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LC–MS analysis, anticancer, antioxidant and antimalarial activities of Cynodon dactylon L. extracts
Industrial Crops and Products 45 (2013) 240–247
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LC–MS analysis, anticancer, antioxidant and antimalarial activities of Cynodon dactylon L. extracts Daycem Khlifi a,b , El Akrem Hayouni c , Alexis Valentin d , Sylvie Cazaux a , Béatrice Moukarzel d , Moktar Hamdi b , Jalloul Bouajila a,∗ a Université de Toulouse, Laboratoire des Interactions Moléculaires et Réactivité Chimique et Photochimique UMR CNRS 5623, Université Paul-Sabatier, 118 route de Narbonne, F-31062 Toulouse, France b Université de Carthage, Laboratoire d’Ecologie et de Technologie Microbienne, Institut National des Sciences Appliquées et de la Technologie (INSAT), B.P. 676, 1080 Tunis, Tunisia c Laboratoire des Substances Biologiquement Actives, Centre de Biotechnologie à l’Ecopark de Borj-Cedria, BP-901, Hamman-Lif, Tunisia d Université de Toulouse, UMR IRD-UPS 152 PHARMA-DEV, Université Paul-Sabatier, Faculté de Pharmacie, 35, Chemin des Maraîchers, F-31062 Toulouse, France
a r t i c l e
i n f o
Article history: Received 1 September 2012 Received in revised form 29 November 2012 Accepted 1 December 2012 Keywords: Cynodon dactylon LC–MS Anthocyanins Antioxidant activity Antimalarial activity Anticancer activity
a b s t r a c t Extracts (petroleum ether, dichloromethane, acetone, methanol/water (3/1) and water) from Cynodon dactylon L. were obtained by sequential method. The chemical composition of phenolics (6.9–57.41 g gallic acid equivalents/kg of dry mass), tannins (2.49–12.32 g catechin equivalent/kg of dry mass), anthocyanins (0–5.98 g cyanidin equivalent/kg of dry mass) and flavonoids (0–5.02 g quercetin equivalent/kg of dry mass) was evaluated. The extracts were evaluated for their antioxidant (DPPH and ABTS assays), antimalarial (Plasmodium falciparum) and anticancer (MCF-7) activities; the strongest activities were obtained by the water extract (IC50 = 57.21 ± 1.47 mg/L), the acetonic extract (IC50 = 38 mg/L) and the petroleum ether extract (IC50 = 39 mg/L), respectively. The results indicate a good correlations between anthocyanins quantity and the antimalarial activity (R2 = 0.79) and also to anticancer activity (R2 = 0.78). The LC–MS analysis of the extracts having the good activities anticancer and antimalarial, has revealed the presence of seven anthocyanins (delphinidin-3-Oacetylglucoside, petunidin-3-O-caffeoylglucoside-5-O-glucoside, petunidin-3-O-coumarylglucoside-5O-glucoside, malvidin-3-O-monoglucoside, delphinidin-3-O-acetylglucoside-pyruvic acid, petunidin-3O-acetylglucoside-5-O-glucoside and cyanidin-3,5-O-diglucoside), identified for the first time in this plant. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction Natural products have served as an important source of drugs since ancient times and a significant part of today’s drugs are somehow derived from natural sources. In the recent years, secondary plant metabolites (phytochemicals), previously with unknown pharmacological activities, have been extensively investigated as a source of medicinal agents (Krishnaraju et al., 2005). Cynodon dactylon L. (Gramineae, Poaceae), commonly known as “Njem” in Tunisia, possesses various medicinal properties such as antiviral, antimicrobial (Dhar et al., 1968), anti-inflammatory (Biswas and Mukherjee, 2003), immunomodulatory activity (Santhi and Annapoorani, 2010) and has significant application in treating dysentery, dropsy (Chopra and Handa, 1982), hypolipidemic and act as hypoglycemic agent (Leporatti and Corradi, 2001). Furthermore, the ethanol extract of aerial part has also marked
∗ Corresponding author. Tel.: +33 562256825; fax: +33 562256826. E-mail address: [email protected] (J. Bouajila).
central nervous system (CNS) depressant (Pal, 2008). In order to search for new molecules having antioxidant, antimalarial or anticancer activities, we have investigated C. dactylon. The objectives of this study were: (i) analysis of chemical composition of various extracts of whole plant C. dactylon (characterization of phenolics, tannins, flavonoids and anthocyanins of their extracts), (ii) investigation of antioxidant, antimalarial and anticancer activities of various extracts, (iii) the search of possible correlations between chemical compositions and antimalarial, anticancer and antioxidant activities and (iv) identification by LC–MS of molecule (s) responsible for the interesting activities while basing itself on best the correlations. 2. Materials and methods 2.1. Chemicals All chemicals used were of analytical reagent grade. All reagents were purchased from Sigma-Aldrich-Fluka (Saint-Quentin France): (sodium acetate (≥99%), potassium chloride (≥99%), gallic acid
0926-6690/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.12.030
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(≥99%), hydrochloric acid (36.5–38.0%), catechin (≥98%), quercetin (≥98%), cyanidin-3-glucoside (≥95%), ascorbic acid (≥99.0%)), (aluminum trichloride (99.99%), 1,1-diphenyl-2-picrylhydrazyl free radical (99%)), (methanol (≥99%), dichloromethane (≥99.5%), petroleum ether (≥98.5%), anhydrous sodium sulfate (≥99.0%), sodium carbonate (≥99.0%), Folin–Ciocalteu reagent 2 N, acetic), (potassium persulfate (≥99.0%), vanillin (≥98.0%), H2 SO4 (≥95%), 2,2 -azinobis-3-ethylbenzothiazoline-6-sulphonate (≥99.0%)). Chloroquine, RPMI 1640 medium (Lonza; Emerainville, France), Hepes, NaHCO3 , doxorubicin, fetal calf serum (Lonza). 2.2. Collection of plant material C. dactylon L. (whole plant) was collected in January 2009 from the central area of Tunisia, precisely from the Sidi Bouzid region. Specimens were identified by Dr. Bousaid Mohamed at the Department of Botany, National Institute of Applied Sciences and Technology (INSAT, Tunis) and voucher specimens were deposited at the Herbarium of the Department of Botany in the cited institute. 2.3. Extraction Plant was dried in air shade at room temperature, and the dry plant was powdered. 50 g of powders was extracted in a soxhlet system with 500 mL of different solvents of croissant polarity was used: petroleum ether (6 h at 40 ◦ C), dichloromethane (6 h at 40 ◦ C), acetone (6 h at 56 ◦ C), methanol/water (3/1) (6 h at 65 ◦ C) and water (6 h at 100 ◦ C). All organic extracts were concentrated by rotary evaporation under vacuum at 35 ◦ C. For the chemical analyses and the antioxidant activities, the extracts of petroleum ether and dichloromethane were re-suspended in the DMSO, whereas extracts of acetone and methanol/water were re-suspended in ethanol. For the antimalarial and anticancer activities, all extracts were re-suspended in the DMSO followed by dilution in the aqueous phase so that the DMSO does not exceed 1%. 2.4. Determination of total phenolic compounds by the Folin–Ciocalteu method The phenolics of each plant extract were determined by Folin and Ciocalteu (1927). The diluted solution of each extract (0.5 mL) was mixed with Folin–Ciocalteu reagent (0.2 N, 2.5 mL). This mixture was allowed to stand at room temperature for 5 min and then sodium carbonate solution (75 g/L in water, 2 mL) was added. After 1 h of incubation, absorbance was measured at 765 nm against appropriate solvent. A standard calibration curve was plotted using gallic acid (0–300 mg/L). The results were expressed as mg of gallic acid equivalents (GAE)/L of dry mass. 2.5. Condensed tannin content Catechins and proanthocyanidins reactive to vanillin were analyzed by the vanillin method of Broadhurst and Jones (1987). One milliliter (1 mL) of each extract solution was placed in a test tube and 2 mL of vanillin (1% in H2 SO4 7 M) in an ice bath and then incubated at 25 ◦ C. After 15 min, the absorbance of the solution was read at 500 nm. Concentrations were calculated as g catechin equivalents (CE)/kg dry mass from a calibration curve. 2.6. Total flavonoids determination The total flavonoids were estimated according to the Dowd method as adapted by Arvouet-Grand et al. (1994). A diluted solution (4 mL) of each extract was mixed with a solution (4 mL) of aluminum trichloride (AlCl3 ) in methanol (2%). The absorbance was
