ANTIPROLIFERATIVE ACTIVITY OF THREE WILD GROWING SPECIES IN TUNISIA: NICOTIANA GLAUCA, ARTEMISIA CAMPESTRIS AND ASTRAGALUS GOMBO

ANTIPROLIFERATIVE ACTIVITY OF THREE WILD GROWING SPECIES IN TUNISIA: NICOTIANA GLAUCA, ARTEMISIA CAMPESTRIS AND ASTRAGALUS GOMBO

Hassen Teyeb ^{* 1}, Wahiba Douki ¹ and José M. Padrón ²

Biochemistry and Toxicology Laboratory ¹, University Hospital of Monastir, University of Monastir, Monastir 5000, Tunisia.

BioLab ², Instituto Universitario de Bio-Orgánica “Antonio González” (IUBO-AG), Centro de Investigaciones Biomédicas de Canarias (CIBICAN), C/ Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain.

ABSTRACT: Tunisian flora contains several wild plants with diverse therapeutic uses. As a contribution to the efforts to select natural sources of antitumor compounds, we investigated in this study the antiproliferative activity of several extracts of the Tunisian species: Astragalus gombo, Nicotiana glauca and Artemisia campestris. Dried aerial part (A. gombo and A. campestris) and leaves (N. glauca) were extracted with ethyl acetate, dichloromethane, and methanol. In the vitro anti-proliferative activity of the extracts was tested against the human solid tumor cell lines: HBL-100, T-47D, and WiDr. Tests were performed using the sulforhodamine B (SRB) assay. All extracts of A. campestris and A. gombo were active against all tested cell lines, with GI₅₀ values between 12 and 93 µg/ml. Dichloromethane extract of A. campestris was the most active extract with GI₅₀ of 12 µg/ml against HBL-100 cell line. This kind of cell seems to be the most sensitive regarding all tested extracts. Our results showed that A. gombo, A. campestris and N. glauca are a promising source of natural antitumor compounds. More detailed studies should be conducted especially for most active extracts.

Nicotiana glauca, Artemisia campestris, Astragalus gombo, Antiproliferative, Extracts

INTRODUCTION: Cancer remains a serious problem for public health. Natural products play a highly significant role in the drug discovery and development process, particularly in the areas of cancer. With the increased resistance to the used anticancer drugs, increasing interest has been shown in research of new compounds from plants. Over 60% of these drugs were shown to be of natural origin ¹.

Several species represent a significant biological potential and have been a source of active compounds for antitumor effects and cancer therapy adjuvant. Among species of Tunisian flora, several were traditionally used for various diseases.

To adapt to different stresses especially in desert zones, plants synthesize several metabolites and are thus a promising source of active molecules. These metabolites confer to these species biological activity responsible for their therapeutic use, like for Artemisia, Nicotiana, and Astragalus genus. Our previous studies about Astragalus gombiformis Pomel showed that this plant has cytotoxic, antibacterial and anticholinesterasic activities ^{2, 3}. Many other investigations suggest that Artemisia campestris could be beneficial for protection against diabetes and its complications, and showed that this plant exhibited antioxidant and anti-bacterial properties ^4-7. Other species of this genus, Artemisia princeps, is a potential anti-endometriotic agent that induces apoptosis of endometrial cells ⁸.

Nicotiana glauca R.A. Graham, also called wild tobacco or tree tobacco, can be a source of benefit molecules such as 7-dehydrocholesterol, vitamin D_3, and other vitamin D₃-related compounds ^{9, 10}. N. glauca was used as medicinal smoke for the treatment of the ear and general skin diseases ¹¹. It has a hepatoprotective effect ¹². This species is toxic for animals and human can be accidentally poisoned ^{13, 14}. Main toxic principles of N. glauca are the two alkaloids nicotine and anabasine, which possess a similar structure. Others alkaloids have been identified in N. glauca such as anatabine, metanicotine and myosmine ^15-17. The anti-oxidant, antimicrobial and antiacetylcholinesterase activities of Tunsisian N. glauca was recently studied by Sellem et al., (2016) ¹⁸.

With many of the studies of the biological activities of plants, they remain dependent to several factors such as the natural environment of growth, the season when the plant was collected, the solvent used for extraction and the experimental conditions. We evaluate in this study the antitumor activity of three Tunisian species, Astragalus gombo, N. glauca, and A. campestris as a contribution to the efforts to select natural sources of anticancer compounds.

