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In vitro antiproliferative activity of selected medicinal plants from the North-West of Morocco on several cancer cell lines
Accepted Manuscript Title: In vitro antiproliferative activity of selected medicinal plants from the North-West of Morocco on several cancer cell lines Authors: Abdelhakim Bouyahya, Youssef Bakri, Abdeslam Et-Touys, In`es Christelle Chadon Assemian, Jamal Abrini, Nadia Dakka PII: DOI: Reference:
S1876-3820(18)30001-5 https://doi.org/10.1016/j.eujim.2018.01.001 EUJIM 757
To appear in: Received date: Revised date: Accepted date:
9-10-2017 31-12-2017 1-1-2018
Please cite this article as: Bouyahya Abdelhakim, Bakri Youssef, Et-Touys Abdeslam, Assemian In`es Christelle Chadon, Abrini Jamal, Dakka Nadia.In vitro antiproliferative activity of selected medicinal plants from the North-West of Morocco on several cancer cell lines.European Journal of Integrative Medicine https://doi.org/10.1016/j.eujim.2018.01.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro antiproliferative activity of selected medicinal plants from the North-West of Morocco on several cancer cell lines
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Abdelhakim Bouyahyaa,b,*, Youssef Bakria, Abdeslam Et-Touysa, Inès Christelle
aLaboratory
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Chadon Assemiana, Jamal Abrinib, Nadia Dakkaa
of Human Pathologies Biology, Department of Biology, Faculty of Sciences,
and health Laboratory, Department of Biology, Faculty of Science, Abdelmalek,
author: Abdelhakim Bouyahya
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*Corresponding
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Essaadi University, Tetouan, Morocco
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bBiology
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and Genomic Center of Human Pathologies, Mohammed V University, Rabat, Morocco
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E-mail: [email protected] ; Phone: +212601350878 Postal address : Bouyahya Abdelhakim, Laboratory of Humain Pathologies Biology, Faculty of Sciences of Rabat, University Mohammed V of Rabat 4, Av. Ibn battouta
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BP1014 Rabat-Morocco.
Abstract Introduction: Ethnomedicinal and ecological approaches have been important in drug discovery, especially for medicinal plants. The North-West of Morocco (Ouezzane) has a Mediterranean climate and is characterized by a good ecological diversity. Traditionally, several medicinal plants that have been used to treat various diseases can be found in this region. The pharmacological valorization of these species could provide
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opportunities for different diseases including cancer. The aim of this study was to screen for anticancer and antioxidative properties of selected medicinal plants from this area of North-West of Morocco.
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Methods: Organic extracts of five selected medicinal plants (Origanum compactum
Benth., Cistus crispus L., Centaurium erytherea Rafin., Myrtus communis L. and Arbutus unedo L.) were tested for their anticancer and antioxidant properties. The anticancer
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activity was tested on three cancerous cell lines L20B, RD and Vero using MTT assay and
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the pharmacological selectivity index (PSI) was calculated using PBMC as the normal cell
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line. The antioxidant activity was evaluated using FRAP and ABTS assays. Results: All plant extracts showed important anticancer activities with some variability.
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The n-hexane extract of C. crispus exhibited remarkable cytotoxic and specific effects on RD (IC50=04.75 µg/mL; PSI=36.48) and L20B cell lines (IC50=07.16 µg/mL; PSI=24.20).
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Moreover, O. compactum n-hexane extract showed a selective antiproliferative activity on Vero cell line (IC50=13.72 µg/mL; PSI=11.46). Regarding the antioxidant activity, M.
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communis methanol extract showed an important antioxidant capacity measured by ABTS and ferric-reducing power assays with IC50 values of 16.59±0.12 µg/mL and
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57.83±0.76 µg/mL, respectively. Conclusions: The findings did not reveal any correlation between antioxidant and anticancer effects thus showing specific targeted action against cancer cell lines. Based on the obtained results, these species could be considered as potential sources of
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anticancer and antioxidant compounds. However, further studies are necessary for the chemical characterization of the bioactive compounds and more investigation on their pharmacological properties is needed. Key words: Cancer; oxidative stress; medicinal plants; anticancer activity; antioxidant activity.
1. Introduction Cancer is a complex disease characterized by various etiologies and multiple stages induced by the deregulation in genetic and epigenetic programs via several factors such as microbial infections, chemical carcinogenesis and hormonal perturbation [1, 2, 3]. When cells become cancerous, they continue to divide eternally without any response of signaling apoptosis or senescence. The treatment is actually based on chemotherapy and
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radiotherapy which target cells with rapid division and therefore cause several side effects on normal cells which are characterized also by rapid division [4]. Today, new approaches targeting tumor cells without affecting normal cells are needed. Targeted
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cancer therapies based molecules from diverse sources that may target specific signaling molecular pathways in tumor cells without affecting normal cells.
On the other hand, oxidative stress caused by the excessive rate of reactive oxygen
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species such as superoxide anion (O2), nitric oxide (NO), hydroxy radical (OH) and
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peroxy radical (ROO) are involving in the pathogenesis of several pathologies including cancer [5]. Indeed, these reactives induce DNA mutations and in the case of deficient
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DNA repairs or DNA damage, it becomes a prognostic or etiological role in cancer [6, 7].
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Targeting oxidative stress by antioxidants may also reduce the risk of cancer genesis. in drug discovery.
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Therefore, the researches of antioxidants from natural sources play an interesting role
Medicinal plants have shown several biological benefits against several diseases such as
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microbial infections, urolithiasis, cancer and oxidative stress-related diseases [8, 9, 10, 11, 12, 13, 14, 15, 16]. Pharmacological properties such as cancer chemopreventive and
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cytotoxic effects of medicinal plants are attributed to various phytochemical compounds such as flavonoids, alkaloids and terpenes present in its metabolites [17]. Polyphenol and flavonoid components are amongst the most widely occurring phytochemicals that have antioxidant properties [13]. Moreover, these compounds may scavenge the ROS by
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their antioxidant capacity and thus prevent the carcinogenesis [14]. In addition, several studies have shown their anticarcinogenic or anticancer activities by their capacity to induce apoptosis in numerous cancer cell lines due to their pro-oxidant effect [18, 19, 20]. The screening of new drugs from medicinal plants leads to the discovery of various bioactive molecules that possess antioxidant and anticancer properties. These
discoveries were usually carried out using three philosophical approaches mainly used for drugs discovery from nature: the geographical approach, the ethnomedicine approach and the randomized approach [21, 22, 23, 24]. The province of Ouezzane (North-West of Morocco) contains a wide variety of vegetation including medicinal plants. This richness of vegetation is due to its ecological variability and its Mediterranean climate. Moreover, we have underlined in a recent study the indigenous knowledge of medicinal plants in this area [25]. We have also shown the importance of
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this ethnomedicine approach to the success of pharmacological properties of selected
medicinal plants [26, 27, 28, 29]. Some of these species showed interesting anticancer
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properties. For example, Origanum compactum extracts inhibited the breast cancer line
MCF7 [10]. The ethyl acetate extract from O. compactum proved cytotoxic effects against two human tumor lines (A549 lung tumor cell line and SMMC-7721 hepatic tumor cell line) [10]. Therefore, based on ecological and ethnomedicinal approaches, the aim of
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this study was the screening of medicinal plants from the province of Ouezzane with
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anticancer and antioxidative properties, to validate the ecological and ethnomedicinal
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approaches in anticancer drugs discovery. The antiproliferative activity was evaluated on two Embryonal Rhabdomyosarcoma cancerous (RD human and L20B rat) and
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Monkey kidney cancerous cell lines (Vero). These cell lines are usually used in our
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laboratory for the preliminary screening of antiproliferative activities. 2. Materials and methods
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2.1. Collection of plant material and preparation of organic extracts Medicinal plants were collected from the province of Ouezzane (North-West of Morocco:
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34° 47’ 50” N and 5° 34’ 56” W). The taxonomic identification was carried out by Pr. Ennabili Abdessalam (PAMSN Laboratory, National Institute of Medicinal and Aromatic Plants, Sidi Mohamed Ben Abdellah University, Fes-Morocco) and the confirmation of some species was later made by means of the literature as described in our previous