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read at 415 nm after 15 min against a blank sample consisting of a methanol (4 mL) and extract (4 mL) without AlCl3 . Quercetin was used as reference compound to produce the standard curve, and the results were expressed as g of quercetin equivalents (QE)/kg of dry mass. 2.7. Determination of total anthocyanin content Total anthocyanin content was measured with the pH differential absorbance method, as described by Cheng and Breen (1991). Briefly, absorbance of the extract was measured at 510 and 700 nm in buffers at pH 1.0 (hydrochloric acid–potassium chloride, 0.2 M) and 4.5 (acetic acid–sodium acetate, 1 M). The wavelength reading was performed after 15 min of incubation. Anthocyanin content was calculated using a molar extinction coefficient (ε) of 29,600 (cyanidin-3-glucoside) and absorbance of A = [(A510 − A700 )pH 1.0 − (A510 − A700 )pH 4.5 ]. Results were expressed as g cyanidin-3-glucoside equivalent (C3GE)/kg of dry mass. 2.8. Free radical scavenging activity DPPH test Antioxidant scavenging activity was studied using 1,1-diphenyl2-picrylhydrazyl free radical (DPPH) as described by Blois (1958) with some modifications; 1.5 mL of various dilutions of the test materials (pure antioxidants or plant extracts) were mixed with 1.5 mL of a 0.2 mM methanolic DPPH solution. After an incubation period of 30 min at 25 ◦ C, the absorbance at 520 nm, the wavelength of maximum absorbance of DPPH, were recorded as A(sample) . A blank experiment was also carried out applying the same procedure to a solution without the test material and the absorbance was recorded as A(blank) . The free radical-scavenging activity of each solution was then calculated as percent inhibition according to the following equation: % inhibition = 100
A(blank) − A(sample) A(blank)
Antioxidant activity extracts was expressed as IC50 , defined as the concentration of the test material required to cause a 50% decrease in initial DPPH concentration. Ascorbic acid was used as a standard. All measurements were performed in triplicate. 2.9. ABTS radical-scavenging test The radical scavenging capacity of the samples for the ABTS (2,2 -azinobis-3-ethylbenzothiazoline-6-sulphonate) radical cation was determined as described by Re et al. (1999). ABTS was generated by mixing a 7 mM of ABTS at pH 7.4 (5 mM NaH2 PO4 , 5 mM Na2 HPO4 and 154 mM NaCl) with 2.5 mM potassium persulfate (final concentration) followed by storage in the dark at room temperature for 16 h before use. The mixture was diluted with ethanol to give an absorbance of 0.70 ± 0.02 units at 734 nm using spectrophotometer. For each sample, diluted methanol solution of the sample (100 L) was allowed to react with fresh ABTS solution (900 L), and then the absorbance was measured 6 min after initial mixing. Ascorbic acid was used as a standard and the capacity of free radical scavenging was expressed by IC50 (mg/L) values calculated denote the concentration required to scavenge 50% of ABTS radicals. The capacity of free radical scavenging IC50 was determined using the same previously used equation for the DPPH method. All measurements were performed in triplicate. 2.10. Antimalarial activity The chloroquine-resistant FcB1-Columbia strain of Plasmodium falciparum (IC50 for chloroquine: 186 nM) was cultured
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Table 1 Chemical composition, antimalarial activity and anticancer activity of Cynodon dactylon extracts. Type of extract
Polyphenols (GAE)a
Petroleum ether Dichloromethane Acetone Methanol (75%) Water Chloroquine Doxorubicin
6.9 23.42 57.41 23.35 44.56
± ± ± ± ±
0.44 0.97 0.46 1.2 0.72
Tannins (CE)a 4.59 12.32 10.19 2.49 2.87
± ± ± ± ±
0.03 0.14 0.06 0.07 0.03
Flavonoids (QE)a
Anthocyanins (C3GE)a
Antimalarial activityb
Anticancer activityb
nd nd 4.80 ± 0.063 5.02 ± 0.09 5.07 ± 0.04
5.64 ± 0.11 nd 5.98 ± 0.08 3.04 ± 0.03 0.16 ± 0.01
48 >100 38 >100 >100 0.10 ± 0.09
39 >100 44 >100 >100 0.22 ± 0.04
Standard deviations (SD) did not exceed 5%. nd: not detect. a g/kg dry mass. b IC50 (mg/L).
continuously according to Trager and Jensen (1976) with modifications described by Benoit et al. (1996). The IC50 values for chloroquine were checked every 2 months, and we observed no significant variations. The parasites were maintained in vitro in human red blood cells (O±; EFS; Toulouse, France), diluted to 4% hematocrit in RPMI 1640 medium (Lonza; Emerainville, France) supplemented with 25 mM Hepes and 30 M NaHCO3 and complemented with 7% human AB + serum (EFS). Parasites cultures were synchronized by combination of magnetic enrichment and by d-sorbitol lysis (5% of d-sorbitol in sterile water). The antimalarial activity of various extracts was evaluated by a radioactive micromethod. Tests were performed in triplicate in 96-well culture plates (TPP) with cultures mostly at ring stages (synchronization interval, 16 h) at 0.5–1% parasitemia (hematocrit, 1.5%). Parasite culture was incubated with each sample for 48 h. Parasite growth was estimated by [3 H]-hypoxanthine (PerkinElmer; Courtaboeuf, France) incorporation, which was added to the Plates 24 h before freezing. After 48 h incubation, plates were frozen-defrosted and each well was harvested on a glass fiber filter. Incorporated [3 H]-hypoxanthine was then determined with a betacounter (1450-Microbeta Trilux; Wallac-Perkin Elmer). The control parasite cultures, free from any sample, was referred to 100% growth. IC50 were determined graphically in concentration versus percent inhibition curves. Chloroquine diphosphate was used as positive control. The antimalarial activity of sample was expressed by IC50 , representing the concentration of drug that induced a 50% parasitemia decrease compared to the positive control culture referred to as 100% parasitemia. According to the literature concerning plant antiplasmodial activities a sample is very active if IC50 < 5 mg/L, active if IC50 between 5 and 50 mg/L, weakly active if IC50 between 50 and 100 mg/L and inactive if IC50 > 100 mg/L (Ouattara et al., 2006). 2.11. Anticancer activity Cytotoxicity of each sample was estimated on human breast cancer cells (MCF7). The cells were cultured in the same conditions as those used for P. falciparum, except for the 10% human serum, which was replaced by 10% fetal calf serum (Lonza). For the determination of pure compound activity, cells were distributed in 96-well plates at 3 × 104 cells/well in 100 L, and then 100 L of culture medium containing sample at various concentrations were added. Cell growth was estimated by (3 H)-hypoxanthine incorporation after 48 h incubation exactly as for the P. falciparum assay. The (3 H)-hypoxanthine incorporation in the presence of sample was compared with that of control cultures without sample (positive control being doxorubicin). 2.12. LC–MS analysis Analyses were performed using an Agilent 1100 liquid chromatograph (Agilent Technologies USA) consisting of a binary pump
and an automatic sampler. The extracts were filtered through a Millex HA 0.45 m filter (Millipore Corp.) before injection (50 L). Chromatographic separation was performed on a Phermerex C18 column (25 cm × 4.6 mm i.d., 5 m particle size) equipped with a 5 m C18 guard column. Column temperature was 25 ◦ C. The mobile phases were (A) formic acid/water (1:99, v/v) and (B) methanol. The gradient used was 0–43 min, 10–90% B; 43–45 min, 90–10% B; 45–50 min, 10% B. The solvent flow rate was 0.9 mL/min. Analysis was achieved by positive ion mode using a Q Trap mass spectrometer (Applied Biosystems/MDS Sciex, USA) with a Turbo ion spray source at 480 ◦ C. The ion spray voltage was set at 5000 V. The declustering potential (DP) was 35 V. The collision energy (CE) was 21 V. Nitrogen was used as nebulizing and collision gas. The data were collected and processed by Analyst 1.6. software (Applied Biosystems/MDS Sciex, USA). 2.13. Statistical analysis All data were expressed as means ± standard deviations of triplicate measurements. The confidence limits were set at P < 0.05. Correlations were carried out using the correlation and regression in the EXEL program. 3. Results and discussion 3.1. Extraction yields The sequential method adapted to obtain different C. dactylon extracts was presented as follows: petroleum ether, dichloromethane, acetone, methanol/water (3/1) and water. The highest yields were achieved by methanol/water (3/1) (8.24%), followed by dichloromethane (2.75%), acetone (2.4%), petroleum ether (1.25%) and the lowest was water (1.2%). One study (Gangwar et al., 2011) is provided in the literature concerning extraction yield for this plant. The extraction was carried out with petroleum ether, chloroform, ethanol and chloroform/water with successive yields of 0.85, 1.25, 2.85 and 3.65%. Our total yield (15.84%) is approximately twice higher than of Gangwar et al. (2011) (8.6%). The yield is affected by several factors such as species, geographical location, harvest time, plant part used and isolation method. 3.2. Chemical composition: phenolics, tannins, flavonoids and anthocyanins This is the first study which recorded the tannins and anthocyanins content of extracts from C. dactylon and from genus Cynodon. All results are presented in Table 1. The amount of total phenolics varied in the different extracts and ranged from 6.9 ± 0.44 to 57.41 ± 0.46 g GAE/kg of dry mass. The highest amount of phenolics was in acetone extract (57.41 ± 0.46 g GAE/kg of dry mass), followed by water extract (44.56 ± 0.72 g GAE/kg of dry mass); petroleum ether was poor in phenolics (6.9 ± 0.44 g GAE/kg of dry mass). This variation was due to the polarity of phenolics. Polar
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Fig. 1. Antioxidant activity of Cynodon dactylon by DPPH assay. 1: petroleum ether extract, 2: dichloromethane extract, 3: acetone extract, 4: methanol/water (3/1) extract, 5: water extract and 6: reference standard: ascorbic acid. Standard deviations (SD) did not exceed 5%.