MATERIAL AND METHODS:

Plant Collection and Extracts Preparation Samples: Aerial parts of A. gombo (February) have been collected on flowering stage from Djerba island in Southeast of Tunisia. Leaves of N. glauca and total aerial part of A. campestris have been collected on October from the region of Ben Gardane in Southeast of Tunisia. After air-drying in shadow, the samples have been powdered and conserved until use.

Solvents: Methanol and ethyl acetate were both from Lab-Scan. Dichloromethane and dimethyl sulphoxide (DMSO) were respectively from Carlo Erba and Sigma (St Louis, MO).

Extract Preparation: Different extracts were prepared by direct overnight maceration with organic solvents Table 1. Then, solvents were evaporated by rotavapor. All residues were dissolved in DMSO at 100 mg/mL concentrations as the stock solution. Both cisplatin and etoposide were used as positive control.

TABLE 1: PREPARATION OF TESTED EXTRACTS WITH DIFFERENT SOLVENT

Antitumor Activity: All starting materials were commercially available research-grade chemicals and used without further purification. RPMI 1640 medium was purchased from Flow Laboratories (Irvine, UK), fetal calf serum (FCS) was from Gibco (Grand Island, NY), trichloroacetic acid (TCA) and glutamine were from Merck (Darmstadt, Germany), and penicillin G, streptomycin, DMSO and sulforhodamine B (SRB) were from Sigma (St Louis, MO).

Cells, Culture and Plating: The human solid tumor cell lines HBL-100 (breast), T-47D (breast) and WiDr (colon) were used in this study. These cell lines were a kind gift from Prof. Godefridus J. Peters (VU Medical Center, Amsterdam, The Netherlands). Cells were maintained in 25 cm² culture flasks in RPMI 1640 supplemented with 5% FBS and 2 mM L-glutamine at 37 ºC, 5% CO₂, 95% humidified air incubator. Exponentially growing cells were trypsinized and resuspended in antibiotic containing medium (100 unit’s penicillin G and 0.1 mg of streptomycin per ml). Single cell suspensions displaying >97% viability by trypan blue dye exclusion were subsequently counted. After counting, dilutions were made to establish the appropriate cell densities for inoculation onto 96-well microtiter plates. Cells were inoculated in a volume of 100 μl per well at densities of 10 000 (HBL-100), 15 000 (T-47D), and 20 000 (WiDr) cells per well, based on their doubling times.

Chemosensitivity Testing: Chemosensitivity tests were performed using the SRB ¹⁹ assay of the NCI with slight modifications. Briefly, plant extracts were initially dissolved in DMSO at 400 times the desired final maximum test concentration of 250 µg/mL. Control cells were exposed to an equivalent concentration of DMSO (0.25% v/v, negative control). Each agent was tested in triplicate at different dilutions in the range of 2.5-250 µg/mL. The drug treatment was started on day 1 after plating.

Drug incubation times were 48 h, after which cells were precipitated with 25 µL ice-cold 50% (w/v) trichloroacetic acid and fixed for 60 min at 4 ºC. The SRB assay was then performed. The optical density (OD) of each well was measured at 492 nm, using BioTek’s Power Wave XS Absorbance Microplate Reader. Values were corrected for background OD from wells only containing a medium. The percentage of growth (PG) was calculated concerning untreated control cells (C) at each drug concentration level based on the difference in OD at the beginning (T₀) and end of drug exposure (T), according to NCI formulas ²⁰.

Therefore, if T is greater than or equal to T₀, the calculation is 100 ´ [(T–T₀)/(C–T₀)]. If T is lower than T₀ denoting cell killing, the calculation is 100 ´ [(T–T₀) / (T₀)]. The effect is defined as the percentage of growth, where 50% growth inhibition (GI₅₀) represents the concentration at which PG is +50. With these calculations, a PG value of 0 corresponds to the amount of cells present at the beginning of drug exposure, while negative PG values denote net cell kill.