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study [25]. Table 1 summarizes the scientific names, vernacular names, collected parts of the plant, medicinal use by the population of Ouezzane and other pharmacological effects related to these species. The collected parts were dried in dark at room temperature and were then ground to obtain the powder. This later has been extracted by maceration using n-hexane, ethanol and methanol. After 72h of maceration, the plant
extracts were filtered and the solvent was eliminated from the filtrate using a rotary evaporator (Heidolph Collegiate, LV28798826, New Jersey, USA). 2.2. Total phenolic and flavonoid contents The total phenolic contents (TPC) and total flavonoid contents were determined using our previously reported method [31, 32, 33, 34]. The TPC was estimated by the Folin Ciocalteu (AOCS, 1990) assay using gallic acid as standard. The TFC was estimated
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by the aluminum chloride (AlCl3) colorimetric assay using quercetin as standard. The results of TPC are expressed as equivalent gallic acid /g of extract and the TFC are
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expressed as equivalent quercetin / g of extract. 2.3. Antiproliferative activity 2.3.1. Cancer cell lines and culture medium
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Tumor cell lines tested in this study were: RD: Human Embryonal Rhabdomyosarcoma cancerous cell lines (ATCC N°CCL-136), L20B: Rat Embryonal Rhabdomyosarcoma
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cancerous cell lines (3T6Swiss albino, ATCC CCL96) and Vero: Monkey kidney cancerous
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cell lines (ATCC N°CCL-81). These three species were obtained from the National
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Institute of Health, Rabat-Morocco. These species were cultivated as described by Aneb et al. [20]. Briefly, cells were grown as monolayers in Minimum Essential Medium
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(MEM) supplemented with 10% of heat-inactivated fetal calf serum and 1% of PenicillinStreptomycin mixture. PBMC (Peripheral Blood Mononuclear Cell) isolated and purified from human blood were used as positive control. Immediately after collection, blood
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samples were diluted with an equal volume of minimal essential medium RPMI and layered onto Ficoll (Pancoll, Biotech) for gradient separation of cells. The buffy coat
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layer was collected after centrifugation at 300 g for 30 min and then washed twice with RPMI. These cells were cultivated in RPMI supplemented with 10% of heat-inactivated fetal calf serum and 1% of Penicillin-Streptomycin mixture. Cultures were maintained at
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37°C in 5% CO2 and 100% of relative humidity atmosphere. 2.3.2. Cytotoxic activity of extracts Before evaluating the anticancer activity, the cellular density of each species was calculated using light microscopy. When cellular density reached a sub-confluence threshold, cells were detached by trypsine-EDTA, washed twice with phosphate buffered saline (PBS) and centrifuged at 150rpm for 15 min (4°C). Cells were then
resuspended in a fresh culture medium (MEM). The enumeration of cells was made by Malassez cell count using the trypan blue as exclusion coloring. Briefly, 100 μL of medium containing 3-4 x 106 cells/mL were placed in each well and cultured at 37°C in 5% CO2/ humidified air for 24 h. After 24h of incubation, cells were treated with crude extracts. Briefly, 100 μL of various concentrations of extracts (from 3.5 to 250 µg/mL; dissolved in 1% DMSO) were added to each well. Each concentration was tested in triplicate. DMSO was used at a final concentration never exceeding 1% which is not toxic
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to cells [35, 37]. Microplates (96 well plates) were then incubated for 48 h at 37°C in air condition of 5% CO2. Sterile PBS and 1% DMSO (vehicle) were used as negative controls.
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After 48h of cells incubation with extracts, the MTT (3-(4,5-diméthylthiazol-2,5diphenyl tetrazolium bromide) (Sigma) assay was used in order to evaluate the cell cytotoxicity. The test based on the biotransformation of the MTT into formazen crystals by mitochondrial deshydrogenases of living cells. The absorbance of the formazen will
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then be quantified by spectrophotometry at 550 nm. Optical density values (OD) are
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directly correlated with the metabolized intracellular MTT concentrations and are
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therefore proportional to the number of living cells. Briefly, 20 µL of MTT (5 mg/mL) were added to each micro-well and incubated for 3h at 37°C in 5% CO2. Tetrazoluim
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salts are cleaved to formazen and the reaction was stopped by addition of 100 µL of 50% (v/v) isopropanol-10% (w/v) sodium dodecyl sulfate (SDS) mixture to each well in
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order to dissolve insoluble formazen formed after tetrazolium dye reduction. After 30 minutes of incubation at room temperature, the absorbance was measured at 550 nm
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using an ELISA plate reader. Cell viability was evaluated by determination of the percentage of cytotoxicity using the followed formula. The concentrations that inhibit
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the half of the cell population (IC50) were obtained by modeling the % of cytotoxicity versus the concentration of extracts. Cytotoxicity (%) = 100 × (Absorbance of untreated cells-Absorbance of treated cells) /
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Absorbance of untreated cells. 2.3.3. Determination of the pharmacological selectivity index The pharmacological selectivity index (PSI) is an interesting parameter that definites the balance between pharmacological effect of compounds and their toxicity. It corresponds to the highest active concentration against cancer cells without toxicity against normal cells. It was expressed as the ratio IC50 normal cells (PBMC) / IC50 cancer cell lines [37].
2.4. Antioxidant activity 2.4.1. ABTS radical scavenging activity ABTS
(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid)
radical-scavenging
activity of the extract was determined according to the method described by El Jemli et al. [38] with some modifications. The ABTS radical cation was produced by the reaction between 5 mL of ABTS stock solution and 5 mL of 2.45 mM potassium persulfate
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(K2S2O8) solution, stored in the dark at room temperature for 16h. Before use, this
solution was diluted with water to get an absorbance of 0.700±0.015 at 734 nm and equilibrated at 30°C. The plant extract at various concentrations were diluted with
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dimethyl sulfoxide (DMSO) to get sample solution. 5 mL of sample solution was
homogenized with 195 mL of ABTS solution, the mixture was incubated at room temperature for 6 min and its absorbance was recorded at 734 nm. Blanks were run in
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each assay. ABTS scavenging ability was expressed as IC50 (µg/mL) and the inhibition
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percentage calculated using the following formula: ABTS scavenging activity (%) = (A0A1)/A0 × 100. Where: A0 is the absorbance of the control, and A1 is the absorbance of the
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4.2.2. Reducing power assay
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sample.
The ferric-reducing antioxidant power of plant extracts was investigated using the
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potassium ferricyanide-ferric chloride method as described by Bey-Ould Si Said et al. [39] and El Jamli et al. [38]. Briefly, one mL of the extract at different dilutions in pure
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methanol (10, 20, 30, 40 and 50 mg/mL) was mixed with phosphate buffer (1 mL, 0.2 M‘w/v’, pH 6.6) and potassium ferri-cyanide [K3Fe (CN)6] (1 mL, 1%‘w/v’). The
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obtained mixture was incubated at 50°C for 20 min to reduce ferricyanide into ferricyanide. A volume of 2.5 mL of trichloroacetic acid (10%) was added to the solution and centrifuged at 1000 rpm for 10 min. The supernatant was gathered and mixed with distilled water (1.5 mL) and FeCl3 (150 µL, 0.1%‘w/v’), and the absorbance was
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recorded at 700 nm. The sample concentration providing 0.5 of absorbance (IC50) was calculated by plotting absorbance against the corresponding sample concentration. Ascorbic acid and Trolox were used as standard compounds. The test was carried out in triplicate and IC50 values were reported as means ± SD. 2. 4. Data analysis
The statistical analysis was performed by a one-way ANOVA analysis of variance. The difference is considered as significant for p ≤ 0.05. 3. Results 3.1. Phenolic and flavonoid contents The TPC and TFC of extracts are summarized in Table 2. All plant extracts possess important amounts of phenolic and flavonoid contents. However, we have noted
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variability between these amounts that depend on the plant extracts and the used solvents. Extracts from A. unedo, O. compactum and M. communis revealed more
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significant contents in TPC and TFC than extracts of C. crispus and C. erythraea. On the other hand, methanol extracts possess higher concentrations in TPC and TFC than ethanol and n-hexane extracts.
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3.2. Anticancer activity
The evaluation of anticancer activity of five Moroccan medicinal plant extracts was
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carried out on three tumor cell lines, namely RD, L20B and Vero. Cancerous cell lines
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were exposed to decreasing concentrations of extracts ranging from 3.5 to 250 μg/mL.