Fig. 2. Antioxidant activity of Cynodon dactylon by ABTS assay. 1: petroleum ether extract, 2: dichloromethane extract, 3: acetone extract, 4: methanol/water (3/1) extract, 5: water extract and 6: reference standard: ascorbic acid. Standard deviations (SD) did not exceed 5%.
fractions had more phenolics than had non-polar fractions. Wegner (2011) quantified phenolics and obtained small quantity (maximum 0.589 mg GAE/g of dry mass). In another work, Mangathayaru et al. (2009) recorded 47 ± 0.33 g GAE/kg in the fresh juice of C. dactylon shoots. Their results showed that the content of phenolics was less important compared to what we found. The difference between our results and their results was probably due to the difference of the part of the plant used, we have used whole plant and they have used shoots. There is no study in the literature which investigated the phenolics content in other species of the genus Cynodon. The amount of tannins in dichloromethane extract was richer (12.32 ± 0.14 g CE/kg of dry mass) followed by acetone extract (10.19 ± 0.06 g CE/kg of dry mass). Methanol 75% was poor in tannins (2.49 ± 0.07 g CE/kg of dry mass). Non-polar fractions had more tannins than had polar fractions. For the amount of flavonoids, the highest quantity was in water extract (5.07 ± 0.04 g QE/kg of dry mass), followed by methanolic 75% extract (5.022 ± 0.09 g QE/kg of dry mass). Petroleum ether and dichloromethane did not contained flavonoids. Wegner (2011) has obtained small quantity for flavonoids (0.123–0.255 mg/g catechin equivalent/g of dry mass). For anthocyanins, the highest amount was obtained by acetonic extract (5.98 ± 0.09 g C3GE/kg of dry mass), followed by petroleum ether extract (5.46 ± 0.11 mg C3GE/kg of dry mass); dichloromethane has not contained anthocyanins. To our knowledge, no study cited the quantification of tannins and or anthocyanins for this specie.
the difference in climate between Tunisia and India, geographical origin, harvesting time and growing conditions.
3.3. Antioxidant activity 3.3.1. DPPH assay The DPPH free radical method determines the antiradical power of antioxidants. The antioxidant activity of water extract (Fig. 1) was superior to all samples tested with an IC50 value of 152.96 ± 2.12 mg/L, followed by methanolic extract (203.29 ± 1.38 mg/L) and by acetonic extract (206.69 ± 2.57 mg/L). On the other hand, extracts prepared with petroleum ether or dichloromethane have exhibited a poor radical-scavenging activity (921.76 ± 2.83 mg/L and 1053.12 ± 1.71 mg/L, respectively). We can deduce, as seen in Fig. 1, that extracts obtained using high polarity solvents were considerably more effective radical-scavengers than were those using low polarity solvents. Similar results for C. dactylon were found by Albert-Baskar and Ignacimuthu (2010), in the same order in terms of solvents polarity. They have found antioxidant activity of methanol extract was superior to all samples tested (IC50 = 63.03 ± 4.82 mg/L), followed by ethyl acetate extract (311.05 ± 23.69 mg/L) and by hexane extract (874.15 ± 66.56 mg/L). The difference between our results and the Albert-Baskar and Ignacimuthu (2010) work is due to: the difference of the plant part used, we have used all plant and they have used the roots; also, the difference of method of extraction and the solvents, we have used the soxhlet, they were used the cold percolation method, and finally
3.3.2. ABTS assay For water extract (Fig. 2), a good antioxidant activity value was found (IC50 = 57.21 ± 1.74 mg/L), followed by acetonic extract (IC50 = 179.77 ± 2.19 mg/L). This result confirmed the agreement between phenolics and antioxidant activity where acetonic and water extracts presented higher total phenolic quantities (57.41 ± 0.46 and 44.56 ± 0.72 g GAE/kg of dry material, respectively). In other work, Auddy et al. (2003) showed that the water infusion of C. dactylon has a moderate antioxidant activity, by ABTS, with IC50 = 273.64 mg/L. This result showed that the antioxidant activity was 4 times less important compared to our results. As a result of the present study, the water extract was found to be effective antioxidant in different in vitro assays. 3.4. Antimalarial activity In the present study, antimalarial activity of acetonic extract was superior to all samples tested with an IC50 = 38 mg/L (Table 1), followed by petroleum ether extract (IC50 = 48 mg/L). By referring to the literature, this result showed an active antimalarial activity of C. dactylon (Ouattara et al., 2006). This may be indicative of a significant potential for isolating pure compounds with much higher antimalarial activity (in term of IC50 ) from acetonic and petroleum ether extracts. Recent study by Bagavan et al. (2011) showed that C. dactylon leaves, ethyl acetate and methanol extracts, were no active up to 100 mg/L against P. falciparum strains 3D7, Dd2 and INDO. The antimalarial activity could come from others organs, not leaves. We found a good correlation between anthocyanins quantity in extracts and the antimalarial activity (R2 = 0.79). El Babili et al. (2010) have obtained also a good correlation between anthocyanins quantity and the antimalarial activity (R2 = 0.82). According to our knowledge, no anthocyanin molecule is quoted in the literature for an antimalarial activity. 3.5. Cytotoxicity C. dactylon extracts showed a significant cytotoxic activity against human breast cancer cells (MCF7). Cytotoxicity of petroleum ether extract was superior to all samples tested with an IC50 = 39 mg/L (Table 1), followed by acetonic extract (IC50 = 44 mg/L). Dichloromethane, water and methanol 75% extracts were not actives (IC50 > 100 mg/L). Albert-Baskar and Ignacimuthu (2010) obtained different results when the ethyl acetate extract of C. dactylon roots was the more active (IC50 = 30 mg/L) against human breast cancer cells (MCF7), followed by hexane extract (IC50 = 40 mg/L) and by methanol extract (IC50 = 55 mg/L). In our work, we found a good correlation between anthocyanins quantity in extract and the anticancer activity (R2 = 0.78).