RESULTS AND DISCUSSION: In total, nine extracts from N. glauca, A. campestris, and A. gombo species were assayed for their anti-proliferative activity against three human solid tumor cell lines: HBL-100 (breast), T-47D (breast), and WiDr (colon).The results are summarized in Table 2. All extracts of A. campestris and A. gombo were active against all cell lines. The GI₅₀ values were in the range 12-93 µg/ml. Dichloromethane extract of aerial part of A. Campestris showed GI₅₀ of 12 µg/mL against HBL-100 cells, and it was the most active. In fact, HBL-100 seems to be the most sensitive regarding all tested extracts. N. glauca exhibited activity only against this cell line. For T-47D and WiDr, this species showed GI₅₀ above 250 µg/mL. It is the first time, that N. glauca and A. gombo Tunisian species were tested for anti-proliferative activity against human solid tumor cell lines. However, A. campestris was previously studied for this activity by ²¹.

TABLE 2: IN-VITRO ANTIPROLIFERATIVE ACTIVITY OF PLANT EXTRACTS AGAINST HUMAN SOLID TUMOR CELL LINES EXPRESSED AS GI₅₀ VALUES GIVEN IN µg/mL

In their study, the plant was collected from Beni-Khedache, a mountainous region in the Southeast of Tunisia at around 150 km from our region (Ben Gardane), which is a few km from the sea. Also, there is also a difference between tested extracts and used cells. These authors showed that A. campestris possess antitumor activity, with a positive correlation between this activity and the antioxidant activity. Using HT-29 human adenocarcinoma cell line, GI₅₀values were more than 100 and 1920 µg/mL for ethanol-water and infusion extracts of aerial part, respectively. Because WiDr is considered a derivative of HT-29 ²², our results indicate that dichloromethane seems better solvent to extract the anti-proliferative compounds present in the aerial parts of A. campestris. The essential oil was found the most active with GI₅₀ of 46.8 µg/mL ²¹. Our results, in agreement with previous data, show the antiproliferative potential of A. campestris ^{4, 5}. Ethanolic leaf extracts of Artemisia campestris var. glutinosa and A. molinieri, showed activity against mosquito Culex pipiens larvae, with low calculated lethal concentrations 50%, after 48 h of exposure ²³.

Concerning N. glauca, few studies investigating the biological activity of this species were reported. Another Tunisian group studied the antifungal activity of N. glauca leaf and flower collected from the region of Monastir. Their results revealed an important antifungal effect of aqueous extracts at all concentrations tested (1, 2, 3 and 4%) ²⁴. A recent study investigating N. glauca from Egyptian flora showed that cytotoxicity of methanol extract of flowers and leaves was 25.5, 34.2, 24.3, and 1.6 % against MCF-7, HCT-116, HepG 2, and A-549 cell lines respectively ²⁵.

In our knowledge, it is the second study about the biological properties of A. gombo after our previous work concerning essential oil of this plant ²⁶. This genus is widely studied. Astragalus-based herbs were known in Chinese Pharmacopoeia. This study focuses on another Astragalus species with antitumor effect. Recent reports were oriented to investigate the mechanism of action of Astragalus metabolites. Kim et al., (2013) ⁸ showed that Astragalus polysaccharides are a potential anti-endometriotic agent. It induces apoptosis of endometrial cells by the modulation of the p38 and NF-kB pathways. Astragalus polysaccharides also improved experimental TNBS-induced colitis in rats through regulation of TNF-a, IL-1b and NFATc4 expression ²⁷.

CONCLUSION: In the frame of selection of interesting wild Tunisian species, this study was focused on the evaluation of the antitumor potential of three wild growing plants. Extracts of A. campestris and A. gombo were active against all tested cell lines, with GI₅₀ under 100 µg/mL. Dichloromethane extract of A. campestris was the most active extract, with GI₅₀ equal to 12 µg/mL against HBL-100 cell line. Our results show that A. gombo, A. campestris and less N. glauca are a promising sources of anticancer natural compounds. Further studies should be performed to investigate the most active extracts.

ACKNOWLEDGEMENT: Co-financed by the European Regional Development Fund (FEDER), the EU Research Potential (FP7-REGPOT-2012-CT2012-31637-IMBRAIN), and the Spanish Instituto de Salud Carlos III (PI11/00840).

CONFLICT OF INTEREST: Nil

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    How to cite this article:

    Teyeb H, Douki W and Padrón JM: Antiproliferative activity of three wild growing species in Tunisia: Nicotiana glauca, Artemisia campestris and Astragalus gombo. Int J Pharmacognosy 2017; 4(9): 294-98. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.4(9).294-98.

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