, was dependent from the extraction method (
Antioxidant activity of the aqueous and ethanolic extracts of the five studied plants
Extracts | Cymbopogon | Crithmum | Hammada | Retama | Zizyphus | |
---|---|---|---|---|---|---|
TAA | Ethanolic | 87.91±0.06e | 89.35±0.08d | 236.00±0.19b | 248.89±0.31a | 179.44±0.21c |
Aqueous | 69.02±0.09e | 82.35±0.05b | 80.90±0.11c | 139.00±0.04a | 73.47±0.05d | |
Antiradical activity | Ethanolic | 0.6.00±0.02a | 0.80±0.01b | 0.60±0.08b | 0.80±0.01a | 0.19±0.08c |
Aqueous | 0.30±0.01c | 0.60±0.01a | 0.45±0.08b | - | 0.24±0.07d | |
Iron reducing | Ethanolic | 0.38±0.01c | 0.46±0.02b | 0.10±0.01e | 0.90±0.01a | 0.30±0.02d |
Aqueous | 0.11±0.01d | 0.40±0.01a | 0.33±0.01c | 0.36±0.01b | 0.40±0.11a | |
Chelating effect | Ethanolic | 68.00±0.08b | 84.00±0.09a | 85.11±0.13a | 8.40±0.02c | 0.11±0.01d |
Aqueous | - | - | - | 5.37±0.01a | 0.16±0.01b |
Data are the mean of three replicates±SD. Different letters represent values that are significantly different between control and treatments at 5% according to the Duncan’s multiple range tests.
Regarding the values of IC50, referred to the antiradical activity (
A total of 20 phenolic compounds were identified in the five assessed plants (
Identification and quantification by reversed-phase high performance liquid chromatography of the main phenolic compounds extracted with ethanol 70% from the five examined plants.
Phenolic compounds | Quantity (%) | ||||
---|---|---|---|---|---|
Cymbopogon | Hammada | Retama raetam | Ziziphus lotus | Crithmum | |
Resorcinol | 5.08 | - | - | - | - |
Caffeic acid | 3.18 | - | - | - | - |
2.5-Dihydroxybenzoic acid | 2.01 | - | - | - | - |
Quercetine-3-o-rhamnoside | 32.82 | 3.64 | - | - | - |
Trans-cinnamic acid | 9.72 | - | - | - | - |
Gallic acid | 8,80 | 2.93 | - | 6.56 | - |
Ferulic acid | 9.83 | - | - | - | - |
Vanillic acid | - | 8.02 | - | 37.04 | 10.37 |
Apigenin-8-C-glucoside | - | 31.17 | - | - | - |
Coumaric acid | - | 3.84 | - | - | - |
Rosmarinic acid | - | 1.93 | - | 4.17 | - |
Chlorogenic acid | - | 7.05 | - | - | 5.39 |
Syringic acid | - | - | 0.2 | - | - |
Kaempferol | - | - | 2.58 | - | - |
3.5-dimethoxy-4- hydroxybenzoic acid | - | - | - | 5.16 | - |
Quercetine-3-galactoside | - | - | - | 10.46 | - |
Epigallocatechin | - | - | - | 35.89 | |
- = not detected
In a recent study, Rocha-Amador and colleagues (38) have reported that the glycosylation of quecetine enhances its bioaccessibility and then the biological effect. The major compound in
The anticancer activity of TMP has not been declared in literature and the high antioxidant activities in these species prompted us to investigate their anti-proliferative capacity. For this reason, the cytotoxicity of each extract was assessed against the human breast cancer cell line MCF-7 using the MTT assay. The results showed that after 48 h of treatment with TMP aqueous and ethanolic extracts, cancer cell proliferation was inhibited in a concentration, solvent and plant tested-dependant manner (
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,
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
The high total phenolic content found in
However, in
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
Based on their anti-proliferative activities against breast cancer cell lines, TMP such as