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Table 2 Anthocyanins and anticancer activity. Name
Delphinidin
Delphinidin 3-O-rutinoside
Delphinidin 3-O-glucoside
Structure
Cancer (human tumor cell lines)
IC50 or % of inhibition
LoVo (colorectal) LoVo/ADR (colorectal) MCF-7 (breast)
11.4a 4.97a 162.4a
MCF-7 (breast) HCT-116 (colon) NCI-H460 (lung) SF-268 (central nervous system) AGS (stomach)
30a 38a 76a 100a
MCF-7 (breast) LNCaP (prostate)
References
Cvorovic et al. (2010) Zhang et al. (2005)
Vareed et al. (2006)
100a
25a
Vareed et al. (2006) a
0.143
˜ Munoz-Espada and Watkins (2006)
HT-29 (colon) HCT-116 (colon) LoVo/ADR (colorectal) LoVo (colorectal) U937 (leukemia)
18.09a 24.41a 7.52a 13.47a 60a
Cyanidin 3-glucoside
HS578 T (breast) HL-60 (leukemia) HT-29 (colon)
6.73a 62.2a 14a
Chen et al. (2005) Dai et al. (2009) Jing et al. (2008)
Cyanidin-3-rutinoside
HL-60 (leukemia)
29.7a
Feng et al. (2007)
Cyanidin-3-O-bglucopyranoside
Jurkat (leukemia) HL-60 (leukemia)
174.9a 16.6a
Fimognaria et al. (2004)
MCF-7 (breast) AGS (stomach) HCT-116 (colon) NCI H 460 (lung) SF-268 (central nervous system) LoVo/ADR (colorectal) LoVo (colorectal) U937 (leukemia)
32.2a 84.7a 71.7a 88.1a 143.3a
Zhang et al. (2005)
Cyanidin
Malvidin
>33.12a >33.12a 40a
Kang et al. (2003) Cvorovic et al. (2010) Hyun and Chung (2004)
Cvorovic et al. (2010) Hyun and Chung (2004)
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Table 2 (Continued) Name
Structure
Petunidin
c
MCF-7 (breast) AGS (stomach)
170.7a 24b , c
MCF-7 (breast) AGS (stomach) SF-268 (central nervous system) NCI H 460 (lung) HCT-116 (colon)
Pelargonidin
a
IC50 or % of inhibition
HS578 T (breast) NCI-H460 (lung) SF-268 (central nervous system) AGS (stomach)
Peonidin 3-glucoside
b
Cancer (human tumor cell lines)
23.17a 50a 60a
References
Zhang et al. (2005)
Chen et al. (2005) Vareed et al. (2006)
a
75
136.5a 176.6a 63b , c
Zhang et al. (2005)
182.1a 154.3a
IC50 (mg/L); Percentage of inhibition (%). Anthocyanins were tested at 100 mg/L.
Previous works have indicated that pure anthocyanins from fruits and vegetables have exhibited anti-proliferative activity toward multiple cancer cell types in vitro. The summary of all the molecules evaluated against the multiple cancer is given in Table 2. We also deduce that our ether petroleum
and acetone extracts can contain an important amount of anthocyanins active against breast cancer. These two extracts contain a small anthocyanins quantity (maximum 5.98 ± 0.08 C3GE g/kg dry mass) and have activities comparable with the pure products.
Fig. 3. TIC (LC–MS) and extract ions (m/z) from acetone extract.
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Table 3 Anthocyanins identified by LC–MS in actives extracts. Compounds
Molecular ion [M–H]− (m/z)
Retention time (min) Petroleum ether
Retention time (min) Acetone
References
Delphinidin-3-O-acetylglucoside Petunidin-3-O-caffeoylglucoside-5-O-glucoside Petunidin-3-O-coumarylglucoside-5-O-glucoside Malvidin-3-O-monoglucoside Delphinidin-3-O-acetylglucoside-pyruvic acid Petunidin-3-O-acetylglucoside-5-O-glucoside Cyanidin-3,5-O-diglucoside
507 803 787 493 575 683 611
– – – – – 40.84 42.75
3.12 17.14 17.54 23.16 25.24 – –
He et al. (2010) He et al. (2010) He et al. (2010) He et al. (2010) He et al. (2012) He et al. (2010) He et al. (2010)
–, not detected.
3.6. Identification of anthocyanins by LC–MS for ether petroleum and acetone extracts These biological activities (antimalarial and anticancer) and the interesting correlations encouraged us to push work to identify anthocyanins by LC–MS in acetone and ether petroleum extracts. We carried out the analysis of the two extracts by LC–MS. We optimized the detection of the mass spectrometer by structures similar to those which we seek (anthocyanins). We applied the mode selected ion monitoring (SIM) by seeking anthocyanins already identified in fruits and vegetables and cited in the literature (He et al., 2012, 2010). Approximately, a hundred molecules were followed and the results given seven anthocyanins (Fig. 3 and Table 3) identified for the first time in this plant: delphinidin-3-Oacetylglucoside, petunidin-3-O-caffeoylglucoside-5-O-glucoside, petunidin-3-O-coumarylglucoside-5-O-glucoside, malvidin-3-Omonoglucoside, delphinidin-3-O-acetylglucoside-pyruvic acid, petunidin-3-O-acetylglucoside-5-O-glucoside and cyanidin-3,5-Odiglucoside. All these molecules were never tested against cancer nor against the malaria. This result confirms the hypothesis of the correlations obtained. 4. Conclusions C. dactylon L. extracts were investigated for their chemical composition, antioxidant antimalarial and anticancer activities. This is the first study that investigated the good antimalarial and anticancer activities of C. dactylon and the genus Cynodon. To confirm correlations between anthocyanins and antimalarial and anticancer activities of this plant, we identified by LC–MS five anthocyanins in acetonic extract and two anthocyanins in ether petroleum extract. The quantity of the anthocyanins by extract do not exceed 0.6%, therefore if the activity comes from these anthocyanins, the IC50 will be more interesting than 250 g/L. It is very interesting to push the work of test of these pure molecules against these diseases and to split the extracts at the same time in order to check if there is presence of others anthocyanins non known in the literature and that one could not be check their presence by LC–MS. Acknowledgments We are very grateful to Pr. Boussaid Mohamed Head of Biotechnology Laboratory, National Institute of Applied Sciences and Technology (INSAT, Tunis) for identification of plant. References Albert-Baskar, A., Ignacimuthu, S., 2010. Chemopreventive effect of Cynodon dactylon (L) Pers. extract against DMH-induced colon carcinogenesis in experimental animals. Exp. Toxicol. Pathol. 62, 423–431. Arvouet-Grand, A., Vennat, B., Pourrat, A., Legret, P., 1994. Standardisation d’un extrait de propolis et identification des principaux constituants. J. Pharm. Belg. 49, 462–468. Auddy, B., Ferreira, M., Blasina, F., Lafon, L., Arredonodo, F., Dajas, F., Tripathi, P.C., Seal, T., Mukherjee, B., 2003. Screening of antioxidant activity of three Indian
