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Inhibitory effects of wild dietary plants on lipid peroxidation and on the proliferation of human cancer cells
Food and Chemical Toxicology 86 (2015) 16e24
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Inhibitory effects of wild dietary plants on lipid peroxidation and on the proliferation of human cancer cells Mariangela Marrelli, Brigida Cristaldi, Francesco Menichini, Filomena Conforti* Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, I-87036 Rende (CS), Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2015 Received in revised form 4 September 2015 Accepted 20 September 2015 Available online 25 September 2015
Thirteen hydroalcoholic extracts of edible plants from Southern Italy were evaluated for their in vitro antioxidant and antiproliferative activity on three human cancer cell lines: breast cancer MCF-7, hepatic cancer HepG2 and colorectal cancer LoVo. After 48 h of incubation the most antiproliferative plant extract was rosemary (Rosmarinus officinalis L.) on LoVo cell line with IC50 of 16.60 mg/ml. Oregano (Origanum vulgare L. subsp. viridulum) showed a selective antiproliferative activity on hepatic cancer with IC50 of 32.59 mg/ml. All the extracts, with the exception of Diplotaxis tenuifolia (L.) DC., exerted antioxidant properties, the most active plants being dewberry (Rubus caesius L.) and “laprista” (Rumex conglomerates Murray) with IC50 of 4.91 and 5.53 mg/ml, respectively. Rumex conglomeratus contained the highest amount of flavonoids (15.5 mg/g) followed by Portulaca oleracea L. (11.8 mg/g). Rosmarinus officinalis contained the highest number of terpenes. Among them ketoursene (14.7%) and aristolone (11.3%) were found to be the major constituents. P. oleracea and Raphanus raphanistrum L. subsp. landra contained the highest number of sterols. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Antioxidant activity Antiproliferative activity Flavonoid content Terpene and sterol composition
1. Introduction Cancer is a growing public problem whose estimated worldwide new incidence is about 6 million cases per year. It is the second major cause of deaths after cardiovascular diseases. It became a general conviction that dietary agents and environmental factors (radiation, sunshine, hormones, viruses, bacteria, and chemicals) have a real impact, either positive or negative, on cancer development affecting the proliferation, angiogenesis and metastasis. Investigations about natural products have recently regained
Abbreviations: AC, Asparagus acutifolius L.; AR, Amaranthus retroflexus L.; AZ, Anchusa azurea Mill.; DT, Diplotaxis tenuifolia (L.) DC.; GC-MS, gas chromatographymass spectrometry; HepG2, hepatic cancer cell line; HPTLC, high performance thin layer chromatography; LoVo, colorectal cancer cells; MCF-7, breast cancer cell line; MS, Mentha spicata L. ssp glabrata (Lej. et Court.) Lebeau; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NPR, natural product reagent; OV, Origanum vulgare L. subsp. viridulum (Martin-Donos) Nyman; PO, Portulaca oleracea L.; RC, Rumex conglomeratus Murray; RO, Rosmarinus officinalis L.; RR, Raphanus raphanistrum L. subsp. landra (DC.) Bonnier & Layens; RUB, Rubus caesius L.; SO, Smyrnium olusatrum L; SV, Silene vulgaris (Moench) Garcke. * Corresponding author. E-mail address: fi[email protected] (F. Conforti). http://dx.doi.org/10.1016/j.fct.2015.09.011 0278-6915/© 2015 Elsevier Ltd. All rights reserved.
prominence with the increasing understanding of their biological significance and increasing recognition of the origin and function of their structural diversity. Plant extracts and derivatives have always been used for various disease treatments and many anticancer agents issued from plants and vegetables are clinically recognized and used all over the world. Currently, more than 50% of all approved drugs (not limited to cancer) are natural products and their derivatives (Ovadje et al., 2015). Over several decades, many studies have shown numerous dietary constituents and nutraceuticals as cancer chemopreventive agents and in the recent past a number of reports have elucidated the cellular mechanisms of prevention and suppression of cancer progression. Anticarcinogenic effects of many plants derivatives, by inhibiting cancer cell proliferation in vitro, were assessed in many scientific reports (Galasso et al., 2014; Petiwala et al., 2014; Mastron et al., 2015). It is commonly accepted that in a situation of oxidative stress, reactive oxygen species (ROS) such as superoxide (O 2 , OOH ), hy droxyl (OH ) and peroxyl (ROO ) radicals are generated. It's known that when the antioxidant control mechanisms are overrun, the cellular redox potential shifts toward oxidative stress. As a
M. Marrelli et al. / Food and Chemical Toxicology 86 (2015) 16e24
consequence, the potential for damaging cellular nucleic acids, lipids, and proteins increases, which is associated to carcinogenesis (Kris-Etherton et al., 2004; Mollazadeh and Hosseinzadeh, 2014). Cancer cells exhibit increased ROS generation that may promote cell proliferation. Antioxidant capacity of some phytochemicals like polyphenols, flavonoids and carotenoids may be involved in cancer €hko € nen et al., prevention by combating oxidative cell damages (Ka 1999; Soobrattee et al., 2005; Velioglu et al., 1998). Epidemiological and experimental studies reveal a negative correlation between the consumption of diets rich in fruit and vegetables and the risks for chronic diseases, such as cardiovascular diseases, arthritis, chronic inflammation and cancers (Chen et al., 2005; Saleem et al., 2002). The present study was undertaken to evaluate the potential cytotoxic activity on cancer cell lines and estimate the antioxidant and protective effect of the following Mediterranean plants: Amaranthus retroflexus L., Anchusa azurea Mill., Asparagus acutifolius L., Diplotaxis tenuifolia (L.) DC., Mentha spicata L. ssp glabrata (Lej. et Court.) Lebeau, Origanum vulgare L. subsp. viridulum (MartinDonos) Nyman, Portulaca oleracea L., Raphanus raphanistrum L. subsp. landra (DC.) Bonnier & Layens, Rosmarinus officinalis L., Rubus caesius L., Rumex conglomeratus Murray, Silene vulgaris (Moench) Garcke, Smyrnium olusatrum L. They are spontaneous edible plants present in the Southern of Italy, a territory characterized by different vegetation belt from the sea level to the highest peak of Sila Mt. (about 1800 m a.s.l.), mostly rich in Mediterranean elements. In this work, 13 extracts obtained from the Italian plants listed before were studied to assess their antioxidant activities and antiproliferative properties against a panel of human cancer cells which, to the best of our knowledge, is not reported for these plants so far. The b-carotene bleaching test was used to evaluate the antioxidant activity. Antiproliferative activity was evaluated against three different human cancer cell lines: breast (MCF-7), hepatic (HepG2) and colorectal (LoVo) cancer cells. Furthermore, the total flavonoid content was determined by a method based on the formation of complex flavonoid-aluminium, the qualitative and quantitative evaluation of some phenolic compounds was determined by HPTLC and the terpene and sterol composition was determined by GC-MS analysis. 2. Materials and methods 2.1. Plant materials The different species studied in this work are reported in Table 1.
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The collected plants were authenticated by Dr. Uzunov from Botanic Garden, University of Calabria, Italy, and the plants were deposited at the Natural History Museum of Calabria. Voucher numbers are also indicated in Table 1.
2.2. Chemicals Methanol, ethanol, H2SO4, n-hexane, ethyl acetate, dichloromethane and DMSO were obtained from VWR International s.r.l. (Milan, Italy). Normal phase glass plates 20 cm 10 cm with glassbacked layers silica gel 60 (2 mm thickness) were purchased from Merck (Darmstadt, Germany). Phosphate buffered saline (PBS), RPMI 1640 medium, DMEM medium, fetal bovine serum, L-glutamine, penicillin/streptomycin, trypan blue, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), propyl gallate, bcarotene, linoleic acid, Tween 20, aluminium chloride and reference substances utilised in HPTLC analyses were obtained from SigmaAldrich S.p.A. (Milan, Italy). Breast cancer cells MCF-7, hepatic cancer cell line HepG2 and colorectal adenocarcinoma cancer cells LoVo were purchased from ATCC no. HTB-22, no. HB-8065 and no. CCL-229, UK. All other reagents, of analytical grade, were Carlo Erba products (Milan, Italy).
2.3. Preparation of samples As previously described (Conforti et al., 2011) the plant material, after dried, was extracted with 70% aqueous EtOH (3 l) through maceration at room temperature. Total flavonoid content was determined for each total extract. Extracts were then partitioned between 100 ml 90% MeOH and n-hexane (100 ml 3 times), and the n-hexane fractions were analysed by gas-chromatographymass spectrometry (GC-MS) in order to identify the terpene and sterol composition. The qualitative and quantitative evaluation of some phenolic compounds was determined by HPTLC. 2.4. Determination of total flavonoid content Total flavonoid content was estimated using a colorimetric method based on the formation of a complex flavonoid-aluminum, having the absorptivity maximum at 430 nm (Conforti et al., 2008). All determinations were made in triplicate and values were calculated from a calibration curve obtained with quercetin, chosen as standard. Final results were expressed as mg of quercetin equivalent per g of extract.
Table 1 Investigated wild dietary plants. Plant Cod.
Botanical name
Family
Plant part
Use
Voucher number
AR AZ AC
Amaranthus retroflexus L. Anchusa azurea Miller Asparagus acutifolius L.
Amaranthaceae Boraginaceae Asparagaceae
Leaves Flowers Stems
21793 21795 21802
DT MS OV
Diplotaxis tenuifolia (L.) DC. Brassicaceae Mentha spicata L. ssp. glabrata (Lej. et Court) Lebeau Lamiaceae Origanum vulgare L. subsp. viridulum (Martin-Donos) Nyman Lamiaceae
Mixed, soup, boil fries Mixed salad, soup, boil, fries Salad of shoots, fries with eggs, under oil, risotto Salad, soup, boil Spice, aromatic, liqueur, salse Spice, condiment, on roast, liqueur
21789 21801 21798
PO RR
Portulaca oleracea L. Raphanus raphanistrum L. subsp. landra (DC.) Bonnier & Layens Rosmarinus officinalis L. Rubus caesius L. Rumex conglomeratus Murray Silene vulgaris (Moench) Garcke Smirnium olusatrum L.