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The MTT assay indicated that extracts revealed different cytotoxic activities towards the tested cancerous cell lines. To compare antiproliferative effects of extracts, we have
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expressed the results as the concentration that proved the half inhibition in cell viability (IC50) compared to control (PBMC). The obtained results are listed in table 3. As summarised, plant extracts showed important cytotoxic activities that were varied
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between plant extracts and between the types of extract in the same plant. Amongst these tested plant extracts, the n-hexane extract of C. crispus and A. unedo showed
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significant growth inhibitory effects in both RD and L20B. Their IC50 values were 12.75±0.82 and 27.83±1.20 μg/mL, respectively on RD cells. For L20B cell line, the IC50 values of the same extracts were 17.16±1.06 and 25.32±1.26 μg/mL, respectively.
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Moreover, O. compactum and A. unedo n-hexane extracts revealed important cytotoxic effects against Vero cell line with IC50 values of 13.72±0.84 and 18.37±1.23 μg/mL, respectively. The cytotoxic activity of extracts against normal cells (PBMC) showed significant toxicity of some extracts such as A. unedo n-hexane extract (IC50=19.36±1.03 μg/mL).
Their pharmacological selectivity indexes were PSI=0.69, PSI=0.76 and
PSI=1.05 on RD, L20B and Vero cell lines, respectively (Table 4). Regarding C. crispus and O. compactum n-hexane extracts, they have shown low cytotoxicity against normal
cells with IC50 values of 173.28±2.13 and 157.28±1.20 μg/mL respectively. Consequently, the C. crispus n-hexane extract had good pharmacological selectivity indexes against RD (PSI=36.48) and L20B (PSI=24.20) (Table 4). Furthermore, O. compactum n-hexane extract showed a high selectivity index toward Vero cells (PSI=11.46). 3.3. Antioxidant activity
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The antioxidant activity of selected medicinal plants from the North-West of Morocco
was estimated using two complementary in vitro antioxidant tests: the FRAP assay that estimates the ferric-reducing capacity of the extracts and the TEAC assay evaluating the
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H donating or radical-scavenging ability of extracts using the ABTS radical cation. The concentrations providing 50% inhibitions (IC50) are listed in table 5. As summarized in table 5, all extracts showed the ability to scavenge ABTS radical cation and ferric-
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reducing power. However, the antioxidant capacity varies depending on the plant, the
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type of extracts and the used method. In ABTS test, the n-hexane extract of C. erytherea showed important antioxidant capacity (32.07±0.53 µg/mL), compared with reference
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antioxidants Trolox and ascorbic acid (p < 0.05), which showed IC50 values of
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37.82±2.75 and 19.62±0.87 µg/mL respectively. The methanol and n-hexane extracts of M. communis and the methanolic extract of C. erytherea showed also important
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antioxidant capacity to scavenge ABTS radical, but their IC50 values were significantly different (p ˃ 0.05) with positive controls (Table 5). Regarding the FRAP assay, a high ferric reductive capacity was obtained with the methanol extract of M. communis
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(16.59±0.12 µg/mL) and n-hexane extract of C. erytherea (17.43±0.07 µg/mL). Their IC50 values were significantly important (p ˃ 0.05) than antioxidant standards (Trolox and
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ascorbic acid). 4. Discussion
Medicinal plants synthesize several bio-organic molecules as secondary metabolites.
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These compounds have various chemical structures such as terpenoids, simple phenols, phenolic acids, flavonoids and alkaloids. Because of their physiological functions in plants, these chemical classes possess attention for several biological properties such as antibacterial, antioxidant, anticancer and anti-inflammatory activities [38, 40, 41, 42]. In recent years, phenolic compounds such as polyphenols and flavonoids extracted from medicinal plants have demonstrated interesting antioxidant and antiproliferative
effects. In this study, five medicinal plants from the North-West of Morocco have been selected based on ethnobotanical approach [25]. The collected plants have subjected to the extraction by maceration using three organic solvents (methanol, ethanol and nhexane). It has shown that these species possessed important charges in polyphenol and flavonoid contents (Table 2). These results are interesting compared with several studies regarding the total phenol and flavonoid contents in medicinal plant extracts [8,
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15]. This may explain the widespread traditional uses of these species. On the other hand, the variability of chemical compounds of medicinal plants depends
on various factors such as the geographical location, the climate, the method of
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extraction, the organ extracted and the phenological stage of the studied plant [25, 43, 44, 45]. These factors could influence and regulate the synthesis of secondary metabolites in medicinal plants. Indeed, it has been shown that the environmental
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factors and developmental stages may induce the modification in genes expression thus inducing the variation in enzymes responsible for secondary metabolite metabolism
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[46].
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Cancer is a complex disease with various etiologies and multiple stages. Actually, the
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treatment is essentially based on chemotherapy and radiotherapy. These treatments target cells with rapid division and therefore cause several side effects on normal cells
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which are also characterized by rapid division [4]. The screening of bioactive molecules from diverse sources that may target specific signaling molecular pathways in tumor cells without affecting normal cells is important. Polyphenol and flavonoid compounds
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from medicinal plants have been known to target specific pathways against cancer cells
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growth [40, 11].
In this context, phenolic extracts of five selected medicinal plants were screened for their anticancer activity against three cancerous cell lines. Extracts showed significant cytotoxicity against tested cells and some cytotoxic effects on normal cells (PBMC).
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However, pharmacological studies on cancer diseases focused today on the specific selectivity of drugs against cancer cells without side effects on normal cells. The anticancer activity of O. compactum n-hexane extract against Vero cell line and n-hexane extract of C. crispus against L20B and RD cell lines characterized by high selectivity indexes (Table 4). The results have indicated that these extracts have specific targets on the tested cancerous cells. Previous studies have demonstrated the anticancer activity of
O. compactum ethyl acetate extract on human tumor cell line A549 and SMMC-7721 [47]. The cytotoxicity of this extract was due to the induction of apoptosis by the activation of caspase enzymes mediated by the modulation of Bcl-2 family proteins. In another early work, Chaouki et al. [48] have shown cytotoxic effects of O. compactum ethyl acetate and chloroform extracts against human breast cancer cell line MCF-7. However, C. crispus has not yet been tested for its anticancer properties. O. compactum and C. crispus possess important charges of phenolic compounds such as flavonoids. Indeed, flavonoids have
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reported having antiradical effects, with high capacity to scavenge free radical as reactive oxygen species [49, 50]. Moreover, ROS have long been known to be implicated
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in the genesis of several diseases including cancer [51]. Antitumor mechanism of flavonoids based on their antioxidant properties is due to their effects of free radicals scavenging, inhibition of enzymes involved in ROS formation, and the prevention of cellular and extracellular compounds oxidation [50]. However, no significant correlation
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has been indicated between antioxidant and cytotoxic activities of plant extracts. This
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observation indicates thus that n-hexane extract of O. compactum and C. crispus have
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other specific targets on RD and Vero cell lines. Indeed, flavonoids possess several cytotoxic mechanisms on cancer cell lines. They may modulate pathways of cancer
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proliferation, arrest the cell cycle, induce the apoptosis and inhibit the angiogenesis [40,
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49, 52].
The antioxidant activity of phenolic compounds such as polyphenols and flavonoids is largely due to their redox properties which make them act as reducing agents, hydrogen
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donors, singlet oxygen quenchers as well as potential metal chelators [53]. Furthermore, the oxidative stress is largely implicated in several human pathologies such as cancer,
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inflammation and neurodegenerative diseases [7]. Our plant extracts were subjected to the evaluation of their antioxidant activity using ABTS and FRAP assays. The screening of this activity leads to remarkable results (Table 5). The ABTS radical scavenging test
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is a good index to reveal the antioxidant activity of the tested samples [54]. The scavenging of the ABTS radical by our extracts was found to be interesting compared with references. The results showed that C. erytherea extracts presented high capacities to scavenge the ABTS radical. Other studies have revealed an interesting capacity of C. erythrea extracts to scavenge DPPH free radical [55, 56]. However, it has been reported that some compounds which have ABTS radical scavenging activity did not show DPPH scavenging activity. In fact, the solubility of extracts in diverse solvents and the
stereoselectivity of the radicals are two factors that significantly affect the ability of extracts to react with radicals [53]. The difference in the antioxidant activity between plant extracts is certainly due to the variability in chemical functionality compounds. However, the difference between antioxidant assays is due to the mechanisms of which an extract reacts with the used method. Indeed, the FRAP assay estimates the ferricreducing capacity while the ABTS assay evaluates the H donating and radical scavenging capacity. It has been reported that phenolic compounds such as flavonoids neutralize
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free radicals by donating one of their own electrons. In fact, phenolic compounds possess hydroxyl groups responsible for their antioxidant properties. These compounds
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have the ability to scavenge free radicals increased with the number of hydroxyl groups
and catechol moieties in the molecule [57]. In addition, the localization of the hydroxyl groups in molecules could influence their antioxidant properties [58]. This study clearly showed that the antioxidant activity of the North-West Moroccan medicinal plants was
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tightly dependent on the polyphenol and flavonoid contents which themselves depend
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on several factors such as plant species, used solvents, antioxidant assays. This should
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be taken into consideration during the preparation of the extracts.