medicinal plants, traditionally used for the management of neurodegenerative diseases. J. Ethnopharmacol. 84, 131–138. Bagavan, A., Rahuman, A., Kaushik, N.K., Sahal, D., 2011. In vitro antimalarial activity of medicinal plant extracts against Plasmodium falciparum. Parasitol. Res. 108, 15–22. Benoit, F., Valentin, A., Pélissier, Y., Diafouka, F., Marion, C., Kone-Bamba, D., Koné, M., Mallié, M., Yapo, A., Bastide, J.M., 1996. In vitro antimalarial activity of vegetal extracts used in west African traditional medicine. Am. J. Trop. Med. Hyg. 54, 67–71. Biswas, T.K., Mukherjee, B., 2003. Plant medicines of Indian origin for wound healing activity. Int. J. Low Extrem. Wounds 2, 25–39. Blois, M.S., 1958. Anioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200. Broadhurst, R.B., Jones, W.T., 1987. Analysis of condensed tannins using acidified vanillin. J. Sci. Food Agric. 28, 788–794. Chen, P.N., Chu, S.C., Chiou, H.L., Chiang, C.L., Yang, S.F., Hsieh, Y.S., 2005. Cyanidin 3-glucoside and peonidin 3-glucoside inhibit tumor cell growth and induce apoptosis in vitro and suppress tumor growth in vivo. Nutr. Cancer 3, 232–243. Cheng, G.W., Breen, P.J., 1991. Activity of phenylalanine ammonialyase (PAL) and concentrations of anthocyanins and phenolics in developing strawberry fruit. J. Am. Soc. Hortic. Sci. 117, 946–950. Chopra, R.N., Handa, K., 1982. Indigenous Drugs of India, 2nd ed. Academic Publishers, India. Cvorovic, J., Tramer, F., Granzotto, M., Candussio, L., Decorti, G., Passamonti, S., 2010. Oxidative stress-based cytotoxicity of delphinidin and cyanidin in colon cancer cells. Arch. Biochem. Biophys. 501, 151–157. Dai, J., Gupte, A., Gates, L., Mumper, R.J., 2009. A comprehensive study of anthocyanin-containing extracts from selected blackberry cultivars: extraction methods, stability, anticancer properties and mechanisms. Food Chem. Toxicol. 47, 837–847. Dhar, M.L., Dhar, M.M., Dhawan, B.N., Ray, C., 1968. Screening of Indian plants for biological activity. Part I. Indian J. Exp. Biol. 6, 232–247. El Babili, F., Bouajila, J., Fouraste, I., Valentin, A., Mauret, S., Moulis, C., 2010. Chemical study, antimalarial and antioxidant activities, and cytotoxicity to human breast cancer cells (MCF7) of Argania spinosa. Phytomedicine 17, 157–160. Feng, R., Min, H., Shiow, N., Wang, Y., Tourkova, I.L., Shurin, M.R., Harad, H., Yin, X.M., 2007. Cyanidin-3-rutinoside, a natural polyphenol antioxidant, selectively kills leukemic cells by induction of oxidative stress. J. Biol. Chem. 282, 13468–13476. Fimognaria, C., Bertia, F., Nüsseb, M., Cantelli-Fortia, G., Hrelia, P., 2004. Induction of apoptosis in two human leukemia cell lines as well as differentiation in human promyelocytic cells by cyanidin-3-O-b-glucopyranoside. Biochem. Pharmacol. 67, 2047–2056. Folin, O., Ciocalteu, V., 1927. On tyrosine and tryptophane determination in proteins. J. Biol. Chem. 27, 627–650. Gangwar, S., Khan, M.A., Saxena, A., Mujeeb, M., Husain, A., 2011. Pharmacognostical and Phytochemical Investigation of Cynodon dactylon L. Pers. leaves. Curr. Pharm. Res. 2 (1), 419–426. He, F., Liang, N.N., Mu, L., Pan, Q.H., Wang, J., Reeves, M.J., Duan, C.Q., 2012. Anthocyanins and their variation in red wines. II. Anthocyanin derived pigments and their color evolution. Review. Molecules 17, 1483–1519. He, F., Mu, L., Yan, G.L., Liang, N.N., Pan, Q.H., Wang, J., Reeves, M.J., Duan, C.Q., 2010. Biosynthesis of anthocyanins and their regulation in colored grapes. Review. Molecules 15, 9057–9091. Hyun, J.W., Chung, H.S., 2004. Cyanidin and malvidin from Oryza sativa cv. Heugjinjubyeo mediate cytotoxicity against human monocytic leukemia cells by arrest of G(2)/M phase and induction of apoptosis. J. Agric. Food Chem. 52, 2213–2217. Jing, P., Bomser, J., Schwartz, S., He, J., Magnuson, B., Giusti, M., 2008. Structure function relationships of anthocyanins from various anthocyanin-rich extracts on the inhibition of colon cancer cell growth. J. Agric. Food Chem. 56, 9391–9398. Kang, S.Y., Seeram, N.P., Nair, M.G., Bourquin, L.D., 2003. Tart cherry anthocyanins inhibit tumor development in ApcMin mice and reduce proliferation of human colon cancer cells. Cancer Lett. 194, 13–19. Krishnaraju, A.V., Rao, T.V.N., Sundararaju, D., 2005. Assessment of bioactivity of Indian medicinal plants using Brine shrimp (Artemia salina) lethality assay. Int. J. Appl. Sci. Eng. 2, 125–134. Leporatti, M.L., Corradi, L., 2001. Ethnopharmacobotanical remarks on the Province of Chieti town (Abruzzo, Central Italy). J. Ethnopharmacol. 74, 17–40. Mangathayaru, K., Umadevi, M., Reddy, C.U., 2009. Evaluation of the immunomodulatory and DNA protective activities of the shoots of Cynodon dactylon. J. Ethnopharmacol. 123, 181–184.
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LC–MS analysis, anticancer, antioxidant and antimalarial activities of Cynodon dactylon L. extracts Daycem Khlifi a,b , El Akrem Hayouni c , Alexis Valentin d , Sylvie Cazaux a , Béatrice Moukarzel d , Moktar Hamdi b , Jalloul Bouajila a,∗ a Université de Toulouse, Laboratoire des Interactions Moléculaires et Réactivité Chimique et Photochimique UMR CNRS 5623, Université Paul-Sabatier, 118 route de Narbonne, F-31062 Toulouse, France b Université de Carthage, Laboratoire d’Ecologie et de Technologie Microbienne, Institut National des Sciences Appliquées et de la Technologie (INSAT), B.P. 676, 1080 Tunis, Tunisia c Laboratoire des Substances Biologiquement Actives, Centre de Biotechnologie à l’Ecopark de Borj-Cedria, BP-901, Hamman-Lif, Tunisia d Université de Toulouse, UMR IRD-UPS 152 PHARMA-DEV, Université Paul-Sabatier, Faculté de Pharmacie, 35, Chemin des Maraîchers, F-31062 Toulouse, France
a r t i c l e
i n f o
Article history: Received 1 September 2012 Received in revised form 29 November 2012 Accepted 1 December 2012 Keywords: Cynodon dactylon LC–MS Anthocyanins Antioxidant activity Antimalarial activity Anticancer activity
a b s t r a c t Extracts (petroleum ether, dichloromethane, acetone, methanol/water (3/1) and water) from Cynodon dactylon L. were obtained by sequential method. The chemical composition of phenolics (6.9–57.41 g gallic acid equivalents/kg of dry mass), tannins (2.49–12.32 g catechin equivalent/kg of dry mass), anthocyanins (0–5.98 g cyanidin equivalent/kg of dry mass) and flavonoids (0–5.02 g quercetin equivalent/kg of dry mass) was evaluated. The extracts were evaluated for their antioxidant (DPPH and ABTS assays), antimalarial (Plasmodium falciparum) and anticancer (MCF-7) activities; the strongest activities were obtained by the water extract (IC50 = 57.21 ± 1.47 mg/L), the acetonic extract (IC50 = 38 mg/L) and the petroleum ether extract (IC50 = 39 mg/L), respectively. The results indicate a good correlations between anthocyanins quantity and the antimalarial activity (R2 = 0.79) and also to anticancer activity (R2 = 0.78). The LC–MS analysis of the extracts having the good activities anticancer and antimalarial, has revealed the presence of seven anthocyanins (delphinidin-3-Oacetylglucoside, petunidin-3-O-caffeoylglucoside-5-O-glucoside, petunidin-3-O-coumarylglucoside-5O-glucoside, malvidin-3-O-monoglucoside, delphinidin-3-O-acetylglucoside-pyruvic acid, petunidin-3O-acetylglucoside-5-O-glucoside and cyanidin-3,5-O-diglucoside), identified for the first time in this plant. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction Natural products have served as an important source of drugs since ancient times and a significant part of today’s drugs are somehow derived from natural sources. In the recent years, secondary plant metabolites (phytochemicals), previously with unknown pharmacological activities, have been extensively investigated as a source of medicinal agents (Krishnaraju et al., 2005). Cynodon dactylon L. (Gramineae, Poaceae), commonly known as “Njem” in Tunisia, possesses various medicinal properties such as antiviral, antimicrobial (Dhar et al., 1968), anti-inflammatory (Biswas and Mukherjee, 2003), immunomodulatory activity (Santhi and Annapoorani, 2010) and has significant application in treating dysentery, dropsy (Chopra and Handa, 1982), hypolipidemic and act as hypoglycemic agent (Leporatti and Corradi, 2001). Furthermore, the ethanol extract of aerial part has also marked
∗ Corresponding author. Tel.: +33 562256825; fax: +33 562256826. E-mail address: [email protected] (J. Bouajila).