Mixed salad, boil Like spinach, deep-dish pizza
21796 21799
Aromatic, spice, aromatic, on roast meat Boil Potage, soup Salad, risotto, fries with eggs Mixed salad, soup
21800 21791 21794 21792 21803
RO RUB RC SV SO
Portulacaceae Brassicaceae
Leaves Leaves Stems, leaves Leaves Leaves
Lamiaceae Rosaceae Polygonaceae Caryophyillaceae Apiaceae
Leaves Leaves Leaves Leaves Leaves
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2.5. GC/MS analysis The n-hexane fractions were analysed by gas chromatographymass spectrometry (GC-MS). A Hewlett-Packard 6890 gas chromatograph equipped with an SE-30 capillary column (100% dimethylpolysiloxane, 30 m length, 0.25 mm in diameter, 0.25 mm film thickness) directly coupled to a selective mass detector (model 5973, Hewlett Packard) was used. Analyses were realized using a programmed temperature from 60 to 280 C (rate 16 min1) and helium as carrier gas. Identification of compounds was based on the comparison of the GC retention factors with those of standards and the comparison of the mass spectra with those present in the Wiley 138 library data of the GC-MS system (Marrelli et al., 2012). 2.6. HPTLC analysis 2.6.1. Apparatus and chromatographic procedure High Performance Thin Layer Chromatography (HPTLC) was chosen to identify phenolic compounds (Marrelli et al., 2014). Normal phase glass plates 20 10 cm (VWR International s.r.l., Milano, Italy) with glass backed layers silica gel 60 (2 mm thickness) were used. Plates were prewashed with methanol and carefully dried for 3 min at 100 C. A CAMAG Linomat 5 sample applicator (Muttenz, Switzerland) connected to a nitrogen tank and to a CAMAG TLC Visualizer was utilized. Syringe delivery speed was 150 nl/s and injection volume was 1 ml. The HPTLC plates were developed with the mobile phase ethyl acetate/dichloromethane/ acetic acid/formic acid/water (100:25:10:10:11, v/v/v/v/v) for the detection of chlorogenic and caffeic acid, quercitrin, catechin, luteolin, naringin, rutin and biapigenin. For the identification of quercetin, kaempferol, ferulic acid and coumaric acid the mobile phase was instead ethyl acetate/ dichloromethane/acetic acid/formic acid/water (100:31.25: 1.25:1.25:1.25, v/v/v/v/v). Band width was 8 mm; distance from bottom and solvent front position were 8 mm and 9 mm, respectively. NPR (1 g diphenylborinic acid aminoethylester in 200 ml of ethyl acetate) and anhysaldehyde (1.5 ml p-anisaldehyde, 2.5 ml H2SO4, 1 ml AcOH in 37 ml EtOH) were used for postchromatographic derivatization. All treated plates were inspected under a UV light at 254 or 366 nm or under white light upper and lower (WRT) before and after derivatization by means of a Camag TLC visualizer. All analyses were carried out in triplicate. 2.6.2. Preparation of samples and standard solutions For the determination of phenolic compounds, the polar fraction of each extract was dissolved in methanol to have a final concentration of 50 mg/ml. This extract was used for TLC fingerprinting and co-chromatography with marker compounds. Sample solutions were applied in triplicate on the TLC plates. Standards have been used at a concentration of 3 mg/ml in methanol (or ethanol for caffeic acid, luteolin, naringin, kaempferol and coumaric acid) for qualitative determinations. For quantitative analyses, working stock solutions were prepared by dilution with methanol (or ethanol for caffeic acid) to give final concentrations of 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0 mg/ml. Standard solutions of each compound were spotted on HPTLC plate to give absolute amounts of 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0 mg/ band. 2.6.3. Quantitative analyses In order to realize quantitative analyses, calibration curves were prepared using absolute amount (mg/band) as independent variable (X) and the peak area of standards as dependent variable (Y) (Marrelli et al., 2013). Quantification of compounds was performed
using regression equations (correlation coefficients R2 > 0.98). Analyses were performed using GraphPad Prism Software (San Diego, CA, USA).
2.7. Antioxidant assay Antioxidant activity was determined by means of the b-carotene bleaching test (Marrelli et al., 2012). In brief, 0.2 ml of samples in methanol at different concentrations (100, 50, 25, 10, 5 and 1 mg/ml) were added to 5 ml of an emulsion of b-carotene and linoleic acid. Tubes were then placed in a water bath at 45 C. The absorbance was measured at 470 nm using a Perkin Elmer Lambda 40 UV/VIS spectrophotometer against a blank, consisting of an emulsion without b-carotene. The measurement was carried out at initial time (t ¼ 0) and successively at 30 and 60 min. Propyl gallate was used as positive control. The antioxidant activity (AA) was measured in terms of successful bleaching of b-carotene. All experiment were realized in triplicate.
2.8. Antiproliferative activity Colorectal adenocarcinoma LoVo cells were cultured in RPMI1640 medium, while hepatocarcinoma cell line HepG2 and breast cancer cells MCF-7 were grown in Dulbecco's Modified Eagle's Medium (DMEM). Both media were supplemented with 1% antibiotic solution (penicillin/streptomycin), 1% L-glutamine and 10% fetal bovine serum (FBS), and incubated at 37 C under 5% CO2. Cell monolayers were subcultured onto 96 well culture plates, used for experiments 24 h later. Cytotoxicity was determined using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously reported (Conforti et al., 2011). Each experiment was performed in quadruplicate. Medium was removed and 100 mL of 0.5% w/v MTT (Sigma, Italy), dissolved in phosphate buffered saline, were added to each well. After 4 h of incubation DMSO was added to dissolve the formazan crystals. Absorbance was measured at 550 nm by means of a microplate reader (GDV DV 990 B/V, Roma, Italy). Treated and control cells were visualized using an inverted microscope (AE20 Motic; Motic Instruments, Inc., VWR, Milano, Italy) and images were captured on a VWR digital camera (VisiCam 3.0 USB camera, Milano, Italy).
2.9. Statistical analysis To evaluate the antiproliferative activity of the extracts experiments were realized with four replication. All other experiments were carried out in triplicate. Data were expressed as means ± S.E. The raw data were fitted through nonlinear regression (GraphPad Prism Software, San Diego, CA, USA) in order to obtain the IC50 parameter which indicate the dose needed to inhibit the 50% of the population. Data were checked for normality (D'Agostino-Pearson test) and tested for homogeneity of variances (Levene's test). The statistical significance of differences among group means were estimated by a one-way analysis of variance (ANOVA) followed by Tukey's posthoc test (P 0.05), using SigmaStat Software (Jantel Scientific Software, San Rafael, CA). Post-hoc comparisons of antiproliferative activity of samples at 100 mg/ml with the control group were performed using Dunnett's test.
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Table 2 Total flavonoid content of investigated plants. Plant Cod.
Sample
mg/g of extract
AR AZ AC DT MS OV PO RR RO RUB RC SV SO
Amaranthus retroflexus L. Anchusa azurea Miller Asparagus acutifolius L. Diplotaxis tenuifolia (L.) DC. Mentha spicata L. ssp. glabrata (Lej. et Court) Lebeau Origanum vulgare L. subsp. viridulum (Martin-Donos) Nyman Portulaca oleracea L. Raphanus raphanistrum L. subsp. landra (DC.) Bonnier & Layens Rosmarinus officinalis L. Rubus caesius L. Rumex conglomeratus Murray Silene vulgaris (Moench) Garcke Smirnium olusatrum L.
8.4 0.9 2.4 2.8 9.6 5.5 11.8 9.1 3.1 9.4 15.5 7.2 5.1
± ± ± ± ± ± ± ± ± ± ± ± ±
0.1d 0.1h 0.1gh 0.1g 0.4c 0.2f 0.4b 0.1c 0.1g 0.1c 0.3a 0.1e 0.1f
Values are expressed as quercetin equivalents/g of extract. Data are expressed as mean ± S.D. (n ¼ 3). Different letters indicate statistically significant differences at P < 0.05 (Tukey's test).
Fig. 1. HPTLC chromatograms of analysed samples and standards. Mobile phase: AcOEt/CH2Cl2/CH3COOH/HCOOH/H2O (100:25:10:10:11; v/v/v/v/v). A, A. azurea Miller (AZ), B, R. caesius L. (RUB); C, O. vulgare subsp. viridulum (Martin-Donos) Nyman (OV); D, A. acutifolius L. (AC); E, M. spicata ssp. glabrata (Lej et Court) Lebeau (MS); F, rutin (Rf ¼ 0.16); G, chlorogenic acid (Rf ¼ 0.35); H, catechin (Rf ¼ 0.88); I, caffeic acid (Rf ¼ 0.95); L, naringin (Rf ¼ 0.25).