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Conclusion
The anticancer and antioxidant activities of organic extracts have shown interesting
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results. Phenolic extracts from some species such as C. crispus and O. compactum showed specific anticancer action against cancer cell lines. The antiproliferative activity was not correlated with the antioxidant effect suggesting thus other specific targets of phenolic
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compounds on cancer cell lines. These findings showed that phenolic extracts from Moroccan medicinal plants can be a good source for screening anticancer drugs.
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However, further investigations are necessary for the isolation of bioactive compounds and the study of their mode of action on cancer cell lines.
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Financial support: None
Conflicts of interests The authors declare that there is no conflict of interests regarding the publication of this article.
Acknowledgements We thank the National Center for Scientific and Technical Research (CNRST) from
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Morocco for the support of the doctoral grant of Bouyahya Abdelhakim.
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Table 1: Medicinal plants studied (scientific name, family, part plant collected, medicinal use and pharmacological property) Vernacular
Voucher specimen )
name
M. communis L. (Myrtaceae, RAB13)
Rihan
Medicinal use in Ouezzane
Pharmacological
collected
province
properties
Leaves
The powder in oil poultice is applied on hair and scalp before sleeping for fall protection [25]
Antibacterial and antileishmanial effects [30, 31]
M Senou
Leaves
The decoction is taken orally as natural antidiabetic drugs [25]
Antibacterial and antioxidant activity [32] Antileishmanial activity [10]
Cistus crispus L. (Cistaceae, RAB40)
Ouekir
Leaves
The poultice is applied on the skin against wounds [25]
Antibacterial and antioxidant activity [33] Antileishmanial activity [10]
Origanum compactum Benth. (Lamiaceae, RAB01)
Za’tar
Flowering top
The decoction is used orally against stomach disorders and febrifuge [25]
Antibacterial and antioxidant activity [34] Antileishmanial activity [10]
Centaurium erytherea Rafin. (Gentianaceae, RAB42 )
Gosset El Haya
Aerial part
The maceration is used against skin diseases by external use [25]
Antibacterial, antileishmanial and antioxidant activity [35]
PT
ED
Arbutus unedo L. (Ericaceae, RAB33)
CC E A
Part plant
A
Species (Family,
I N U SC R
ED
Arbutus unedo
CC E
PT
Origanum compactum
A
Cistus crispus
Centaurium erytherea
Extract MeOH
TPC 137.46±0.35
TFP 27.24±0.83
EtOH
126.41±1.03
18.41±0.43
n-hexane
122.72±1.16
31.24±1.23
MeOH
141.72±0.56
31.61±0.59
EtOH
133.61±0.45
21.61±1.65
n-hexane
94.51±0.08
24.76±0.70
MeOH
153.27±0.68
52.72±0.72
EtOH
117.60±1.12
27.60±0.32
n-hexane
105.54±0.35
29.64±0.85
MeOH
53.27±0.68
11.72±0.72
EtOH
37.60±1.12
07.60±0.32
n-hexane
35.54±0.35
09.64±0.85
MeOH
105.54±0.84
34.27±1.17
EtOH
69.42±0.55
29.42±1.05
n-hexane
95.61±0.64
38.43±1.30
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Species Myrtus communis
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Table 2: Total Phenol Content (TPC) and Total Flavonoid Content (TFC) References [31]
[32]
[34]
[33]
[35]
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Table 3: Proliferation inhibition concentration (IC50 in μg/mL) values of medicinal plant extracts towards RD, L20B, Vero cancerous cell lines and PBMC (control) as determined by the MTT assay Extracts
Myrtus communis
L20B
Vero
PBMC
78.18±2.51b
142.65±3.22a
283.18±4.72a
263.06±3.17a
˃250
˃250
˃250
˃250
n-hexane
39.42±1.25c
172.83±3.04a
˃250
102.72±2.59a
MeOH
214.72±4.06a
˃250
˃250
˃250
EtOH
174.28±3.74a
˃250
226.82±3.14a
243.83±3.26a
n-hexane
27.83±1.20c
25.32±1.26c
18.37±1.23c
19.36±1.03c
MeOH
70.63±2.17b
113.32±3.28a
27.36±2.05c
153.19±2.31a
EtOH
˃250
179.24±3.54a
232.56±5.37a
243.64±4.12a
n-hexane
42.82±1.57c
73.97±2.77b
13.72±0.84c
157.28±1.20a
MeOH
218.62±3.46a
161.48±2.34a
˃250
227.56±3.41a
EtOH
84.69±2.73b
203.35±4.72a
˃250
246.81±3.06a
n-hexane
12.75±0.82c
17.16±1.06 c
137.82±2.56a
173.28±2.13a
MeOH
218.47±3.91a
219.83±3.25a
248.52±2.73 a
˃250
EtOH
˃250
˃250
˃250
˃250
210.67±4.48a
˃250
174.56±3.24a
MeOH
PT
ED
M
EtOH Arbutus unedo
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Origanum compactum
A
Cistus crispus
Centaurium erytherea
IC50 (µg/mL)
RD
A
Species
n-hexane ˃250 Different letters indicate significant differences (p ≤ 0.05; n = 3).
Table 4: Pharmacological selectivity index (PSI) of medicinal plant extracts towards RD, L20B and Vero cell lines. Extracts
Centaurium erythraea
3.36
1.844
0.928
EtOH
nd
nd
nd
n-hexane
2.60
0.594
nd
MeOH
nd
nd
nd
EtOH
1.39
nd
n-hexane
0.69
0.76
MeOH
2.16
EtOH
nd
n-hexane
3.67
MeOH
1.04
EtOH
2.91
n-hexane
36.48
MeOH EtOH
ED
n-hexane
A
CC E
PT
nd: not determined
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MeOH
SC R
Cistus crispus
Vero
1.07 1.05
1.35
5.59
1.35
1.04
2.12
11.46
1.40
nd
1.21
nd
24.20
1.25
nd
nd
nd
nd
nd
nd
nd
nd
nd
U
Origanum compactum
L20B
N
Arbutus unedo
RD
A
Myrtus communis
Pharmacological selectivity index (PSI)
M
Species
Table 5: IC50 values (µg/mL) of plant extracts and controls (ascorbic acid and Trolox)
Origanum compactum
Cistus crispus
MeOH
16.59±0.12c
57.83±0.76b
EtOH
41.97±0.64b
142.32±3.04b
n-hexane
23.08±1.56c
48.42±1.24b
MeOH
135.02±1.08a
188.73±2.44a
EtOH
˃250
n-hexane
132.76±2.56a
MeOH
124.83±1.22a
EtOH
˃250
˃250
n-hexane
182.11±0.06a
138.45±1.19a
MeOH
119.47±0.84a
156.26±3.55a
EtOH
173.94±2.68a
˃250
n-hexane
˃250
˃250
27.28±0.74c
63.48±2.14b
98.63±2.82c
232.75±8.63
17.43±0.07c
32.07±0.53c
85.45±1.36b
37.82±2.75c
47.63±0.58b
19.62±0.87c
MeOH
A
Centaurium erytherea
ABTS
Trolox
ED
Positive controls
M
EtOH n-hexane
Ascorbic acid
CC E
PT
Different letters indicate significant differences (p ≤ 0.05; n = 3).