central nervous system (CNS) depressant (Pal, 2008). In order to search for new molecules having antioxidant, antimalarial or anticancer activities, we have investigated C. dactylon. The objectives of this study were: (i) analysis of chemical composition of various extracts of whole plant C. dactylon (characterization of phenolics, tannins, flavonoids and anthocyanins of their extracts), (ii) investigation of antioxidant, antimalarial and anticancer activities of various extracts, (iii) the search of possible correlations between chemical compositions and antimalarial, anticancer and antioxidant activities and (iv) identification by LC–MS of molecule (s) responsible for the interesting activities while basing itself on best the correlations. 2. Materials and methods 2.1. Chemicals All chemicals used were of analytical reagent grade. All reagents were purchased from Sigma-Aldrich-Fluka (Saint-Quentin France): (sodium acetate (≥99%), potassium chloride (≥99%), gallic acid
0926-6690/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.12.030
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(≥99%), hydrochloric acid (36.5–38.0%), catechin (≥98%), quercetin (≥98%), cyanidin-3-glucoside (≥95%), ascorbic acid (≥99.0%)), (aluminum trichloride (99.99%), 1,1-diphenyl-2-picrylhydrazyl free radical (99%)), (methanol (≥99%), dichloromethane (≥99.5%), petroleum ether (≥98.5%), anhydrous sodium sulfate (≥99.0%), sodium carbonate (≥99.0%), Folin–Ciocalteu reagent 2 N, acetic), (potassium persulfate (≥99.0%), vanillin (≥98.0%), H2 SO4 (≥95%), 2,2 -azinobis-3-ethylbenzothiazoline-6-sulphonate (≥99.0%)). Chloroquine, RPMI 1640 medium (Lonza; Emerainville, France), Hepes, NaHCO3 , doxorubicin, fetal calf serum (Lonza). 2.2. Collection of plant material C. dactylon L. (whole plant) was collected in January 2009 from the central area of Tunisia, precisely from the Sidi Bouzid region. Specimens were identified by Dr. Bousaid Mohamed at the Department of Botany, National Institute of Applied Sciences and Technology (INSAT, Tunis) and voucher specimens were deposited at the Herbarium of the Department of Botany in the cited institute. 2.3. Extraction Plant was dried in air shade at room temperature, and the dry plant was powdered. 50 g of powders was extracted in a soxhlet system with 500 mL of different solvents of croissant polarity was used: petroleum ether (6 h at 40 ◦ C), dichloromethane (6 h at 40 ◦ C), acetone (6 h at 56 ◦ C), methanol/water (3/1) (6 h at 65 ◦ C) and water (6 h at 100 ◦ C). All organic extracts were concentrated by rotary evaporation under vacuum at 35 ◦ C. For the chemical analyses and the antioxidant activities, the extracts of petroleum ether and dichloromethane were re-suspended in the DMSO, whereas extracts of acetone and methanol/water were re-suspended in ethanol. For the antimalarial and anticancer activities, all extracts were re-suspended in the DMSO followed by dilution in the aqueous phase so that the DMSO does not exceed 1%. 2.4. Determination of total phenolic compounds by the Folin–Ciocalteu method The phenolics of each plant extract were determined by Folin and Ciocalteu (1927). The diluted solution of each extract (0.5 mL) was mixed with Folin–Ciocalteu reagent (0.2 N, 2.5 mL). This mixture was allowed to stand at room temperature for 5 min and then sodium carbonate solution (75 g/L in water, 2 mL) was added. After 1 h of incubation, absorbance was measured at 765 nm against appropriate solvent. A standard calibration curve was plotted using gallic acid (0–300 mg/L). The results were expressed as mg of gallic acid equivalents (GAE)/L of dry mass. 2.5. Condensed tannin content Catechins and proanthocyanidins reactive to vanillin were analyzed by the vanillin method of Broadhurst and Jones (1987). One milliliter (1 mL) of each extract solution was placed in a test tube and 2 mL of vanillin (1% in H2 SO4 7 M) in an ice bath and then incubated at 25 ◦ C. After 15 min, the absorbance of the solution was read at 500 nm. Concentrations were calculated as g catechin equivalents (CE)/kg dry mass from a calibration curve. 2.6. Total flavonoids determination The total flavonoids were estimated according to the Dowd method as adapted by Arvouet-Grand et al. (1994). A diluted solution (4 mL) of each extract was mixed with a solution (4 mL) of aluminum trichloride (AlCl3 ) in methanol (2%). The absorbance was
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read at 415 nm after 15 min against a blank sample consisting of a methanol (4 mL) and extract (4 mL) without AlCl3 . Quercetin was used as reference compound to produce the standard curve, and the results were expressed as g of quercetin equivalents (QE)/kg of dry mass. 2.7. Determination of total anthocyanin content Total anthocyanin content was measured with the pH differential absorbance method, as described by Cheng and Breen (1991). Briefly, absorbance of the extract was measured at 510 and 700 nm in buffers at pH 1.0 (hydrochloric acid–potassium chloride, 0.2 M) and 4.5 (acetic acid–sodium acetate, 1 M). The wavelength reading was performed after 15 min of incubation. Anthocyanin content was calculated using a molar extinction coefficient (ε) of 29,600 (cyanidin-3-glucoside) and absorbance of A = [(A510 − A700 )pH 1.0 − (A510 − A700 )pH 4.5 ]. Results were expressed as g cyanidin-3-glucoside equivalent (C3GE)/kg of dry mass. 2.8. Free radical scavenging activity DPPH test Antioxidant scavenging activity was studied using 1,1-diphenyl2-picrylhydrazyl free radical (DPPH) as described by Blois (1958) with some modifications; 1.5 mL of various dilutions of the test materials (pure antioxidants or plant extracts) were mixed with 1.5 mL of a 0.2 mM methanolic DPPH solution. After an incubation period of 30 min at 25 ◦ C, the absorbance at 520 nm, the wavelength of maximum absorbance of DPPH, were recorded as A(sample) . A blank experiment was also carried out applying the same procedure to a solution without the test material and the absorbance was recorded as A(blank) . The free radical-scavenging activity of each solution was then calculated as percent inhibition according to the following equation: % inhibition = 100
A(blank) − A(sample) A(blank)
Antioxidant activity extracts was expressed as IC50 , defined as the concentration of the test material required to cause a 50% decrease in initial DPPH concentration. Ascorbic acid was used as a standard. All measurements were performed in triplicate. 2.9. ABTS radical-scavenging test The radical scavenging capacity of the samples for the ABTS (2,2 -azinobis-3-ethylbenzothiazoline-6-sulphonate) radical cation was determined as described by Re et al. (1999). ABTS was generated by mixing a 7 mM of ABTS at pH 7.4 (5 mM NaH2 PO4 , 5 mM Na2 HPO4 and 154 mM NaCl) with 2.5 mM potassium persulfate (final concentration) followed by storage in the dark at room temperature for 16 h before use. The mixture was diluted with ethanol to give an absorbance of 0.70 ± 0.02 units at 734 nm using spectrophotometer. For each sample, diluted methanol solution of the sample (100 L) was allowed to react with fresh ABTS solution (900 L), and then the absorbance was measured 6 min after initial mixing. Ascorbic acid was used as a standard and the capacity of free radical scavenging was expressed by IC50 (mg/L) values calculated denote the concentration required to scavenge 50% of ABTS radicals. The capacity of free radical scavenging IC50 was determined using the same previously used equation for the DPPH method. All measurements were performed in triplicate. 2.10. Antimalarial activity The chloroquine-resistant FcB1-Columbia strain of Plasmodium falciparum (IC50 for chloroquine: 186 nM) was cultured
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Table 1 Chemical composition, antimalarial activity and anticancer activity of Cynodon dactylon extracts. Type of extract
Polyphenols (GAE)a
Petroleum ether Dichloromethane Acetone Methanol (75%) Water Chloroquine Doxorubicin
6.9 23.42 57.41 23.35 44.56
± ± ± ± ±
0.44 0.97 0.46 1.2 0.72
Tannins (CE)a 4.59 12.32 10.19 2.49 2.87
± ± ± ± ±
0.03 0.14 0.06 0.07 0.03
Flavonoids (QE)a
Anthocyanins (C3GE)a
Antimalarial activityb
Anticancer activityb
nd nd 4.80 ± 0.063 5.02 ± 0.09 5.07 ± 0.04
5.64 ± 0.11 nd 5.98 ± 0.08 3.04 ± 0.03 0.16 ± 0.01
48 >100 38 >100 >100 0.10 ± 0.09
39 >100 44 >100 >100 0.22 ± 0.04
Standard deviations (SD) did not exceed 5%. nd: not detect. a g/kg dry mass. b IC50 (mg/L).