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3. Results and discussion 3.1. Phytochemical content Phenolic compounds have been reported to exhibit antioxidant activity due to the reactivity of the phenol moiety and have the ability to scavenge free radicals via hydrogen donation or electron donation (Shahidi and Wanasundra, 1992). Table 2 reports the results of total flavonoids analyses. It should be emphasized that these results are estimation of total flavonoid content in their chemical equivalents (quercetin), since different compounds contribute differently to the readings using the complex flavonoidaluminum for total flavonoids results. In our previous study we have determined the amounts of total phenolics which varied widely in the different analysed extracts and ranged from 482 mg/g for R. caesius to 29 mg/g for Diplotaxis tenuifolia (Conforti et al., 2011). Among plant extracts, R. conglomeratus contained the highest amount of flavonoids (15.5 mg/g) followed by P. oleracea (11.8 mg/g), whereas the lowest level was found in Anchusa azurea (0.9 mg/g). Many studies have shown that many flavonoids and related polyphenols contribute significantly to the total antioxidant activity of many fruits and vegetables (Luo et al., 2002; Calado et al., 2015). The presence of five phenols, i.e., rutin, chlorogenic acid, catechin, caffeic acid and naringin was verified in various samples by means of HPTLC. Results are showed in Fig. 1, which reports the chromatographic profiles of investigated samples and their components identified by comparison with selected standards. Four constituents were identified in A. azurea (AZ) polar fraction: rutin, chlorogenic acid, catechin and caffeic acid. Also MS appeared to contain different phenolic compounds: caffeic acid, catechin and rutin. This last compound was also identified, to a minor extent, in AC polar fraction, together with naringin (Fig. 1D). RUB extract was demonstrated to contain chlorogenic acid and caffeic acid (Fig. 1B). Caffeic acid was also identified in OV sample. Fingerprints of investigated samples are reported in Fig. 2, where the presence of various constituents is shown by the use of corresponding standards. AZ and RUB samples are mainly constituted by chlorogenic acid, as indicated by the typical blue (in the web version) spots (Fig. 2A). Caffeic acid is clearly recognizable in AZ and OV samples and, to a minor extent, in RUB polar fraction. The flavonoid glycoside rutin, characterized by a yellow (in the web version) spot, is recognizable in AZ sample (Fig. 2A) and is also present in AC and MS samples, as evidenced in Fig. 2B at 254 nm. In this figure the spots corresponding to catechin and caffeic acid are also recognizable in MS extract, while the presence of naringin is also clear in AC. Quantification of identified phenolic compounds was performed using regression equations. As evidenced in Fig. 3, AZ sample showed the greatest amount of catechin (121.26 ± 4.36 mg/g of polar fraction), which was abundant also in MS, with an amount of 69.60 ± 1.04 mg/g of extract. A. azurea polar fraction (AZ) contained also the greatest quantity of chlorogenic acid (81.70 ± 1.60 mg/g), which was identified also in RUB sample, but to a minor extent (47.28 ± 0.95 mg/g). Caffeic acid, identified in four different fractions, was always present in concentration ranging from 20.90 ± 0.50 (OV) to 27.92 ± 0.74 mg/g (RUB sample). Regarding rutin, this flavonoid glycoside was more abundant in MS sample than in AZ and AC polar fractions (40.90 ± 1.08, 24.54 ± 0.92 and 24.26 ± 0.60 mg/g, respectively). AC sample also contained 37.72 ± 0.86 mg/g of naringin. As regard the apolar constituents of investigated samples, our previous studies dealt with the fatty acid composition and, in particular, the different content of linoleic acid and linolenic acid,
Fig. 2. HPTLC analysis of analysed samples and standards. Mobile phase: ethyl acetate/ dichloromethane/acetic acid/formic acid/water (100:25:10:10:11; v/v/v/v/v). Derivatisation: NPR. Visualisation: 366 nm (A) and 254 (B). Tracks: AZ, A. azurea Miller; RC, R. conglomeratus Murray; SV, S. vulgaris (Moench) Garcke; RUB, R. caesius L.; AR, A. retroflexus L.; OV, O. vulgare subsp. viridulum (Martin-Donos) Nyman; PO, P. oleracea L.; RO, R. officinalis L.; AC, A. acutifolius L.; DT, D. tenuifolia (L.) DC.; RR, R. raphanistrum subsp. landra (DC.) Bonnier & Layens; SO, S. olusatrum L.; MS, M. spicata ssp. Glabrata (Lej. et Court) Lebeau; 1, chlorogenic acid; 2, quercitrin; 3, catechin; 4, caffeic acid; 5, luteolin; 6, naringin; 7, rutin; 8, biapigenin.
determined by GC-MS (Conforti et al., 2011). A. retroflexus (AR) showed the highest content of linoleic acid (4.2%), while the highest content of linolenic acid (9.4%) was recognized in Raphanus
Fig. 3. Quantitative analysis of identified phenolic compounds. Data are expressed as mean ± SD (n ¼ 3).
M. Marrelli et al. / Food and Chemical Toxicology 86 (2015) 16e24
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the highest inhibition of linoleic acid oxidation (IC50 ¼ 4.91 mg/ml and 5.53 mg/ml, respectively, Table 4). A good activity was also observed for Mentha spicata L. (MS) extract, with an IC50 value of 11.24 mg/ml. These results might be related to the high content of flavonoids found in these species, as an high content of these compounds was detected in RC and MS samples, and a moderate content was find in RUB extract. Hydroalcoholic extracts from P. oleracea L. (PO), Smirnium olusatrum L. (SO), O. vulgare L. subsp. viridulum (Martin-Donos) Nyman (OV) and A. acutifolius L. (AC) showed also inhibitory activity, even if significantly lower (IC50 values ranging from 20.07 to 25.90 mg/ml). The lowest activity was exhibited by Diplotaxis tenuifolia (DT) extract (IC50 > 100 mg/ml). The antioxidant activity of the extracts decreased during the reaction time. Anyway, after 60 min incubation, the IC50 values of most active ones (R. caesius (RUB) and R. conglomeratus (RC)) were 11.1 and 13.1 mg/ml, respectively. As reference, IC50 of propyl gallate was 1 mg/ml, both after 30 and 60 min incubation.
raphanistrum subsp. landra (RR) sample. The presence of tocopherols were also evidenced in M. spicata and S. olusatrum samples. In the present study terpenes and phytosterols compositions were investigated. GC-MS analysis of the n-hexane fractions of hydroalcoholic extracts showed the presence of twenty terpenes and twelve sterols (Table 3). Among terpenes three are monoterpenes, twelve are sesquiterpenes, two are diterpenes and five are triterpenes. Rosmarinus officinalis L. (RO) contained the highest number of terpenes. Among them ketoursene (14.7%) and aristolone (11.3%) were found to be the major constituents. P. oleracea (PO) and Raphanus raphanistrum L. subsp. landra (RR) contained the highest number of sterols while R. caesius (RUB) contained the predominant phytosterol (33.7% of stigmastenol). 3.2. Antioxidant activity The antioxidant activity was determined by the b-carotene bleaching method. Inhibition of the breakdown of lipid hydroperoxides to unwanted volatile products allowed us to determine secondary antioxidants in related mechanisms. In the absence of antioxidants, oxidation products (lipid hydroperoxides, conjugated dienes and volatile byproducts) of linoleic acid simultaneously attack on b-carotene, resulting in bleaching of its characteristic yellow colour in ethanolic solution. In presence of the total extracts oxidation products were scavenged and bleaching was prevented. At the b-carotene bleaching test after 30 min incubation, R. caesius (RUB) and R. conglomeratus Murray (RC) extracts showed
3.3. Antiproliferative activity According to the criteria of the American National Cancer Institute (NCI), the IC50 values of less than 20 mg/ml, 20e100 mg/ml and more than 100 mg/ml are regarded as active (A), moderately active (MA) and inactive (IA), respectively (Homan, 1972; Skehan et al., 1990).
Table 3 Terpenes and sterols composition of the n-hexane fractions of analysed plant. Terpenesa
RTb
RAPc AC
AZ
AR
DT
MS
OV
PO
RR
RO
RUB
RC
SV
SO
1.1 0.2 0.9 0.1 0.3 0.2 0.1
e e e e e e e e e e e e e 2.4 e e e e e tr
e e e e e e e e e e e e e 0.5 e e e e e e
e e e e e e e e e e e e tr 0.9 e e e e e e
e e e e 0.9 e e e e 6.2 e e
e 7.2 e e e e e e tr 3.7 e e
e e e e e e 1.0 e e 1.8 e e
Endo-Borneol Delta-carene Endobornyl acetate a-Ylangene a-Copaene Aromadendrene Eremophilene Longifolene b-Guaiene Valencene b-Bisabolene g-Cadinene b-Selinene Neophytadiene Ferruginol Aristolone b-Amyrin Oleanene Ketoursene a-Amyrin Phytosterolsa
10.992 11.335 12.490 13.496 13.541 14.244 14.700 14.730 14.798 14.804 14.833 14.964 15.644 17.650 21.411 34.316 34.981 34.996 35.597 36.368
e e e e e e e e e e e e e e e e e e e e
e e e e e e e e e e e e e trd e e tr e e e
e e e e e e e e e e e e e 5.2 e e e e e e
e e e e e e e e e e e e e e e e tr e e e
e e e e e e e e e e e e e tr e e 1.9 tr e 2.2
e e e e tr e e tr tr tr tr tr e e e e e e e e
e e e e e e e e e e e e e 2.2 e e tr 0.8 e e
e e e e e e e e e e e e e 1.0 e e e e e 0.6
14.7 2.0
e e e e e e e e e e e e e 1.9 e e 2.9 e e 2.8
Cholestenedione Ergostatrienol Cholestenol Ergostadienol Ergostenol Norcholestadienol Stigmastadienol Dimethylcolesterol Ethylcholestenol Stigmastenol Stigmastadienone Stigmastenone
29.064 29.087 29.481 31.601 31.773 32.476 32.489 33.985 34.008 34.093 36.740 38.077
e e e e 3.1 e e e e 12.4 5.6 e
e e e e tr e tr e e tr e e
e e e e e e 5.8 e e e e e
e e e e e e e e e 1.7 1.8 e
e e e e e e e e e 3.7 e e
e e e e e e e 4.7 tr tr e e
e e e e 2.1 1.2 e e e 9.1 e 0.7
e e 0.7 0.3 2.9 e 0.4 e e 7.2 e e
1.1 e e e e e e e e tr e e
e e e e e e e e e 33.7 e e
a b c d
Compounds listed in order of elution from SE30 MS column. Retention time (as minutes). Relative area percentage (peak area relative to total peak area %). Compositional values less than 0.1% are denoted as traces.
0.1 e e 0.3 tr 0.9 0.9 11.3 2.5
22
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Table 4 Antioxidant activity of wild dietary plants. IC50 (mg/mL)
Sample
30 min AR AZ AC DT MS OV PO RR RO RUB RC SV SO
Amaranthus retroflexus L. Anchusa azurea Miller Asparagus acutifolius L. Diplotaxis tenuifolia (L.) DC. Mentha spicata L. ssp. Glabrata (Lej. et Court) Lebeau Origanum vulgare L. subsp. viridulum (Martin-Donos) Nyman Portulaca oleracea L. Raphanus raphanistrum L. subsp. landra (DC.) Bonnier & Layens Rosmarinus officinalis L. Rubus caesius L. Rumex conglomeratus Murray Silene vulgaris (Moench) Garcke Smirnium olusatrum L.