A
IP T
Arbutus unedo
FRAP
SC R
Myrtus communis
IC50 (µg/mL)
U
Extracts
N
Species
˃250
166.13±3.52a 169.06±3.27a
S1876-3820(18)30001-5 https://doi.org/10.1016/j.eujim.2018.01.001 EUJIM 757
To appear in: Received date: Revised date: Accepted date:
9-10-2017 31-12-2017 1-1-2018
Please cite this article as: Bouyahya Abdelhakim, Bakri Youssef, Et-Touys Abdeslam, Assemian In`es Christelle Chadon, Abrini Jamal, Dakka Nadia.In vitro antiproliferative activity of selected medicinal plants from the North-West of Morocco on several cancer cell lines.European Journal of Integrative Medicine https://doi.org/10.1016/j.eujim.2018.01.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
In vitro antiproliferative activity of selected medicinal plants from the North-West of Morocco on several cancer cell lines
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Abdelhakim Bouyahyaa,b,*, Youssef Bakria, Abdeslam Et-Touysa, Inès Christelle
aLaboratory
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Chadon Assemiana, Jamal Abrinib, Nadia Dakkaa
of Human Pathologies Biology, Department of Biology, Faculty of Sciences,
and health Laboratory, Department of Biology, Faculty of Science, Abdelmalek,
author: Abdelhakim Bouyahya
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*Corresponding
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Essaadi University, Tetouan, Morocco
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bBiology
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and Genomic Center of Human Pathologies, Mohammed V University, Rabat, Morocco
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E-mail: [email protected] ; Phone: +212601350878 Postal address : Bouyahya Abdelhakim, Laboratory of Humain Pathologies Biology, Faculty of Sciences of Rabat, University Mohammed V of Rabat 4, Av. Ibn battouta
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BP1014 Rabat-Morocco.
Abstract Introduction: Ethnomedicinal and ecological approaches have been important in drug discovery, especially for medicinal plants. The North-West of Morocco (Ouezzane) has a Mediterranean climate and is characterized by a good ecological diversity. Traditionally, several medicinal plants that have been used to treat various diseases can be found in this region. The pharmacological valorization of these species could provide
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opportunities for different diseases including cancer. The aim of this study was to screen for anticancer and antioxidative properties of selected medicinal plants from this area of North-West of Morocco.
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Methods: Organic extracts of five selected medicinal plants (Origanum compactum
Benth., Cistus crispus L., Centaurium erytherea Rafin., Myrtus communis L. and Arbutus unedo L.) were tested for their anticancer and antioxidant properties. The anticancer
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activity was tested on three cancerous cell lines L20B, RD and Vero using MTT assay and
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the pharmacological selectivity index (PSI) was calculated using PBMC as the normal cell
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line. The antioxidant activity was evaluated using FRAP and ABTS assays. Results: All plant extracts showed important anticancer activities with some variability.
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The n-hexane extract of C. crispus exhibited remarkable cytotoxic and specific effects on RD (IC50=04.75 µg/mL; PSI=36.48) and L20B cell lines (IC50=07.16 µg/mL; PSI=24.20).
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Moreover, O. compactum n-hexane extract showed a selective antiproliferative activity on Vero cell line (IC50=13.72 µg/mL; PSI=11.46). Regarding the antioxidant activity, M.
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communis methanol extract showed an important antioxidant capacity measured by ABTS and ferric-reducing power assays with IC50 values of 16.59±0.12 µg/mL and
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57.83±0.76 µg/mL, respectively. Conclusions: The findings did not reveal any correlation between antioxidant and anticancer effects thus showing specific targeted action against cancer cell lines. Based on the obtained results, these species could be considered as potential sources of
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anticancer and antioxidant compounds. However, further studies are necessary for the chemical characterization of the bioactive compounds and more investigation on their pharmacological properties is needed. Key words: Cancer; oxidative stress; medicinal plants; anticancer activity; antioxidant activity.
1. Introduction Cancer is a complex disease characterized by various etiologies and multiple stages induced by the deregulation in genetic and epigenetic programs via several factors such as microbial infections, chemical carcinogenesis and hormonal perturbation [1, 2, 3]. When cells become cancerous, they continue to divide eternally without any response of signaling apoptosis or senescence. The treatment is actually based on chemotherapy and
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radiotherapy which target cells with rapid division and therefore cause several side effects on normal cells which are characterized also by rapid division [4]. Today, new approaches targeting tumor cells without affecting normal cells are needed. Targeted
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cancer therapies based molecules from diverse sources that may target specific signaling molecular pathways in tumor cells without affecting normal cells.
On the other hand, oxidative stress caused by the excessive rate of reactive oxygen
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species such as superoxide anion (O2), nitric oxide (NO), hydroxy radical (OH) and
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peroxy radical (ROO) are involving in the pathogenesis of several pathologies including cancer [5]. Indeed, these reactives induce DNA mutations and in the case of deficient
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DNA repairs or DNA damage, it becomes a prognostic or etiological role in cancer [6, 7].
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Targeting oxidative stress by antioxidants may also reduce the risk of cancer genesis. in drug discovery.
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Therefore, the researches of antioxidants from natural sources play an interesting role
Medicinal plants have shown several biological benefits against several diseases such as
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microbial infections, urolithiasis, cancer and oxidative stress-related diseases [8, 9, 10, 11, 12, 13, 14, 15, 16]. Pharmacological properties such as cancer chemopreventive and
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cytotoxic effects of medicinal plants are attributed to various phytochemical compounds such as flavonoids, alkaloids and terpenes present in its metabolites [17]. Polyphenol and flavonoid components are amongst the most widely occurring phytochemicals that have antioxidant properties [13]. Moreover, these compounds may scavenge the ROS by
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their antioxidant capacity and thus prevent the carcinogenesis [14]. In addition, several studies have shown their anticarcinogenic or anticancer activities by their capacity to induce apoptosis in numerous cancer cell lines due to their pro-oxidant effect [18, 19, 20]. The screening of new drugs from medicinal plants leads to the discovery of various bioactive molecules that possess antioxidant and anticancer properties. These
discoveries were usually carried out using three philosophical approaches mainly used for drugs discovery from nature: the geographical approach, the ethnomedicine approach and the randomized approach [21, 22, 23, 24]. The province of Ouezzane (North-West of Morocco) contains a wide variety of vegetation including medicinal plants. This richness of vegetation is due to its ecological variability and its Mediterranean climate. Moreover, we have underlined in a recent study the indigenous knowledge of medicinal plants in this area [25]. We have also shown the importance of
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this ethnomedicine approach to the success of pharmacological properties of selected
medicinal plants [26, 27, 28, 29]. Some of these species showed interesting anticancer
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properties. For example, Origanum compactum extracts inhibited the breast cancer line
MCF7 [10]. The ethyl acetate extract from O. compactum proved cytotoxic effects against two human tumor lines (A549 lung tumor cell line and SMMC-7721 hepatic tumor cell line) [10]. Therefore, based on ecological and ethnomedicinal approaches, the aim of
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this study was the screening of medicinal plants from the province of Ouezzane with
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anticancer and antioxidative properties, to validate the ecological and ethnomedicinal
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approaches in anticancer drugs discovery. The antiproliferative activity was evaluated on two Embryonal Rhabdomyosarcoma cancerous (RD human and L20B rat) and
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Monkey kidney cancerous cell lines (Vero). These cell lines are usually used in our
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laboratory for the preliminary screening of antiproliferative activities. 2. Materials and methods
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2.1. Collection of plant material and preparation of organic extracts Medicinal plants were collected from the province of Ouezzane (North-West of Morocco:
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34° 47’ 50” N and 5° 34’ 56” W). The taxonomic identification was carried out by Pr. Ennabili Abdessalam (PAMSN Laboratory, National Institute of Medicinal and Aromatic Plants, Sidi Mohamed Ben Abdellah University, Fes-Morocco) and the confirmation of some species was later made by means of the literature as described in our previous