continuously according to Trager and Jensen (1976) with modifications described by Benoit et al. (1996). The IC50 values for chloroquine were checked every 2 months, and we observed no significant variations. The parasites were maintained in vitro in human red blood cells (O±; EFS; Toulouse, France), diluted to 4% hematocrit in RPMI 1640 medium (Lonza; Emerainville, France) supplemented with 25 mM Hepes and 30 M NaHCO3 and complemented with 7% human AB + serum (EFS). Parasites cultures were synchronized by combination of magnetic enrichment and by d-sorbitol lysis (5% of d-sorbitol in sterile water). The antimalarial activity of various extracts was evaluated by a radioactive micromethod. Tests were performed in triplicate in 96-well culture plates (TPP) with cultures mostly at ring stages (synchronization interval, 16 h) at 0.5–1% parasitemia (hematocrit, 1.5%). Parasite culture was incubated with each sample for 48 h. Parasite growth was estimated by [3 H]-hypoxanthine (PerkinElmer; Courtaboeuf, France) incorporation, which was added to the Plates 24 h before freezing. After 48 h incubation, plates were frozen-defrosted and each well was harvested on a glass fiber filter. Incorporated [3 H]-hypoxanthine was then determined with a betacounter (1450-Microbeta Trilux; Wallac-Perkin Elmer). The control parasite cultures, free from any sample, was referred to 100% growth. IC50 were determined graphically in concentration versus percent inhibition curves. Chloroquine diphosphate was used as positive control. The antimalarial activity of sample was expressed by IC50 , representing the concentration of drug that induced a 50% parasitemia decrease compared to the positive control culture referred to as 100% parasitemia. According to the literature concerning plant antiplasmodial activities a sample is very active if IC50 < 5 mg/L, active if IC50 between 5 and 50 mg/L, weakly active if IC50 between 50 and 100 mg/L and inactive if IC50 > 100 mg/L (Ouattara et al., 2006). 2.11. Anticancer activity Cytotoxicity of each sample was estimated on human breast cancer cells (MCF7). The cells were cultured in the same conditions as those used for P. falciparum, except for the 10% human serum, which was replaced by 10% fetal calf serum (Lonza). For the determination of pure compound activity, cells were distributed in 96-well plates at 3 × 104 cells/well in 100 L, and then 100 L of culture medium containing sample at various concentrations were added. Cell growth was estimated by (3 H)-hypoxanthine incorporation after 48 h incubation exactly as for the P. falciparum assay. The (3 H)-hypoxanthine incorporation in the presence of sample was compared with that of control cultures without sample (positive control being doxorubicin). 2.12. LC–MS analysis Analyses were performed using an Agilent 1100 liquid chromatograph (Agilent Technologies USA) consisting of a binary pump
and an automatic sampler. The extracts were filtered through a Millex HA 0.45 m filter (Millipore Corp.) before injection (50 L). Chromatographic separation was performed on a Phermerex C18 column (25 cm × 4.6 mm i.d., 5 m particle size) equipped with a 5 m C18 guard column. Column temperature was 25 ◦ C. The mobile phases were (A) formic acid/water (1:99, v/v) and (B) methanol. The gradient used was 0–43 min, 10–90% B; 43–45 min, 90–10% B; 45–50 min, 10% B. The solvent flow rate was 0.9 mL/min. Analysis was achieved by positive ion mode using a Q Trap mass spectrometer (Applied Biosystems/MDS Sciex, USA) with a Turbo ion spray source at 480 ◦ C. The ion spray voltage was set at 5000 V. The declustering potential (DP) was 35 V. The collision energy (CE) was 21 V. Nitrogen was used as nebulizing and collision gas. The data were collected and processed by Analyst 1.6. software (Applied Biosystems/MDS Sciex, USA). 2.13. Statistical analysis All data were expressed as means ± standard deviations of triplicate measurements. The confidence limits were set at P < 0.05. Correlations were carried out using the correlation and regression in the EXEL program. 3. Results and discussion 3.1. Extraction yields The sequential method adapted to obtain different C. dactylon extracts was presented as follows: petroleum ether, dichloromethane, acetone, methanol/water (3/1) and water. The highest yields were achieved by methanol/water (3/1) (8.24%), followed by dichloromethane (2.75%), acetone (2.4%), petroleum ether (1.25%) and the lowest was water (1.2%). One study (Gangwar et al., 2011) is provided in the literature concerning extraction yield for this plant. The extraction was carried out with petroleum ether, chloroform, ethanol and chloroform/water with successive yields of 0.85, 1.25, 2.85 and 3.65%. Our total yield (15.84%) is approximately twice higher than of Gangwar et al. (2011) (8.6%). The yield is affected by several factors such as species, geographical location, harvest time, plant part used and isolation method. 3.2. Chemical composition: phenolics, tannins, flavonoids and anthocyanins This is the first study which recorded the tannins and anthocyanins content of extracts from C. dactylon and from genus Cynodon. All results are presented in Table 1. The amount of total phenolics varied in the different extracts and ranged from 6.9 ± 0.44 to 57.41 ± 0.46 g GAE/kg of dry mass. The highest amount of phenolics was in acetone extract (57.41 ± 0.46 g GAE/kg of dry mass), followed by water extract (44.56 ± 0.72 g GAE/kg of dry mass); petroleum ether was poor in phenolics (6.9 ± 0.44 g GAE/kg of dry mass). This variation was due to the polarity of phenolics. Polar
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Fig. 1. Antioxidant activity of Cynodon dactylon by DPPH assay. 1: petroleum ether extract, 2: dichloromethane extract, 3: acetone extract, 4: methanol/water (3/1) extract, 5: water extract and 6: reference standard: ascorbic acid. Standard deviations (SD) did not exceed 5%.
Fig. 2. Antioxidant activity of Cynodon dactylon by ABTS assay. 1: petroleum ether extract, 2: dichloromethane extract, 3: acetone extract, 4: methanol/water (3/1) extract, 5: water extract and 6: reference standard: ascorbic acid. Standard deviations (SD) did not exceed 5%.
fractions had more phenolics than had non-polar fractions. Wegner (2011) quantified phenolics and obtained small quantity (maximum 0.589 mg GAE/g of dry mass). In another work, Mangathayaru et al. (2009) recorded 47 ± 0.33 g GAE/kg in the fresh juice of C. dactylon shoots. Their results showed that the content of phenolics was less important compared to what we found. The difference between our results and their results was probably due to the difference of the part of the plant used, we have used whole plant and they have used shoots. There is no study in the literature which investigated the phenolics content in other species of the genus Cynodon. The amount of tannins in dichloromethane extract was richer (12.32 ± 0.14 g CE/kg of dry mass) followed by acetone extract (10.19 ± 0.06 g CE/kg of dry mass). Methanol 75% was poor in tannins (2.49 ± 0.07 g CE/kg of dry mass). Non-polar fractions had more tannins than had polar fractions. For the amount of flavonoids, the highest quantity was in water extract (5.07 ± 0.04 g QE/kg of dry mass), followed by methanolic 75% extract (5.022 ± 0.09 g QE/kg of dry mass). Petroleum ether and dichloromethane did not contained flavonoids. Wegner (2011) has obtained small quantity for flavonoids (0.123–0.255 mg/g catechin equivalent/g of dry mass). For anthocyanins, the highest amount was obtained by acetonic extract (5.98 ± 0.09 g C3GE/kg of dry mass), followed by petroleum ether extract (5.46 ± 0.11 mg C3GE/kg of dry mass); dichloromethane has not contained anthocyanins. To our knowledge, no study cited the quantification of tannins and or anthocyanins for this specie.
the difference in climate between Tunisia and India, geographical origin, harvesting time and growing conditions.