60 min e
40.19 ± 0.96 66.39 ± 0.92h 25.90 ± 0.17d >100 11.24 ± 0.27b 25.45 ± 0.23d 20.07 ± 0.34c 56.70 ± 0.94g 26.89 ± 0.29d 4.91 ± 0.09a 5.53 ± 0.68a 51.15 ± 1.94f 23.11 ± 0.31 cd
87.39 >100 51.37 >100 26.42 41.84 47.32 >100 61.39 11.10 13.11 >100 49.70
± 1.74i ± 1.94f ± 0.34d ± 0.65e ± 0.59f ± 1.14g ± 0.29b ± 0.41b ± 1.03f
Data are expressed as mean ± S. E. (n ¼ 3). Different letters along and between columns indicate statistically significant differences at P < 0.05 (Tukey's test). Propyl gallate (IC50 ¼ 1.00 ± 0.02 mg/mL) was used as positive control.
In a first phase of the study, a screening of all species was carried out, in order to highlight the most interest samples on which realize further analysis. The effects of all extracts were then verified on all cell lines at a concentration of 100 mg/ml. Obtained results are summarized in Fig. 4. Almost all samples significantly inhibited cell proliferation compared to untreated cells (Dunnett's test). The highest inhibitory activity was showed by Rosmarinus officinalis L. hydroalcoholic extract (RO), which induced 94.74% of inhibition of cell viability on LoVo cells and 63.12% on HepG2 cell lines. O. vulgare L. subsp. viridulum (Martin-Donos) Nyman (OV) also showed a good activity (66.59% on LoVo cells and 44.86% on breast cancer cell line MCF-7). Samples SV, AR, DT and RUB were able to induce an inhibitory activity higher than 40%. Particularly, Silene vulgaris extract against LoVo and HepG2 cells caused a percent of inhibition of 48 and 44 at a concentration of 100 mg/ml, respectively (Fig. 4). Diplotaxis tenuifolia (L.) DC., Mentha spicata L. ssp. glabrata (Lej. et Court) Lebeau and Smirnium olusatrum L. samples were able to affect cell viabilty of two cell lines utilized, while only one line was affected by A. retroflexus L. and P. oleracea L. A. acutifolius L. sample showed no activity. Overall, HepG2 and LoVo cell lines were more responsive compared to MCF-7 cells. Samples that at a concentration of 100 mg/ml induced a
percentage of inhibition higher than 50% were tested at different concentrations (100e2.5 mg/ml) in order to determine IC50 values. After 48 h of incubation the most antiproliferative plant extract was rosemary (Rosmarinus officinalis L.) on LoVo cell lines with an IC50 of 16.60 mg/ml (Fig. 5a). This species showed antiproliferative activity on hepatocarcinoma cell line HepG2 (IC50 ¼ 83.44 mg/ml, Fig. 5b). Oregano (O. vulgare subsp. viridulum) showed a selective antiproliferative activity on hepatic cancer with IC50 of 32.59 mg/ml (Fig. 5c). The same sample showed also weak activity against MCF-7 cells with a percent of inhibition of 45 at a concentration of 100 mg/ ml. Changes in treated cells viability were visualized using an inverted microscope and captured on a VWR digital camera. As evidenced in Fig. 6, the incubation of cell cultures in the presence of a concentration of 100 mg/mL of each sample significantly affected cell viability. In this study, MTT assay was used to provide qualitative preliminary data on the antiproliferative activity of edible plant extracts. This activity is thought to be related to the antioxidant properties of the extracts; several works on natural antioxidants in vitro activities against cancer cells have been cited in literature s-Linares et al., 2015; Eberhardt et al., 2000; Kaur et al., 2014; (Borra Pacifico et al., 2012; Erol et al., 2012). However, some scientific
Fig. 4. Inhibition of cell proliferation induced by samples at 100 mg/ml on MCF-7, HepG2 and LoVo cell lines. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control (Dunnett's test).
M. Marrelli et al. / Food and Chemical Toxicology 86 (2015) 16e24
23
Fig. 5. Dose depending effect of rosemary (RO) and oregano (OV) extracts on the proliferation of cancer cells. a) Effect of Rosmarinus officinalis L. on LoVo cell line; b) effect of rosemary on HepG2 cells; c) effect of Origanum vulgare L. subsp. viridulum on HepG2 cell viability.
Fig. 6. Changes in cell viability induced by OV and RO samples on HepG2 and cell lines. a) HepG2 cell line, control, cells in EtOH 70% (0.5%, v/v), without sample; b) HepG2 cell line, OV sample, 100 mg/ml; c) HepG2 cells, RO sample, 100 mg/ml; d) LoVo cell line, control cells in EtOH 70% (0.5%, v/v), without sample; e) LoVo cells, RO sample, 100 mg/ml.
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reports have highlighted the fact that there might be an interference between some antioxidant molecules and the MTT assay. Wang et al. (2010) reported that it could be possible that polyphenols present in green tea may interfere with formazan formation, critical to the MTT assay, by changing the activity of succinate dehydrogenase or interact with MTT directly. Their results indicate that using MTT assay in presence of ECGC (green tea polyphenol) could underestimate the antiproliferative activity of the extract and show more viable cells but not in presence of apigenin (flavonoid). Furthermore, this possible interference could be considered when the half maximal inhibitory concentration (IC50) has to be estimated not for the simple assessment of qualitative antiproliferative activity. 4. Conclusion Natural products play a leading role in the discovery and the development of drugs for the treatment of human diseases. The discovery of the anticancer activities of so many traditional medicines and natural products need to be supported by scientific evidence and validation. More importantly, natural products as a complex polychemical mixture of pharmacologically active compounds may target multiple vulnerabilities of cancer cells, without toxicity to the noncancerous cells. The data presented in this study demonstrated that almost all the reported species possess antioxidant activity. The observed in vitro antioxidant activity suggest that the investigated plant extracts could exert effects also against human cancer proliferation. With this respect, the most promising plants appear to be O. vulgare L. subsp. viridulum and Rosmarinus officinalis L. Further studies are needed for these plants, to obtain the isolation and structural elucidation of bioactive compounds and about their in vivo toxic effects in experimental animals to formulate a new drug for regular practice. In conclusion, this work reveals that the Italian flora can be an interesting source of anti-proliferative and antioxidant principles, with a potential use in pharmaceutical fields and demonstrates for the first time the selective cytotoxic capacity of O. vulgare L. subsp. viridulum and Rosmarinus officinalis L., confirming their medicinal plant feature. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2015.09.011. References s-Linares, I., Pe rez-Sa nchez, A., Lozano-Sa nchez, J., Barrajo n-Catala n, E., Borra ez-Rom Arra an, D., Cifuentes, A., Micol, V., Carretero, A.S., 2015. A bioguided identification of the active compounds that contribute to the antiproliferative/ cytotoxic effects of rosemary extract on colon cancer cells. Food Chem. Toxicol. 80, 215e222. ~o, P.A., de Oliveira, E.A., Letra, M.H.S., Sawaya, A.C.H.F., Calado, J.C.P., Alberta Marcucci, M.C., 2015. Flavonoid contents and antioxidant activity in fruit, vegetables and other types of food. Agric. Sci. 6, 426e435. Chen, C.C., Liu, L.K., Hsu, J.D., Huang, H.P., Yang, M.Y., Wang, C.J., 2005. Mulberry extract inhibits the development of atherosclerosis in cholesterol-fed rabbits. Food Chem. 91, 601e607. Conforti, F., Marrelli, M., Colica, C., Menichini, F., Perri, V., Uzunov, D., Statti, G.A., Duez, P., Menichini, F., 2011. Bioactive phytonutrients (omega fatty acids, tocopherols, polyphenols), in vitro inhibition of nitric oxide production and free
radical scavenging activity of non-cultivated Mediterranean vegetables. Food Chem. 129, 1413e1419. Conforti, F., Sosa, S., Marrelli, M., Menichini, F., Statti, G.A., Uzunov, D., Tubaro, A., Menichini, F., Della Loggia, R., 2008. In vivo anti-inflammatory and in vitro antioxidant activities of Mediterranean dietary plants. J. Ethnopharmacol. 116, 144e151. Eberhardt, M.V., Lee, C.Y., Liu, R.H., 2000. Antioxidant activity of fresh apples. Nature 405, 903e904. Erol, O., Arda, A., Erdem, G., 2012. Phenols of virgin olive oil protects nuclear DNA against oxidative damage in HeLa cells. Food Chem. Toxicol. 50, 3475e3479. Galasso, S., Pacifico, S., Kretschmer, N., Pan, S.P., Marciano, S., Piccolella, S., Monaco, P., Bauer, R., 2014. Influence of seasonal variation on Thymus longicaulis C. Presl chemical composition and its antioxidant and anti-inflammatory properties. Phytochemistry 107, 80e90. Homan, E.R., 1972. Quantitative relationships between toxic doses of antitumor chemotherapeutic agents in animals and man. Cancer Chemother. Rep. 3, 13e19. €hko €nen, M.P., Hopia, A.I., Vuorela, H.J., Rauha, J.P., Pihlaja, K., Kujala, T.S., Ka Heinonen, M., 1999. Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem. 47, 3954e3962. Kaur, P., Kaur, V., Kumar, M., Kaur, S., 2014. Suppression of SOS response in E. coli PQ 37, antioxidant potential and antiproliferative action of methanolic extract of Pteris vittata L. on human MCF-7 breast cancer cells. Food Chem. Toxicol. 74, 326e333. Kris-Etherton, P.M., Lefevre, M., Beecher, G.R., Gross, M.D., Keen, C.L., Etherton, T.D., 2004. Bioactive compounds in nutrition and health-research methodologies for establishing biological function: the antioxidant and anti-inflammatory effects of flavonoids on atherosclerosis. Annu. Rev. Nutr. 24, 511e538. Luo, X.D., Basile, M.J., Kennelly, E.J., 2002. Polyphenolic antioxidants from the fruits of Chrysophyllum crinito L. (star apple). J. Agric. Food Chem. 50, 1379e1382. Marrelli, M., Menichini, F., Statti, G.A., Bonesi, M., Duez, P., Menichini, F., Conforti, F., 2012. Changes in the phenolic and lipophilic composition, in the enzyme inhibition and antiproliferative activity of Ficus carica L. cultivar Dottato fruits during maturation. Food Chem. Toxicol. 