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study [25]. Table 1 summarizes the scientific names, vernacular names, collected parts of the plant, medicinal use by the population of Ouezzane and other pharmacological effects related to these species. The collected parts were dried in dark at room temperature and were then ground to obtain the powder. This later has been extracted by maceration using n-hexane, ethanol and methanol. After 72h of maceration, the plant
extracts were filtered and the solvent was eliminated from the filtrate using a rotary evaporator (Heidolph Collegiate, LV28798826, New Jersey, USA). 2.2. Total phenolic and flavonoid contents The total phenolic contents (TPC) and total flavonoid contents were determined using our previously reported method [31, 32, 33, 34]. The TPC was estimated by the Folin Ciocalteu (AOCS, 1990) assay using gallic acid as standard. The TFC was estimated
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by the aluminum chloride (AlCl3) colorimetric assay using quercetin as standard. The results of TPC are expressed as equivalent gallic acid /g of extract and the TFC are
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expressed as equivalent quercetin / g of extract. 2.3. Antiproliferative activity 2.3.1. Cancer cell lines and culture medium
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Tumor cell lines tested in this study were: RD: Human Embryonal Rhabdomyosarcoma cancerous cell lines (ATCC N°CCL-136), L20B: Rat Embryonal Rhabdomyosarcoma
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cancerous cell lines (3T6Swiss albino, ATCC CCL96) and Vero: Monkey kidney cancerous
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cell lines (ATCC N°CCL-81). These three species were obtained from the National
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Institute of Health, Rabat-Morocco. These species were cultivated as described by Aneb et al. [20]. Briefly, cells were grown as monolayers in Minimum Essential Medium
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(MEM) supplemented with 10% of heat-inactivated fetal calf serum and 1% of PenicillinStreptomycin mixture. PBMC (Peripheral Blood Mononuclear Cell) isolated and purified from human blood were used as positive control. Immediately after collection, blood
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samples were diluted with an equal volume of minimal essential medium RPMI and layered onto Ficoll (Pancoll, Biotech) for gradient separation of cells. The buffy coat
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layer was collected after centrifugation at 300 g for 30 min and then washed twice with RPMI. These cells were cultivated in RPMI supplemented with 10% of heat-inactivated fetal calf serum and 1% of Penicillin-Streptomycin mixture. Cultures were maintained at
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37°C in 5% CO2 and 100% of relative humidity atmosphere. 2.3.2. Cytotoxic activity of extracts Before evaluating the anticancer activity, the cellular density of each species was calculated using light microscopy. When cellular density reached a sub-confluence threshold, cells were detached by trypsine-EDTA, washed twice with phosphate buffered saline (PBS) and centrifuged at 150rpm for 15 min (4°C). Cells were then
resuspended in a fresh culture medium (MEM). The enumeration of cells was made by Malassez cell count using the trypan blue as exclusion coloring. Briefly, 100 μL of medium containing 3-4 x 106 cells/mL were placed in each well and cultured at 37°C in 5% CO2/ humidified air for 24 h. After 24h of incubation, cells were treated with crude extracts. Briefly, 100 μL of various concentrations of extracts (from 3.5 to 250 µg/mL; dissolved in 1% DMSO) were added to each well. Each concentration was tested in triplicate. DMSO was used at a final concentration never exceeding 1% which is not toxic
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to cells [35, 37]. Microplates (96 well plates) were then incubated for 48 h at 37°C in air condition of 5% CO2. Sterile PBS and 1% DMSO (vehicle) were used as negative controls.
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After 48h of cells incubation with extracts, the MTT (3-(4,5-diméthylthiazol-2,5diphenyl tetrazolium bromide) (Sigma) assay was used in order to evaluate the cell cytotoxicity. The test based on the biotransformation of the MTT into formazen crystals by mitochondrial deshydrogenases of living cells. The absorbance of the formazen will
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then be quantified by spectrophotometry at 550 nm. Optical density values (OD) are
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directly correlated with the metabolized intracellular MTT concentrations and are
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therefore proportional to the number of living cells. Briefly, 20 µL of MTT (5 mg/mL) were added to each micro-well and incubated for 3h at 37°C in 5% CO2. Tetrazoluim
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salts are cleaved to formazen and the reaction was stopped by addition of 100 µL of 50% (v/v) isopropanol-10% (w/v) sodium dodecyl sulfate (SDS) mixture to each well in
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order to dissolve insoluble formazen formed after tetrazolium dye reduction. After 30 minutes of incubation at room temperature, the absorbance was measured at 550 nm
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using an ELISA plate reader. Cell viability was evaluated by determination of the percentage of cytotoxicity using the followed formula. The concentrations that inhibit
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the half of the cell population (IC50) were obtained by modeling the % of cytotoxicity versus the concentration of extracts. Cytotoxicity (%) = 100 × (Absorbance of untreated cells-Absorbance of treated cells) /
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Absorbance of untreated cells. 2.3.3. Determination of the pharmacological selectivity index The pharmacological selectivity index (PSI) is an interesting parameter that definites the balance between pharmacological effect of compounds and their toxicity. It corresponds to the highest active concentration against cancer cells without toxicity against normal cells. It was expressed as the ratio IC50 normal cells (PBMC) / IC50 cancer cell lines [37].
2.4. Antioxidant activity 2.4.1. ABTS radical scavenging activity ABTS
(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid)
radical-scavenging
activity of the extract was determined according to the method described by El Jemli et al. [38] with some modifications. The ABTS radical cation was produced by the reaction between 5 mL of ABTS stock solution and 5 mL of 2.45 mM potassium persulfate
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(K2S2O8) solution, stored in the dark at room temperature for 16h. Before use, this
solution was diluted with water to get an absorbance of 0.700±0.015 at 734 nm and equilibrated at 30°C. The plant extract at various concentrations were diluted with
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dimethyl sulfoxide (DMSO) to get sample solution. 5 mL of sample solution was
homogenized with 195 mL of ABTS solution, the mixture was incubated at room temperature for 6 min and its absorbance was recorded at 734 nm. Blanks were run in
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each assay. ABTS scavenging ability was expressed as IC50 (µg/mL) and the inhibition
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percentage calculated using the following formula: ABTS scavenging activity (%) = (A0A1)/A0 × 100. Where: A0 is the absorbance of the control, and A1 is the absorbance of the
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4.2.2. Reducing power assay
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sample.
The ferric-reducing antioxidant power of plant extracts was investigated using the
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potassium ferricyanide-ferric chloride method as described by Bey-Ould Si Said et al. [39] and El Jamli et al. [38]. Briefly, one mL of the extract at different dilutions in pure
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methanol (10, 20, 30, 40 and 50 mg/mL) was mixed with phosphate buffer (1 mL, 0.2 M‘w/v’, pH 6.6) and potassium ferri-cyanide [K3Fe (CN)6] (1 mL, 1%‘w/v’). The
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obtained mixture was incubated at 50°C for 20 min to reduce ferricyanide into ferricyanide. A volume of 2.5 mL of trichloroacetic acid (10%) was added to the solution and centrifuged at 1000 rpm for 10 min. The supernatant was gathered and mixed with distilled water (1.5 mL) and FeCl3 (150 µL, 0.1%‘w/v’), and the absorbance was
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recorded at 700 nm. The sample concentration providing 0.5 of absorbance (IC50) was calculated by plotting absorbance against the corresponding sample concentration. Ascorbic acid and Trolox were used as standard compounds. The test was carried out in triplicate and IC50 values were reported as means ± SD. 2. 4. Data analysis
The statistical analysis was performed by a one-way ANOVA analysis of variance. The difference is considered as significant for p ≤ 0.05. 3. Results 3.1. Phenolic and flavonoid contents The TPC and TFC of extracts are summarized in Table 2. All plant extracts possess important amounts of phenolic and flavonoid contents. However, we have noted
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variability between these amounts that depend on the plant extracts and the used solvents. Extracts from A. unedo, O. compactum and M. communis revealed more
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significant contents in TPC and TFC than extracts of C. crispus and C. erythraea. On the other hand, methanol extracts possess higher concentrations in TPC and TFC than ethanol and n-hexane extracts.
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3.2. Anticancer activity
The evaluation of anticancer activity of five Moroccan medicinal plant extracts was
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carried out on three tumor cell lines, namely RD, L20B and Vero. Cancerous cell lines
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were exposed to decreasing concentrations of extracts ranging from 3.5 to 250 μg/mL.