50, 726e733. Marrelli, M., Loizzo, M.R., Nicoletti, M., Menichini, F., Conforti, F., 2013. Inhibition of key enzymes linked to obesity by preparations from mediterranean dietary plants: effects on a-amylase and pancreatic lipase activities. Plant Foods Hum. Nutr. 68, 340e346. Marrelli, M., Conforti, F., Toniolo, C., Nicoletti, M., Statti, G., Menichini, F., 2014. Hypericum perforatum: influences of the habitat on chemical composition, photo-induced cytotoxicity, and antiradical activity. Pharm. Biol. 52, 909e918. Mastron, J.K., Siveen, K.S., Sethi, G., Bishayee, A., 2015. Silymarin and hepatocellular carcinoma: a systematic, comprehensive, and critical review. Anticancer Drugs 26, 475e486. http://dx.doi.org/10.1097/CAD.0000000000000211. Jan 19. Mollazadeh, H., Hosseinzadeh, H., 2014. The protective effect of Nigella sativa against liver injury: a review. Iran. J. Basic Med. Sci. 17, 958e966. Ovadje, P., Roma, A., Steckle, M., Nicoletti, L., Arnason, J.T., Pandey, S., 2015. Advances in the research and development of natural health products as main stream cancer therapeutics. Evid. Based Complement. Altern. Med. 2015 http:// dx.doi.org/10.1155/2015/751348. ID 751348. Pacifico, S., Gallicchio, M., Fiorentino, A., Fischer, A., Meyer, U., 2012. Antioxidant properties and cytotoxic effects on human cancer cell lines of aqueous fermented and lipophilic quince (Cydoniaoblonga Mill.) preparations. Food Chem. Toxicol. 50, 4130e4135. Petiwala, S.M., Berhe, S., Li, G., Puthenveetil, A.G., Rahman, O., Nonn, L., Johnson, J.J., 2014. Rosemary (Rosmarinus officinalis) extract modulates CHOP/GADD153 to promote androgen receptor degradation and decreases xenograft tumor growth. PLoS One 9, e89772. Saleem, A., Husheem, M., Harkonen, P., Pihlaja, K., 2002. Inhibition of cancer cell growth by crude extract and the phenolics of Terminalia chebula retz. fruit. J. Ethnopharmacol. 81, 327e336. Shahidi, F., Wanasundra, P.K., 1992. Phenolic antioxidants. Crit. Rev. Food Sci. 32, 67e103. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Varren, J.T., Bokesch, H., Kenney, S., Boyd, M., 1990. New colorimetric cytotoxicity assay for anticancerdrug screening. J. Natl. Cancer Inst. 82, 1107e1112. Soobrattee, M.A., Neergheen, V.S., Luximon-Ramma, A., Aruoma, O.I., Bahorun, T., 2005. Phenolics as potential antioxidant therapeutic agents: mechanism and actions. Mutat. Res. 579, 200e213. Velioglu, Y.S., Mazza, G., Gao, L., Oomah, B.D., 1998. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. J. Agric. Food Chem. 46, 4113e4117. Wang, P., Henning, S.M., Heber, D., 2010. Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols. PLoS One 5 (4), e10202.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Inhibitory effects of wild dietary plants on lipid peroxidation and on the proliferation of human cancer cells Mariangela Marrelli, Brigida Cristaldi, Francesco Menichini, Filomena Conforti* Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, I-87036 Rende (CS), Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 May 2015 Received in revised form 4 September 2015 Accepted 20 September 2015 Available online 25 September 2015
Thirteen hydroalcoholic extracts of edible plants from Southern Italy were evaluated for their in vitro antioxidant and antiproliferative activity on three human cancer cell lines: breast cancer MCF-7, hepatic cancer HepG2 and colorectal cancer LoVo. After 48 h of incubation the most antiproliferative plant extract was rosemary (Rosmarinus officinalis L.) on LoVo cell line with IC50 of 16.60 mg/ml. Oregano (Origanum vulgare L. subsp. viridulum) showed a selective antiproliferative activity on hepatic cancer with IC50 of 32.59 mg/ml. All the extracts, with the exception of Diplotaxis tenuifolia (L.) DC., exerted antioxidant properties, the most active plants being dewberry (Rubus caesius L.) and “laprista” (Rumex conglomerates Murray) with IC50 of 4.91 and 5.53 mg/ml, respectively. Rumex conglomeratus contained the highest amount of flavonoids (15.5 mg/g) followed by Portulaca oleracea L. (11.8 mg/g). Rosmarinus officinalis contained the highest number of terpenes. Among them ketoursene (14.7%) and aristolone (11.3%) were found to be the major constituents. P. oleracea and Raphanus raphanistrum L. subsp. landra contained the highest number of sterols. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Antioxidant activity Antiproliferative activity Flavonoid content Terpene and sterol composition
1. Introduction Cancer is a growing public problem whose estimated worldwide new incidence is about 6 million cases per year. It is the second major cause of deaths after cardiovascular diseases. It became a general conviction that dietary agents and environmental factors (radiation, sunshine, hormones, viruses, bacteria, and chemicals) have a real impact, either positive or negative, on cancer development affecting the proliferation, angiogenesis and metastasis. Investigations about natural products have recently regained
Abbreviations: AC, Asparagus acutifolius L.; AR, Amaranthus retroflexus L.; AZ, Anchusa azurea Mill.; DT, Diplotaxis tenuifolia (L.) DC.; GC-MS, gas chromatographymass spectrometry; HepG2, hepatic cancer cell line; HPTLC, high performance thin layer chromatography; LoVo, colorectal cancer cells; MCF-7, breast cancer cell line; MS, Mentha spicata L. ssp glabrata (Lej. et Court.) Lebeau; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NPR, natural product reagent; OV, Origanum vulgare L. subsp. viridulum (Martin-Donos) Nyman; PO, Portulaca oleracea L.; RC, Rumex conglomeratus Murray; RO, Rosmarinus officinalis L.; RR, Raphanus raphanistrum L. subsp. landra (DC.) Bonnier & Layens; RUB, Rubus caesius L.; SO, Smyrnium olusatrum L; SV, Silene vulgaris (Moench) Garcke. * Corresponding author. E-mail address: fi[email protected] (F. Conforti). http://dx.doi.org/10.1016/j.fct.2015.09.011 0278-6915/© 2015 Elsevier Ltd. All rights reserved.
prominence with the increasing understanding of their biological significance and increasing recognition of the origin and function of their structural diversity. Plant extracts and derivatives have always been used for various disease treatments and many anticancer agents issued from plants and vegetables are clinically recognized and used all over the world. Currently, more than 50% of all approved drugs (not limited to cancer) are natural products and their derivatives (Ovadje et al., 2015). Over several decades, many studies have shown numerous dietary constituents and nutraceuticals as cancer chemopreventive agents and in the recent past a number of reports have elucidated the cellular mechanisms of prevention and suppression of cancer progression. Anticarcinogenic effects of many plants derivatives, by inhibiting cancer cell proliferation in vitro, were assessed in many scientific reports (Galasso et al., 2014; Petiwala et al., 2014; Mastron et al., 2015). It is commonly accepted that in a situation of oxidative stress, reactive oxygen species (ROS) such as superoxide (O 2 , OOH ), hy droxyl (OH ) and peroxyl (ROO ) radicals are generated. It's known that when the antioxidant control mechanisms are overrun, the cellular redox potential shifts toward oxidative stress. As a
M. Marrelli et al. / Food and Chemical Toxicology 86 (2015) 16e24
consequence, the potential for damaging cellular nucleic acids, lipids, and proteins increases, which is associated to carcinogenesis (Kris-Etherton et al., 2004; Mollazadeh and Hosseinzadeh, 2014). Cancer cells exhibit increased ROS generation that may promote cell proliferation. Antioxidant capacity of some phytochemicals like polyphenols, flavonoids and carotenoids may be involved in cancer €hko € nen et al., prevention by combating oxidative cell damages (Ka 1999; Soobrattee et al., 2005; Velioglu et al., 1998). Epidemiological and experimental studies reveal a negative correlation between the consumption of diets rich in fruit and vegetables and the risks for chronic diseases, such as cardiovascular diseases, arthritis, chronic inflammation and cancers (Chen et al., 2005; Saleem et al., 2002). The present study was undertaken to evaluate the potential cytotoxic activity on cancer cell lines and estimate the antioxidant and protective effect of the following Mediterranean plants: Amaranthus retroflexus L., Anchusa azurea Mill., Asparagus acutifolius L., Diplotaxis tenuifolia (L.) DC., Mentha spicata L. ssp glabrata (Lej. et Court.) Lebeau, Origanum vulgare L. subsp. viridulum (MartinDonos) Nyman, Portulaca oleracea L., Raphanus raphanistrum L. subsp. landra (DC.) Bonnier & Layens, Rosmarinus officinalis L., Rubus caesius L., Rumex conglomeratus Murray, Silene vulgaris (Moench) Garcke, Smyrnium olusatrum L. They are spontaneous edible plants present in the Southern of Italy, a territory characterized by different vegetation belt from the sea level to the highest peak of Sila Mt. (about 1800 m a.s.l.), mostly rich in Mediterranean elements. In this work, 13 extracts obtained from the Italian plants listed before were studied to assess their antioxidant activities and antiproliferative properties against a panel of human cancer cells which, to the best of our knowledge, is not reported for these plants so far. The b-carotene bleaching test was used to evaluate the antioxidant activity. Antiproliferative activity was evaluated against three different human cancer cell lines: breast (MCF-7), hepatic (HepG2) and colorectal (LoVo) cancer cells. Furthermore, the total flavonoid content was determined by a method based on the formation of complex flavonoid-aluminium, the qualitative and quantitative evaluation of some phenolic compounds was determined by HPTLC and the terpene and sterol composition was determined by GC-MS analysis. 2. Materials and methods 2.1. Plant materials The different species studied in this work are reported in Table 1.
17
The collected plants were authenticated by Dr. Uzunov from Botanic Garden, University of Calabria, Italy, and the plants were deposited at the Natural History Museum of Calabria. Voucher numbers are also indicated in Table 1.
2.2. Chemicals Methanol, ethanol, H2SO4, n-hexane, ethyl acetate, dichloromethane and DMSO were obtained from VWR International s.r.l. (Milan, Italy). Normal phase glass plates 20 cm 10 cm with glassbacked layers silica gel 60 (2 mm thickness) were purchased from Merck (Darmstadt, Germany). Phosphate buffered saline (PBS), RPMI 1640 medium, DMEM medium, fetal bovine serum, L-glutamine, penicillin/streptomycin, trypan blue, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT), propyl gallate, bcarotene, linoleic acid, Tween 20, aluminium chloride and reference substances utilised in HPTLC analyses were obtained from SigmaAldrich S.p.A. (Milan, Italy). Breast cancer cells MCF-7, hepatic cancer cell line HepG2 and colorectal adenocarcinoma cancer cells LoVo were purchased from ATCC no. HTB-22, no. HB-8065 and no. CCL-229, UK. All other reagents, of analytical grade, were Carlo Erba products (Milan, Italy).
2.3. Preparation of samples As previously described (Conforti et al., 2011) the plant material, after dried, was extracted with 70% aqueous EtOH (3 l) through maceration at room temperature. Total flavonoid content was determined for each total extract. Extracts were then partitioned between 100 ml 90% MeOH and n-hexane (100 ml 3 times), and the n-hexane fractions were analysed by gas-chromatographymass spectrometry (GC-MS) in order to identify the terpene and sterol composition. The qualitative and quantitative evaluation of some phenolic compounds was determined by HPTLC. 2.4. Determination of total flavonoid content Total flavonoid content was estimated using a colorimetric method based on the formation of a complex flavonoid-aluminum, having the absorptivity maximum at 430 nm (Conforti et al., 2008). All determinations were made in triplicate and values were calculated from a calibration curve obtained with quercetin, chosen as standard. Final results were expressed as mg of quercetin equivalent per g of extract.
Table 1 Investigated wild dietary plants. Plant Cod.
Botanical name
Family
Plant part
Use
Voucher number
AR AZ AC
Amaranthus retroflexus L. Anchusa azurea Miller Asparagus acutifolius L.
Amaranthaceae Boraginaceae Asparagaceae
Leaves Flowers Stems
21793 21795 21802
DT MS OV
Diplotaxis tenuifolia (L.) DC. Brassicaceae Mentha spicata L. ssp. glabrata (Lej. et Court) Lebeau Lamiaceae Origanum vulgare L. subsp. viridulum (Martin-Donos) Nyman Lamiaceae
Mixed, soup, boil fries Mixed salad, soup, boil, fries Salad of shoots, fries with eggs, under oil, risotto Salad, soup, boil Spice, aromatic, liqueur, salse Spice, condiment, on roast, liqueur
21789 21801 21798
PO RR
Portulaca oleracea L. Raphanus raphanistrum L. subsp. landra (DC.) Bonnier & Layens Rosmarinus officinalis L. Rubus caesius L. Rumex conglomeratus Murray Silene vulgaris (Moench) Garcke Smirnium olusatrum L.
Mixed salad, boil Like spinach, deep-dish pizza
21796 21799
Aromatic, spice, aromatic, on roast meat Boil Potage, soup Salad, risotto, fries with eggs Mixed salad, soup
21800 21791 21794 21792 21803
RO RUB RC SV SO
Portulacaceae Brassicaceae
Leaves Leaves Stems, leaves Leaves Leaves
Lamiaceae Rosaceae Polygonaceae Caryophyillaceae Apiaceae
Leaves Leaves Leaves Leaves Leaves
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2.5. GC/MS analysis The n-hexane fractions were analysed by gas chromatographymass spectrometry (GC-MS). A Hewlett-Packard 6890 gas chromatograph equipped with an SE-30 capillary column (100% dimethylpolysiloxane, 30 m length, 0.25 mm in diameter, 0.25 mm film thickness) directly coupled to a selective mass detector (model 5973, Hewlett Packard) was used. Analyses were realized using a programmed temperature from 60 to 280 C (rate 16 min1) and helium as carrier gas. Identification of compounds was based on the comparison of the GC retention factors with those of standards and the comparison of the mass spectra with those present in the Wiley 138 library data of the GC-MS system (Marrelli et al., 2012). 2.6. HPTLC analysis 2.6.1. Apparatus and chromatographic procedure High Performance Thin Layer Chromatography (HPTLC) was chosen to identify phenolic compounds (Marrelli et al., 2014). Normal phase glass plates 20 10 cm (VWR International s.r.l., Milano, Italy) with glass backed layers silica gel 60 (2 mm thickness) were used. Plates were prewashed with methanol and carefully dried for 3 min at 100 C. A CAMAG Linomat 5 sample applicator (Muttenz, Switzerland) connected to a nitrogen tank and to a CAMAG TLC Visualizer was utilized. Syringe delivery speed was 150 nl/s and injection volume was 1 ml. The HPTLC plates were developed with the mobile phase ethyl acetate/dichloromethane/ acetic acid/formic acid/water (100:25:10:10:11, v/v/v/v/v) for the detection of chlorogenic and caffeic acid, quercitrin, catechin, luteolin, naringin, rutin and biapigenin. For the identification of quercetin, kaempferol, ferulic acid and coumaric acid the mobile phase was instead ethyl acetate/ dichloromethane/acetic acid/formic acid/water (100:31.25: 1.25:1.25:1.25, v/v/v/v/v). Band width was 8 mm; distance from bottom and solvent front position were 8 mm and 9 mm, respectively. NPR (1 g diphenylborinic acid aminoethylester in 200 ml of ethyl acetate) and anhysaldehyde (1.5 ml p-anisaldehyde, 2.5 ml H2SO4, 1 ml AcOH in 37 ml EtOH) were used for postchromatographic derivatization. All treated plates were inspected under a UV light at 254 or 366 nm or under white light upper and lower (WRT) before and after derivatization by means of a Camag TLC visualizer. All analyses were carried out in triplicate. 2.6.2. Preparation of samples and standard solutions For the determination of phenolic compounds, the polar fraction of each extract was dissolved in methanol to have a final concentration of 50 mg/ml. This extract was used for TLC fingerprinting and co-chromatography with marker compounds. Sample solutions were applied in triplicate on the TLC plates. Standards have been used at a concentration of 3 mg/ml in methanol (or ethanol for caffeic acid, luteolin, naringin, kaempferol and coumaric acid) for qualitative determinations. For quantitative analyses, working stock solutions were prepared by dilution with methanol (or ethanol for caffeic acid) to give final concentrations of 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0 mg/ml. Standard solutions of each compound were spotted on HPTLC plate to give absolute amounts of 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0 mg/ band. 2.6.3. Quantitative analyses In order to realize quantitative analyses, calibration curves were prepared using absolute amount (mg/band) as independent variable (X) and the peak area of standards as dependent variable (Y) (Marrelli et al., 2013). Quantification of compounds was performed
using regression equations (correlation coefficients R2 > 0.98). Analyses were performed using GraphPad Prism Software (San Diego, CA, USA).
2.7. Antioxidant assay Antioxidant activity was determined by means of the b-carotene bleaching test (Marrelli et al., 2012). In brief, 0.2 ml of samples in methanol at different concentrations (100, 50, 25, 10, 5 and 1 mg/ml) were added to 5 ml of an emulsion of b-carotene and linoleic acid. Tubes were then placed in a water bath at 45 C. The absorbance was measured at 470 nm using a Perkin Elmer Lambda 40 UV/VIS spectrophotometer against a blank, consisting of an emulsion without b-carotene. The measurement was carried out at initial time (t ¼ 0) and successively at 30 and 60 min. Propyl gallate was used as positive control. The antioxidant activity (AA) was measured in terms of successful bleaching of b-carotene. All experiment were realized in triplicate.
2.8. Antiproliferative activity Colorectal adenocarcinoma LoVo cells were cultured in RPMI1640 medium, while hepatocarcinoma cell line HepG2 and breast cancer cells MCF-7 were grown in Dulbecco's Modified Eagle's Medium (DMEM). Both media were supplemented with 1% antibiotic solution (penicillin/streptomycin), 1% L-glutamine and 10% fetal bovine serum (FBS), and incubated at 37 C under 5% CO2. Cell monolayers were subcultured onto 96 well culture plates, used for experiments 24 h later. Cytotoxicity was determined using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously reported (Conforti et al., 2011). Each experiment was performed in quadruplicate. Medium was removed and 100 mL of 0.5% w/v MTT (Sigma, Italy), dissolved in phosphate buffered saline, were added to each well. After 4 h of incubation DMSO was added to dissolve the formazan crystals. Absorbance was measured at 550 nm by means of a microplate reader (GDV DV 990 B/V, Roma, Italy). Treated and control cells were visualized using an inverted microscope (AE20 Motic; Motic Instruments, Inc., VWR, Milano, Italy) and images were captured on a VWR digital camera (VisiCam 3.0 USB camera, Milano, Italy).
2.9. Statistical analysis To evaluate the antiproliferative activity of the extracts experiments were realized with four replication. All other experiments were carried out in triplicate. Data were expressed as means ± S.E. The raw data were fitted through nonlinear regression (GraphPad Prism Software, San Diego, CA, USA) in order to obtain the IC50 parameter which indicate the dose needed to inhibit the 50% of the population. Data were checked for normality (D'Agostino-Pearson test) and tested for homogeneity of variances (Levene's test). The statistical significance of differences among group means were estimated by a one-way analysis of variance (ANOVA) followed by Tukey's posthoc test (P 0.05), using SigmaStat Software (Jantel Scientific Software, San Rafael, CA). Post-hoc comparisons of antiproliferative activity of samples at 100 mg/ml with the control group were performed using Dunnett's test.
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Table 2 Total flavonoid content of investigated plants. Plant Cod.
Sample
mg/g of extract
AR AZ AC DT MS OV PO RR RO RUB RC SV SO
Amaranthus retroflexus L. Anchusa azurea Miller Asparagus acutifolius L. Diplotaxis tenuifolia (L.) DC. Mentha spicata L. ssp. glabrata (Lej. et Court) Lebeau Origanum vulgare L. subsp. viridulum (Martin-Donos) Nyman Portulaca oleracea L. Raphanus raphanistrum L. subsp. landra (DC.) Bonnier & Layens Rosmarinus officinalis L. Rubus caesius L. Rumex conglomeratus Murray Silene vulgaris (Moench) Garcke Smirnium olusatrum L.