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The MTT assay indicated that extracts revealed different cytotoxic activities towards the tested cancerous cell lines. To compare antiproliferative effects of extracts, we have
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expressed the results as the concentration that proved the half inhibition in cell viability (IC50) compared to control (PBMC). The obtained results are listed in table 3. As summarised, plant extracts showed important cytotoxic activities that were varied
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between plant extracts and between the types of extract in the same plant. Amongst these tested plant extracts, the n-hexane extract of C. crispus and A. unedo showed
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significant growth inhibitory effects in both RD and L20B. Their IC50 values were 12.75±0.82 and 27.83±1.20 μg/mL, respectively on RD cells. For L20B cell line, the IC50 values of the same extracts were 17.16±1.06 and 25.32±1.26 μg/mL, respectively.
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Moreover, O. compactum and A. unedo n-hexane extracts revealed important cytotoxic effects against Vero cell line with IC50 values of 13.72±0.84 and 18.37±1.23 μg/mL, respectively. The cytotoxic activity of extracts against normal cells (PBMC) showed significant toxicity of some extracts such as A. unedo n-hexane extract (IC50=19.36±1.03 μg/mL).
Their pharmacological selectivity indexes were PSI=0.69, PSI=0.76 and
PSI=1.05 on RD, L20B and Vero cell lines, respectively (Table 4). Regarding C. crispus and O. compactum n-hexane extracts, they have shown low cytotoxicity against normal
cells with IC50 values of 173.28±2.13 and 157.28±1.20 μg/mL respectively. Consequently, the C. crispus n-hexane extract had good pharmacological selectivity indexes against RD (PSI=36.48) and L20B (PSI=24.20) (Table 4). Furthermore, O. compactum n-hexane extract showed a high selectivity index toward Vero cells (PSI=11.46). 3.3. Antioxidant activity
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The antioxidant activity of selected medicinal plants from the North-West of Morocco
was estimated using two complementary in vitro antioxidant tests: the FRAP assay that estimates the ferric-reducing capacity of the extracts and the TEAC assay evaluating the
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H donating or radical-scavenging ability of extracts using the ABTS radical cation. The concentrations providing 50% inhibitions (IC50) are listed in table 5. As summarized in table 5, all extracts showed the ability to scavenge ABTS radical cation and ferric-
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reducing power. However, the antioxidant capacity varies depending on the plant, the
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type of extracts and the used method. In ABTS test, the n-hexane extract of C. erytherea showed important antioxidant capacity (32.07±0.53 µg/mL), compared with reference
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antioxidants Trolox and ascorbic acid (p < 0.05), which showed IC50 values of
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37.82±2.75 and 19.62±0.87 µg/mL respectively. The methanol and n-hexane extracts of M. communis and the methanolic extract of C. erytherea showed also important
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antioxidant capacity to scavenge ABTS radical, but their IC50 values were significantly different (p ˃ 0.05) with positive controls (Table 5). Regarding the FRAP assay, a high ferric reductive capacity was obtained with the methanol extract of M. communis
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(16.59±0.12 µg/mL) and n-hexane extract of C. erytherea (17.43±0.07 µg/mL). Their IC50 values were significantly important (p ˃ 0.05) than antioxidant standards (Trolox and
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ascorbic acid). 4. Discussion
Medicinal plants synthesize several bio-organic molecules as secondary metabolites.
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These compounds have various chemical structures such as terpenoids, simple phenols, phenolic acids, flavonoids and alkaloids. Because of their physiological functions in plants, these chemical classes possess attention for several biological properties such as antibacterial, antioxidant, anticancer and anti-inflammatory activities [38, 40, 41, 42]. In recent years, phenolic compounds such as polyphenols and flavonoids extracted from medicinal plants have demonstrated interesting antioxidant and antiproliferative
effects. In this study, five medicinal plants from the North-West of Morocco have been selected based on ethnobotanical approach [25]. The collected plants have subjected to the extraction by maceration using three organic solvents (methanol, ethanol and nhexane). It has shown that these species possessed important charges in polyphenol and flavonoid contents (Table 2). These results are interesting compared with several studies regarding the total phenol and flavonoid contents in medicinal plant extracts [8,
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15]. This may explain the widespread traditional uses of these species. On the other hand, the variability of chemical compounds of medicinal plants depends
on various factors such as the geographical location, the climate, the method of
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extraction, the organ extracted and the phenological stage of the studied plant [25, 43, 44, 45]. These factors could influence and regulate the synthesis of secondary metabolites in medicinal plants. Indeed, it has been shown that the environmental
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factors and developmental stages may induce the modification in genes expression thus inducing the variation in enzymes responsible for secondary metabolite metabolism
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[46].
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Cancer is a complex disease with various etiologies and multiple stages. Actually, the
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treatment is essentially based on chemotherapy and radiotherapy. These treatments target cells with rapid division and therefore cause several side effects on normal cells
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which are also characterized by rapid division [4]. The screening of bioactive molecules from diverse sources that may target specific signaling molecular pathways in tumor cells without affecting normal cells is important. Polyphenol and flavonoid compounds
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from medicinal plants have been known to target specific pathways against cancer cells
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growth [40, 11].
In this context, phenolic extracts of five selected medicinal plants were screened for their anticancer activity against three cancerous cell lines. Extracts showed significant cytotoxicity against tested cells and some cytotoxic effects on normal cells (PBMC).
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However, pharmacological studies on cancer diseases focused today on the specific selectivity of drugs against cancer cells without side effects on normal cells. The anticancer activity of O. compactum n-hexane extract against Vero cell line and n-hexane extract of C. crispus against L20B and RD cell lines characterized by high selectivity indexes (Table 4). The results have indicated that these extracts have specific targets on the tested cancerous cells. Previous studies have demonstrated the anticancer activity of
O. compactum ethyl acetate extract on human tumor cell line A549 and SMMC-7721 [47]. The cytotoxicity of this extract was due to the induction of apoptosis by the activation of caspase enzymes mediated by the modulation of Bcl-2 family proteins. In another early work, Chaouki et al. [48] have shown cytotoxic effects of O. compactum ethyl acetate and chloroform extracts against human breast cancer cell line MCF-7. However, C. crispus has not yet been tested for its anticancer properties. O. compactum and C. crispus possess important charges of phenolic compounds such as flavonoids. Indeed, flavonoids have
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reported having antiradical effects, with high capacity to scavenge free radical as reactive oxygen species [49, 50]. Moreover, ROS have long been known to be implicated
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in the genesis of several diseases including cancer [51]. Antitumor mechanism of flavonoids based on their antioxidant properties is due to their effects of free radicals scavenging, inhibition of enzymes involved in ROS formation, and the prevention of cellular and extracellular compounds oxidation [50]. However, no significant correlation
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has been indicated between antioxidant and cytotoxic activities of plant extracts. This
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observation indicates thus that n-hexane extract of O. compactum and C. crispus have
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other specific targets on RD and Vero cell lines. Indeed, flavonoids possess several cytotoxic mechanisms on cancer cell lines. They may modulate pathways of cancer
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proliferation, arrest the cell cycle, induce the apoptosis and inhibit the angiogenesis [40,
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49, 52].
The antioxidant activity of phenolic compounds such as polyphenols and flavonoids is largely due to their redox properties which make them act as reducing agents, hydrogen
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donors, singlet oxygen quenchers as well as potential metal chelators [53]. Furthermore, the oxidative stress is largely implicated in several human pathologies such as cancer,
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inflammation and neurodegenerative diseases [7]. Our plant extracts were subjected to the evaluation of their antioxidant activity using ABTS and FRAP assays. The screening of this activity leads to remarkable results (Table 5). The ABTS radical scavenging test
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is a good index to reveal the antioxidant activity of the tested samples [54]. The scavenging of the ABTS radical by our extracts was found to be interesting compared with references. The results showed that C. erytherea extracts presented high capacities to scavenge the ABTS radical. Other studies have revealed an interesting capacity of C. erythrea extracts to scavenge DPPH free radical [55, 56]. However, it has been reported that some compounds which have ABTS radical scavenging activity did not show DPPH scavenging activity. In fact, the solubility of extracts in diverse solvents and the
stereoselectivity of the radicals are two factors that significantly affect the ability of extracts to react with radicals [53]. The difference in the antioxidant activity between plant extracts is certainly due to the variability in chemical functionality compounds. However, the difference between antioxidant assays is due to the mechanisms of which an extract reacts with the used method. Indeed, the FRAP assay estimates the ferricreducing capacity while the ABTS assay evaluates the H donating and radical scavenging capacity. It has been reported that phenolic compounds such as flavonoids neutralize
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free radicals by donating one of their own electrons. In fact, phenolic compounds possess hydroxyl groups responsible for their antioxidant properties. These compounds
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have the ability to scavenge free radicals increased with the number of hydroxyl groups
and catechol moieties in the molecule [57]. In addition, the localization of the hydroxyl groups in molecules could influence their antioxidant properties [58]. This study clearly showed that the antioxidant activity of the North-West Moroccan medicinal plants was
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tightly dependent on the polyphenol and flavonoid contents which themselves depend
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on several factors such as plant species, used solvents, antioxidant assays. This should
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be taken into consideration during the preparation of the extracts.