8.4 0.9 2.4 2.8 9.6 5.5 11.8 9.1 3.1 9.4 15.5 7.2 5.1
± ± ± ± ± ± ± ± ± ± ± ± ±
0.1d 0.1h 0.1gh 0.1g 0.4c 0.2f 0.4b 0.1c 0.1g 0.1c 0.3a 0.1e 0.1f
Values are expressed as quercetin equivalents/g of extract. Data are expressed as mean ± S.D. (n ¼ 3). Different letters indicate statistically significant differences at P < 0.05 (Tukey's test).
Fig. 1. HPTLC chromatograms of analysed samples and standards. Mobile phase: AcOEt/CH2Cl2/CH3COOH/HCOOH/H2O (100:25:10:10:11; v/v/v/v/v). A, A. azurea Miller (AZ), B, R. caesius L. (RUB); C, O. vulgare subsp. viridulum (Martin-Donos) Nyman (OV); D, A. acutifolius L. (AC); E, M. spicata ssp. glabrata (Lej et Court) Lebeau (MS); F, rutin (Rf ¼ 0.16); G, chlorogenic acid (Rf ¼ 0.35); H, catechin (Rf ¼ 0.88); I, caffeic acid (Rf ¼ 0.95); L, naringin (Rf ¼ 0.25).
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3. Results and discussion 3.1. Phytochemical content Phenolic compounds have been reported to exhibit antioxidant activity due to the reactivity of the phenol moiety and have the ability to scavenge free radicals via hydrogen donation or electron donation (Shahidi and Wanasundra, 1992). Table 2 reports the results of total flavonoids analyses. It should be emphasized that these results are estimation of total flavonoid content in their chemical equivalents (quercetin), since different compounds contribute differently to the readings using the complex flavonoidaluminum for total flavonoids results. In our previous study we have determined the amounts of total phenolics which varied widely in the different analysed extracts and ranged from 482 mg/g for R. caesius to 29 mg/g for Diplotaxis tenuifolia (Conforti et al., 2011). Among plant extracts, R. conglomeratus contained the highest amount of flavonoids (15.5 mg/g) followed by P. oleracea (11.8 mg/g), whereas the lowest level was found in Anchusa azurea (0.9 mg/g). Many studies have shown that many flavonoids and related polyphenols contribute significantly to the total antioxidant activity of many fruits and vegetables (Luo et al., 2002; Calado et al., 2015). The presence of five phenols, i.e., rutin, chlorogenic acid, catechin, caffeic acid and naringin was verified in various samples by means of HPTLC. Results are showed in Fig. 1, which reports the chromatographic profiles of investigated samples and their components identified by comparison with selected standards. Four constituents were identified in A. azurea (AZ) polar fraction: rutin, chlorogenic acid, catechin and caffeic acid. Also MS appeared to contain different phenolic compounds: caffeic acid, catechin and rutin. This last compound was also identified, to a minor extent, in AC polar fraction, together with naringin (Fig. 1D). RUB extract was demonstrated to contain chlorogenic acid and caffeic acid (Fig. 1B). Caffeic acid was also identified in OV sample. Fingerprints of investigated samples are reported in Fig. 2, where the presence of various constituents is shown by the use of corresponding standards. AZ and RUB samples are mainly constituted by chlorogenic acid, as indicated by the typical blue (in the web version) spots (Fig. 2A). Caffeic acid is clearly recognizable in AZ and OV samples and, to a minor extent, in RUB polar fraction. The flavonoid glycoside rutin, characterized by a yellow (in the web version) spot, is recognizable in AZ sample (Fig. 2A) and is also present in AC and MS samples, as evidenced in Fig. 2B at 254 nm. In this figure the spots corresponding to catechin and caffeic acid are also recognizable in MS extract, while the presence of naringin is also clear in AC. Quantification of identified phenolic compounds was performed using regression equations. As evidenced in Fig. 3, AZ sample showed the greatest amount of catechin (121.26 ± 4.36 mg/g of polar fraction), which was abundant also in MS, with an amount of 69.60 ± 1.04 mg/g of extract. A. azurea polar fraction (AZ) contained also the greatest quantity of chlorogenic acid (81.70 ± 1.60 mg/g), which was identified also in RUB sample, but to a minor extent (47.28 ± 0.95 mg/g). Caffeic acid, identified in four different fractions, was always present in concentration ranging from 20.90 ± 0.50 (OV) to 27.92 ± 0.74 mg/g (RUB sample). Regarding rutin, this flavonoid glycoside was more abundant in MS sample than in AZ and AC polar fractions (40.90 ± 1.08, 24.54 ± 0.92 and 24.26 ± 0.60 mg/g, respectively). AC sample also contained 37.72 ± 0.86 mg/g of naringin. As regard the apolar constituents of investigated samples, our previous studies dealt with the fatty acid composition and, in particular, the different content of linoleic acid and linolenic acid,
Fig. 2. HPTLC analysis of analysed samples and standards. Mobile phase: ethyl acetate/ dichloromethane/acetic acid/formic acid/water (100:25:10:10:11; v/v/v/v/v). Derivatisation: NPR. Visualisation: 366 nm (A) and 254 (B). Tracks: AZ, A. azurea Miller; RC, R. conglomeratus Murray; SV, S. vulgaris (Moench) Garcke; RUB, R. caesius L.; AR, A. retroflexus L.; OV, O. vulgare subsp. viridulum (Martin-Donos) Nyman; PO, P. oleracea L.; RO, R. officinalis L.; AC, A. acutifolius L.; DT, D. tenuifolia (L.) DC.; RR, R. raphanistrum subsp. landra (DC.) Bonnier & Layens; SO, S. olusatrum L.; MS, M. spicata ssp. Glabrata (Lej. et Court) Lebeau; 1, chlorogenic acid; 2, quercitrin; 3, catechin; 4, caffeic acid; 5, luteolin; 6, naringin; 7, rutin; 8, biapigenin.
determined by GC-MS (Conforti et al., 2011). A. retroflexus (AR) showed the highest content of linoleic acid (4.2%), while the highest content of linolenic acid (9.4%) was recognized in Raphanus
Fig. 3. Quantitative analysis of identified phenolic compounds. Data are expressed as mean ± SD (n ¼ 3).
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21
the highest inhibition of linoleic acid oxidation (IC50 ¼ 4.91 mg/ml and 5.53 mg/ml, respectively, Table 4). A good activity was also observed for Mentha spicata L. (MS) extract, with an IC50 value of 11.24 mg/ml. These results might be related to the high content of flavonoids found in these species, as an high content of these compounds was detected in RC and MS samples, and a moderate content was find in RUB extract. Hydroalcoholic extracts from P. oleracea L. (PO), Smirnium olusatrum L. (SO), O. vulgare L. subsp. viridulum (Martin-Donos) Nyman (OV) and A. acutifolius L. (AC) showed also inhibitory activity, even if significantly lower (IC50 values ranging from 20.07 to 25.90 mg/ml). The lowest activity was exhibited by Diplotaxis tenuifolia (DT) extract (IC50 > 100 mg/ml). The antioxidant activity of the extracts decreased during the reaction time. Anyway, after 60 min incubation, the IC50 values of most active ones (R. caesius (RUB) and R. conglomeratus (RC)) were 11.1 and 13.1 mg/ml, respectively. As reference, IC50 of propyl gallate was 1 mg/ml, both after 30 and 60 min incubation.
raphanistrum subsp. landra (RR) sample. The presence of tocopherols were also evidenced in M. spicata and S. olusatrum samples. In the present study terpenes and phytosterols compositions were investigated. GC-MS analysis of the n-hexane fractions of hydroalcoholic extracts showed the presence of twenty terpenes and twelve sterols (Table 3). Among terpenes three are monoterpenes, twelve are sesquiterpenes, two are diterpenes and five are triterpenes. Rosmarinus officinalis L. (RO) contained the highest number of terpenes. Among them ketoursene (14.7%) and aristolone (11.3%) were found to be the major constituents. P. oleracea (PO) and Raphanus raphanistrum L. subsp. landra (RR) contained the highest number of sterols while R. caesius (RUB) contained the predominant phytosterol (33.7% of stigmastenol). 3.2. Antioxidant activity The antioxidant activity was determined by the b-carotene bleaching method. Inhibition of the breakdown of lipid hydroperoxides to unwanted volatile products allowed us to determine secondary antioxidants in related mechanisms. In the absence of antioxidants, oxidation products (lipid hydroperoxides, conjugated dienes and volatile byproducts) of linoleic acid simultaneously attack on b-carotene, resulting in bleaching of its characteristic yellow colour in ethanolic solution. In presence of the total extracts oxidation products were scavenged and bleaching was prevented. At the b-carotene bleaching test after 30 min incubation, R. caesius (RUB) and R. conglomeratus Murray (RC) extracts showed
3.3. Antiproliferative activity According to the criteria of the American National Cancer Institute (NCI), the IC50 values of less than 20 mg/ml, 20e100 mg/ml and more than 100 mg/ml are regarded as active (A), moderately active (MA) and inactive (IA), respectively (Homan, 1972; Skehan et al., 1990).