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Conclusion
The anticancer and antioxidant activities of organic extracts have shown interesting
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results. Phenolic extracts from some species such as C. crispus and O. compactum showed specific anticancer action against cancer cell lines. The antiproliferative activity was not correlated with the antioxidant effect suggesting thus other specific targets of phenolic
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compounds on cancer cell lines. These findings showed that phenolic extracts from Moroccan medicinal plants can be a good source for screening anticancer drugs.
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However, further investigations are necessary for the isolation of bioactive compounds and the study of their mode of action on cancer cell lines.
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Financial support: None
Conflicts of interests The authors declare that there is no conflict of interests regarding the publication of this article.
Acknowledgements We thank the National Center for Scientific and Technical Research (CNRST) from
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Morocco for the support of the doctoral grant of Bouyahya Abdelhakim.
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Table 1: Medicinal plants studied (scientific name, family, part plant collected, medicinal use and pharmacological property) Vernacular
Voucher specimen )
name
M. communis L. (Myrtaceae, RAB13)
Rihan
Medicinal use in Ouezzane
Pharmacological
collected
province
properties
Leaves
The powder in oil poultice is applied on hair and scalp before sleeping for fall protection [25]
Antibacterial and antileishmanial effects [30, 31]
M Senou
Leaves
The decoction is taken orally as natural antidiabetic drugs [25]
Antibacterial and antioxidant activity [32] Antileishmanial activity [10]
Cistus crispus L. (Cistaceae, RAB40)
Ouekir
Leaves
The poultice is applied on the skin against wounds [25]
Antibacterial and antioxidant activity [33] Antileishmanial activity [10]
Origanum compactum Benth. (Lamiaceae, RAB01)
Za’tar
Flowering top
The decoction is used orally against stomach disorders and febrifuge [25]
Antibacterial and antioxidant activity [34] Antileishmanial activity [10]
Centaurium erytherea Rafin. (Gentianaceae, RAB42 )
Gosset El Haya
Aerial part
The maceration is used against skin diseases by external use [25]
Antibacterial, antileishmanial and antioxidant activity [35]
PT
ED
Arbutus unedo L. (Ericaceae, RAB33)
CC E A
Part plant
A
Species (Family,
I N U SC R
ED
Arbutus unedo
CC E
PT
Origanum compactum
A
Cistus crispus
Centaurium erytherea
Extract MeOH
TPC 137.46±0.35
TFP 27.24±0.83
EtOH
126.41±1.03
18.41±0.43
n-hexane
122.72±1.16
31.24±1.23
MeOH
141.72±0.56
31.61±0.59
EtOH
133.61±0.45
21.61±1.65
n-hexane
94.51±0.08
24.76±0.70
MeOH
153.27±0.68
52.72±0.72
EtOH
117.60±1.12
27.60±0.32
n-hexane
105.54±0.35
29.64±0.85
MeOH
53.27±0.68
11.72±0.72
EtOH
37.60±1.12
07.60±0.32
n-hexane
35.54±0.35
09.64±0.85
MeOH
105.54±0.84
34.27±1.17
EtOH
69.42±0.55
29.42±1.05
n-hexane
95.61±0.64
38.43±1.30
M
Species Myrtus communis
A
Table 2: Total Phenol Content (TPC) and Total Flavonoid Content (TFC) References [31]
[32]
[34]
[33]
[35]
I N U SC R
Table 3: Proliferation inhibition concentration (IC50 in μg/mL) values of medicinal plant extracts towards RD, L20B, Vero cancerous cell lines and PBMC (control) as determined by the MTT assay Extracts
Myrtus communis
L20B
Vero
PBMC
78.18±2.51b
142.65±3.22a
283.18±4.72a
263.06±3.17a
˃250
˃250
˃250
˃250
n-hexane
39.42±1.25c
172.83±3.04a
˃250
102.72±2.59a
MeOH
214.72±4.06a
˃250
˃250
˃250
EtOH
174.28±3.74a
˃250
226.82±3.14a
243.83±3.26a
n-hexane
27.83±1.20c
25.32±1.26c
18.37±1.23c
19.36±1.03c
MeOH
70.63±2.17b
113.32±3.28a
27.36±2.05c
153.19±2.31a
EtOH
˃250
179.24±3.54a
232.56±5.37a
243.64±4.12a
n-hexane
42.82±1.57c
73.97±2.77b
13.72±0.84c
157.28±1.20a
MeOH
218.62±3.46a
161.48±2.34a
˃250
227.56±3.41a
EtOH
84.69±2.73b
203.35±4.72a
˃250
246.81±3.06a
n-hexane
12.75±0.82c
17.16±1.06 c
137.82±2.56a
173.28±2.13a
MeOH
218.47±3.91a
219.83±3.25a
248.52±2.73 a
˃250
EtOH
˃250
˃250
˃250
˃250
210.67±4.48a
˃250
174.56±3.24a
MeOH
PT
ED
M
EtOH Arbutus unedo
CC E
Origanum compactum
A
Cistus crispus
Centaurium erytherea
IC50 (µg/mL)
RD
A
Species
n-hexane ˃250 Different letters indicate significant differences (p ≤ 0.05; n = 3).
Table 4: Pharmacological selectivity index (PSI) of medicinal plant extracts towards RD, L20B and Vero cell lines. Extracts
Centaurium erythraea
3.36
1.844
0.928
EtOH
nd
nd
nd
n-hexane
2.60
0.594
nd
MeOH
nd
nd
nd
EtOH
1.39
nd
n-hexane
0.69
0.76
MeOH
2.16
EtOH
nd
n-hexane
3.67
MeOH
1.04
EtOH
2.91
n-hexane
36.48
MeOH EtOH
ED
n-hexane
A
CC E
PT
nd: not determined
IP T
MeOH
SC R
Cistus crispus
Vero
1.07 1.05
1.35
5.59
1.35
1.04
2.12
11.46
1.40
nd
1.21
nd
24.20
1.25
nd
nd
nd
nd
nd
nd
nd
nd
nd
U
Origanum compactum
L20B
N
Arbutus unedo
RD
A
Myrtus communis
Pharmacological selectivity index (PSI)
M
Species
Table 5: IC50 values (µg/mL) of plant extracts and controls (ascorbic acid and Trolox)
Origanum compactum
Cistus crispus
MeOH
16.59±0.12c
57.83±0.76b
EtOH
41.97±0.64b
142.32±3.04b
n-hexane
23.08±1.56c
48.42±1.24b
MeOH
135.02±1.08a
188.73±2.44a
EtOH
˃250
n-hexane
132.76±2.56a
MeOH
124.83±1.22a
EtOH
˃250
˃250
n-hexane
182.11±0.06a
138.45±1.19a
MeOH
119.47±0.84a
156.26±3.55a
EtOH
173.94±2.68a
˃250
n-hexane
˃250
˃250
27.28±0.74c
63.48±2.14b
98.63±2.82c
232.75±8.63
17.43±0.07c
32.07±0.53c
85.45±1.36b
37.82±2.75c
47.63±0.58b
19.62±0.87c
MeOH
A
Centaurium erytherea
ABTS
Trolox
ED
Positive controls
M
EtOH n-hexane
Ascorbic acid
CC E
PT
Different letters indicate significant differences (p ≤ 0.05; n = 3).
A
IP T
Arbutus unedo
FRAP
SC R
Myrtus communis
IC50 (µg/mL)
U
Extracts
N
Species
˃250
166.13±3.52a 169.06±3.27a