Table 3 Terpenes and sterols composition of the n-hexane fractions of analysed plant. Terpenesa
RTb
RAPc AC
AZ
AR
DT
MS
OV
PO
RR
RO
RUB
RC
SV
SO
1.1 0.2 0.9 0.1 0.3 0.2 0.1
e e e e e e e e e e e e e 2.4 e e e e e tr
e e e e e e e e e e e e e 0.5 e e e e e e
e e e e e e e e e e e e tr 0.9 e e e e e e
e e e e 0.9 e e e e 6.2 e e
e 7.2 e e e e e e tr 3.7 e e
e e e e e e 1.0 e e 1.8 e e
Endo-Borneol Delta-carene Endobornyl acetate a-Ylangene a-Copaene Aromadendrene Eremophilene Longifolene b-Guaiene Valencene b-Bisabolene g-Cadinene b-Selinene Neophytadiene Ferruginol Aristolone b-Amyrin Oleanene Ketoursene a-Amyrin Phytosterolsa
10.992 11.335 12.490 13.496 13.541 14.244 14.700 14.730 14.798 14.804 14.833 14.964 15.644 17.650 21.411 34.316 34.981 34.996 35.597 36.368
e e e e e e e e e e e e e e e e e e e e
e e e e e e e e e e e e e trd e e tr e e e
e e e e e e e e e e e e e 5.2 e e e e e e
e e e e e e e e e e e e e e e e tr e e e
e e e e e e e e e e e e e tr e e 1.9 tr e 2.2
e e e e tr e e tr tr tr tr tr e e e e e e e e
e e e e e e e e e e e e e 2.2 e e tr 0.8 e e
e e e e e e e e e e e e e 1.0 e e e e e 0.6
14.7 2.0
e e e e e e e e e e e e e 1.9 e e 2.9 e e 2.8
Cholestenedione Ergostatrienol Cholestenol Ergostadienol Ergostenol Norcholestadienol Stigmastadienol Dimethylcolesterol Ethylcholestenol Stigmastenol Stigmastadienone Stigmastenone
29.064 29.087 29.481 31.601 31.773 32.476 32.489 33.985 34.008 34.093 36.740 38.077
e e e e 3.1 e e e e 12.4 5.6 e
e e e e tr e tr e e tr e e
e e e e e e 5.8 e e e e e
e e e e e e e e e 1.7 1.8 e
e e e e e e e e e 3.7 e e
e e e e e e e 4.7 tr tr e e
e e e e 2.1 1.2 e e e 9.1 e 0.7
e e 0.7 0.3 2.9 e 0.4 e e 7.2 e e
1.1 e e e e e e e e tr e e
e e e e e e e e e 33.7 e e
a b c d
Compounds listed in order of elution from SE30 MS column. Retention time (as minutes). Relative area percentage (peak area relative to total peak area %). Compositional values less than 0.1% are denoted as traces.
0.1 e e 0.3 tr 0.9 0.9 11.3 2.5
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Table 4 Antioxidant activity of wild dietary plants. IC50 (mg/mL)
Sample
30 min AR AZ AC DT MS OV PO RR RO RUB RC SV SO
Amaranthus retroflexus L. Anchusa azurea Miller Asparagus acutifolius L. Diplotaxis tenuifolia (L.) DC. Mentha spicata L. ssp. Glabrata (Lej. et Court) Lebeau Origanum vulgare L. subsp. viridulum (Martin-Donos) Nyman Portulaca oleracea L. Raphanus raphanistrum L. subsp. landra (DC.) Bonnier & Layens Rosmarinus officinalis L. Rubus caesius L. Rumex conglomeratus Murray Silene vulgaris (Moench) Garcke Smirnium olusatrum L.
60 min e
40.19 ± 0.96 66.39 ± 0.92h 25.90 ± 0.17d >100 11.24 ± 0.27b 25.45 ± 0.23d 20.07 ± 0.34c 56.70 ± 0.94g 26.89 ± 0.29d 4.91 ± 0.09a 5.53 ± 0.68a 51.15 ± 1.94f 23.11 ± 0.31 cd
87.39 >100 51.37 >100 26.42 41.84 47.32 >100 61.39 11.10 13.11 >100 49.70
± 1.74i ± 1.94f ± 0.34d ± 0.65e ± 0.59f ± 1.14g ± 0.29b ± 0.41b ± 1.03f
Data are expressed as mean ± S. E. (n ¼ 3). Different letters along and between columns indicate statistically significant differences at P < 0.05 (Tukey's test). Propyl gallate (IC50 ¼ 1.00 ± 0.02 mg/mL) was used as positive control.
In a first phase of the study, a screening of all species was carried out, in order to highlight the most interest samples on which realize further analysis. The effects of all extracts were then verified on all cell lines at a concentration of 100 mg/ml. Obtained results are summarized in Fig. 4. Almost all samples significantly inhibited cell proliferation compared to untreated cells (Dunnett's test). The highest inhibitory activity was showed by Rosmarinus officinalis L. hydroalcoholic extract (RO), which induced 94.74% of inhibition of cell viability on LoVo cells and 63.12% on HepG2 cell lines. O. vulgare L. subsp. viridulum (Martin-Donos) Nyman (OV) also showed a good activity (66.59% on LoVo cells and 44.86% on breast cancer cell line MCF-7). Samples SV, AR, DT and RUB were able to induce an inhibitory activity higher than 40%. Particularly, Silene vulgaris extract against LoVo and HepG2 cells caused a percent of inhibition of 48 and 44 at a concentration of 100 mg/ml, respectively (Fig. 4). Diplotaxis tenuifolia (L.) DC., Mentha spicata L. ssp. glabrata (Lej. et Court) Lebeau and Smirnium olusatrum L. samples were able to affect cell viabilty of two cell lines utilized, while only one line was affected by A. retroflexus L. and P. oleracea L. A. acutifolius L. sample showed no activity. Overall, HepG2 and LoVo cell lines were more responsive compared to MCF-7 cells. Samples that at a concentration of 100 mg/ml induced a
percentage of inhibition higher than 50% were tested at different concentrations (100e2.5 mg/ml) in order to determine IC50 values. After 48 h of incubation the most antiproliferative plant extract was rosemary (Rosmarinus officinalis L.) on LoVo cell lines with an IC50 of 16.60 mg/ml (Fig. 5a). This species showed antiproliferative activity on hepatocarcinoma cell line HepG2 (IC50 ¼ 83.44 mg/ml, Fig. 5b). Oregano (O. vulgare subsp. viridulum) showed a selective antiproliferative activity on hepatic cancer with IC50 of 32.59 mg/ml (Fig. 5c). The same sample showed also weak activity against MCF-7 cells with a percent of inhibition of 45 at a concentration of 100 mg/ ml. Changes in treated cells viability were visualized using an inverted microscope and captured on a VWR digital camera. As evidenced in Fig. 6, the incubation of cell cultures in the presence of a concentration of 100 mg/mL of each sample significantly affected cell viability. In this study, MTT assay was used to provide qualitative preliminary data on the antiproliferative activity of edible plant extracts. This activity is thought to be related to the antioxidant properties of the extracts; several works on natural antioxidants in vitro activities against cancer cells have been cited in literature s-Linares et al., 2015; Eberhardt et al., 2000; Kaur et al., 2014; (Borra Pacifico et al., 2012; Erol et al., 2012). However, some scientific
Fig. 4. Inhibition of cell proliferation induced by samples at 100 mg/ml on MCF-7, HepG2 and LoVo cell lines. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control (Dunnett's test).
M. Marrelli et al. / Food and Chemical Toxicology 86 (2015) 16e24
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Fig. 5. Dose depending effect of rosemary (RO) and oregano (OV) extracts on the proliferation of cancer cells. a) Effect of Rosmarinus officinalis L. on LoVo cell line; b) effect of rosemary on HepG2 cells; c) effect of Origanum vulgare L. subsp. viridulum on HepG2 cell viability.
Fig. 6. Changes in cell viability induced by OV and RO samples on HepG2 and cell lines. a) HepG2 cell line, control, cells in EtOH 70% (0.5%, v/v), without sample; b) HepG2 cell line, OV sample, 100 mg/ml; c) HepG2 cells, RO sample, 100 mg/ml; d) LoVo cell line, control cells in EtOH 70% (0.5%, v/v), without sample; e) LoVo cells, RO sample, 100 mg/ml.
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reports have highlighted the fact that there might be an interference between some antioxidant molecules and the MTT assay. Wang et al. (2010) reported that it could be possible that polyphenols present in green tea may interfere with formazan formation, critical to the MTT assay, by changing the activity of succinate dehydrogenase or interact with MTT directly. Their results indicate that using MTT assay in presence of ECGC (green tea polyphenol) could underestimate the antiproliferative activity of the extract and show more viable cells but not in presence of apigenin (flavonoid). Furthermore, this possible interference could be considered when the half maximal inhibitory concentration (IC50) has to be estimated not for the simple assessment of qualitative antiproliferative activity. 4. Conclusion Natural products play a leading role in the discovery and the development of drugs for the treatment of human diseases. The discovery of the anticancer activities of so many traditional medicines and natural products need to be supported by scientific evidence and validation. More importantly, natural products as a complex polychemical mixture of pharmacologically active compounds may target multiple vulnerabilities of cancer cells, without toxicity to the noncancerous cells. The data presented in this study demonstrated that almost all the reported species possess antioxidant activity. The observed in vitro antioxidant activity suggest that the investigated plant extracts could exert effects also against human cancer proliferation. With this respect, the most promising plants appear to be O. vulgare L. subsp. viridulum and Rosmarinus officinalis L. Further studies are needed for these plants, to obtain the isolation and structural elucidation of bioactive compounds and about their in vivo toxic effects in experimental animals to formulate a new drug for regular practice. In conclusion, this work reveals that the Italian flora can be an interesting source of anti-proliferative and antioxidant principles, with a potential use in pharmaceutical fields and demonstrates for the first time the selective cytotoxic capacity of O. vulgare L. subsp. viridulum and Rosmarinus officinalis L., confirming their medicinal plant feature. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2015.09.011. References s-Linares, I., Pe rez-Sa nchez, A., Lozano-Sa nchez, J., Barrajo n-Catala n, E., Borra ez-Rom Arra an, D., Cifuentes, A., Micol, V., Carretero, A.S., 2015. A bioguided identification of the active compounds that contribute to the antiproliferative/ cytotoxic effects of rosemary extract on colon cancer cells. Food Chem. Toxicol. 80, 215e222. ~o, P.A., de Oliveira, E.A., Letra, M.H.S., Sawaya, A.C.H.F., Calado, J.C.P., Alberta Marcucci, M.C., 2015. Flavonoid contents and antioxidant activity in fruit, vegetables and other types of food. Agric. Sci. 6, 426e435. Chen, C.C., Liu, L.K., Hsu, J.D., Huang, H.P., Yang, M.Y., Wang, C.J., 2005. Mulberry extract inhibits the development of atherosclerosis in cholesterol-fed rabbits. Food Chem. 91, 601e607. Conforti, F., Marrelli, M., Colica, C., Menichini, F., Perri, V., Uzunov, D., Statti, G.A., Duez, P., Menichini, F., 2011. Bioactive phytonutrients (omega fatty acids, tocopherols, polyphenols), in vitro inhibition of nitric oxide production and free
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