3.3. Antioxidant activity 3.3.1. DPPH assay The DPPH free radical method determines the antiradical power of antioxidants. The antioxidant activity of water extract (Fig. 1) was superior to all samples tested with an IC50 value of 152.96 ± 2.12 mg/L, followed by methanolic extract (203.29 ± 1.38 mg/L) and by acetonic extract (206.69 ± 2.57 mg/L). On the other hand, extracts prepared with petroleum ether or dichloromethane have exhibited a poor radical-scavenging activity (921.76 ± 2.83 mg/L and 1053.12 ± 1.71 mg/L, respectively). We can deduce, as seen in Fig. 1, that extracts obtained using high polarity solvents were considerably more effective radical-scavengers than were those using low polarity solvents. Similar results for C. dactylon were found by Albert-Baskar and Ignacimuthu (2010), in the same order in terms of solvents polarity. They have found antioxidant activity of methanol extract was superior to all samples tested (IC50 = 63.03 ± 4.82 mg/L), followed by ethyl acetate extract (311.05 ± 23.69 mg/L) and by hexane extract (874.15 ± 66.56 mg/L). The difference between our results and the Albert-Baskar and Ignacimuthu (2010) work is due to: the difference of the plant part used, we have used all plant and they have used the roots; also, the difference of method of extraction and the solvents, we have used the soxhlet, they were used the cold percolation method, and finally
3.3.2. ABTS assay For water extract (Fig. 2), a good antioxidant activity value was found (IC50 = 57.21 ± 1.74 mg/L), followed by acetonic extract (IC50 = 179.77 ± 2.19 mg/L). This result confirmed the agreement between phenolics and antioxidant activity where acetonic and water extracts presented higher total phenolic quantities (57.41 ± 0.46 and 44.56 ± 0.72 g GAE/kg of dry material, respectively). In other work, Auddy et al. (2003) showed that the water infusion of C. dactylon has a moderate antioxidant activity, by ABTS, with IC50 = 273.64 mg/L. This result showed that the antioxidant activity was 4 times less important compared to our results. As a result of the present study, the water extract was found to be effective antioxidant in different in vitro assays. 3.4. Antimalarial activity In the present study, antimalarial activity of acetonic extract was superior to all samples tested with an IC50 = 38 mg/L (Table 1), followed by petroleum ether extract (IC50 = 48 mg/L). By referring to the literature, this result showed an active antimalarial activity of C. dactylon (Ouattara et al., 2006). This may be indicative of a significant potential for isolating pure compounds with much higher antimalarial activity (in term of IC50 ) from acetonic and petroleum ether extracts. Recent study by Bagavan et al. (2011) showed that C. dactylon leaves, ethyl acetate and methanol extracts, were no active up to 100 mg/L against P. falciparum strains 3D7, Dd2 and INDO. The antimalarial activity could come from others organs, not leaves. We found a good correlation between anthocyanins quantity in extracts and the antimalarial activity (R2 = 0.79). El Babili et al. (2010) have obtained also a good correlation between anthocyanins quantity and the antimalarial activity (R2 = 0.82). According to our knowledge, no anthocyanin molecule is quoted in the literature for an antimalarial activity. 3.5. Cytotoxicity C. dactylon extracts showed a significant cytotoxic activity against human breast cancer cells (MCF7). Cytotoxicity of petroleum ether extract was superior to all samples tested with an IC50 = 39 mg/L (Table 1), followed by acetonic extract (IC50 = 44 mg/L). Dichloromethane, water and methanol 75% extracts were not actives (IC50 > 100 mg/L). Albert-Baskar and Ignacimuthu (2010) obtained different results when the ethyl acetate extract of C. dactylon roots was the more active (IC50 = 30 mg/L) against human breast cancer cells (MCF7), followed by hexane extract (IC50 = 40 mg/L) and by methanol extract (IC50 = 55 mg/L). In our work, we found a good correlation between anthocyanins quantity in extract and the anticancer activity (R2 = 0.78).
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Table 2 Anthocyanins and anticancer activity. Name
Delphinidin
Delphinidin 3-O-rutinoside
Delphinidin 3-O-glucoside
Structure
Cancer (human tumor cell lines)
IC50 or % of inhibition
LoVo (colorectal) LoVo/ADR (colorectal) MCF-7 (breast)
11.4a 4.97a 162.4a
MCF-7 (breast) HCT-116 (colon) NCI-H460 (lung) SF-268 (central nervous system) AGS (stomach)
30a 38a 76a 100a
MCF-7 (breast) LNCaP (prostate)
References
Cvorovic et al. (2010) Zhang et al. (2005)
Vareed et al. (2006)
100a
25a
Vareed et al. (2006) a
0.143
˜ Munoz-Espada and Watkins (2006)
HT-29 (colon) HCT-116 (colon) LoVo/ADR (colorectal) LoVo (colorectal) U937 (leukemia)
18.09a 24.41a 7.52a 13.47a 60a
Cyanidin 3-glucoside
HS578 T (breast) HL-60 (leukemia) HT-29 (colon)
6.73a 62.2a 14a
Chen et al. (2005) Dai et al. (2009) Jing et al. (2008)
Cyanidin-3-rutinoside
HL-60 (leukemia)
29.7a
Feng et al. (2007)
Cyanidin-3-O-bglucopyranoside
Jurkat (leukemia) HL-60 (leukemia)
174.9a 16.6a
Fimognaria et al. (2004)
MCF-7 (breast) AGS (stomach) HCT-116 (colon) NCI H 460 (lung) SF-268 (central nervous system) LoVo/ADR (colorectal) LoVo (colorectal) U937 (leukemia)
32.2a 84.7a 71.7a 88.1a 143.3a
Zhang et al. (2005)
Cyanidin
Malvidin
>33.12a >33.12a 40a
Kang et al. (2003) Cvorovic et al. (2010) Hyun and Chung (2004)
Cvorovic et al. (2010) Hyun and Chung (2004)
D. Khlifi et al. / Industrial Crops and Products 45 (2013) 240–247
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Table 2 (Continued) Name
Structure
Petunidin
c
MCF-7 (breast) AGS (stomach)
170.7a 24b , c
MCF-7 (breast) AGS (stomach) SF-268 (central nervous system) NCI H 460 (lung) HCT-116 (colon)
Pelargonidin
a
IC50 or % of inhibition
HS578 T (breast) NCI-H460 (lung) SF-268 (central nervous system) AGS (stomach)
Peonidin 3-glucoside
b
Cancer (human tumor cell lines)
23.17a 50a 60a
References
Zhang et al. (2005)
Chen et al. (2005) Vareed et al. (2006)
a
75
136.5a 176.6a 63b , c
Zhang et al. (2005)
182.1a 154.3a
IC50 (mg/L); Percentage of inhibition (%). Anthocyanins were tested at 100 mg/L.
Previous works have indicated that pure anthocyanins from fruits and vegetables have exhibited anti-proliferative activity toward multiple cancer cell types in vitro. The summary of all the molecules evaluated against the multiple cancer is given in Table 2. We also deduce that our ether petroleum
and acetone extracts can contain an important amount of anthocyanins active against breast cancer. These two extracts contain a small anthocyanins quantity (maximum 5.98 ± 0.08 C3GE g/kg dry mass) and have activities comparable with the pure products.
Fig. 3. TIC (LC–MS) and extract ions (m/z) from acetone extract.
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Table 3 Anthocyanins identified by LC–MS in actives extracts. Compounds
Molecular ion [M–H]− (m/z)
Retention time (min) Petroleum ether
Retention time (min) Acetone
References
Delphinidin-3-O-acetylglucoside Petunidin-3-O-caffeoylglucoside-5-O-glucoside Petunidin-3-O-coumarylglucoside-5-O-glucoside Malvidin-3-O-monoglucoside Delphinidin-3-O-acetylglucoside-pyruvic acid Petunidin-3-O-acetylglucoside-5-O-glucoside Cyanidin-3,5-O-diglucoside
507 803 787 493 575 683 611
– – – – – 40.84 42.75
3.12 17.14 17.54 23.16 25.24 – –
He et al. (2010) He et al. (2010) He et al. (2010) He et al. (2010) He et al. (2012) He et al. (2010) He et al. (2010)
–, not detected.
3.6. Identification of anthocyanins by LC–MS for ether petroleum and acetone extracts These biological activities (antimalarial and anticancer) and the interesting correlations encouraged us to push work to identify anthocyanins by LC–MS in acetone and ether petroleum extracts. We carried out the analysis of the two extracts by LC–MS. We optimized the detection of the mass spectrometer by structures similar to those which we seek (anthocyanins). We applied the mode selected ion monitoring (SIM) by seeking anthocyanins already identified in fruits and vegetables and cited in the literature (He et al., 2012, 2010). Approximately, a hundred molecules were followed and the results given seven anthocyanins (Fig. 3 and Table 3) identified for the first time in this plant: delphinidin-3-Oacetylglucoside, petunidin-3-O-caffeoylglucoside-5-O-glucoside, petunidin-3-O-coumarylglucoside-5-O-glucoside, malvidin-3-Omonoglucoside, delphinidin-3-O-acetylglucoside-pyruvic acid, petunidin-3-O-acetylglucoside-5-O-glucoside and cyanidin-3,5-Odiglucoside. All these molecules were never tested against cancer nor against the malaria. This result confirms the hypothesis of the correlations obtained. 4. Conclusions C. dactylon L. extracts were investigated for their chemical composition, antioxidant antimalarial and anticancer activities. This is the first study that investigated the good antimalarial and anticancer activities of C. dactylon and the genus Cynodon. To confirm correlations between anthocyanins and antimalarial and anticancer activities of this plant, we identified by LC–MS five anthocyanins in acetonic extract and two anthocyanins in ether petroleum extract. The quantity of the anthocyanins by extract do not exceed 0.6%, therefore if the activity comes from these anthocyanins, the IC50 will be more interesting than 250 g/L. It is very interesting to push the work of test of these pure molecules against these diseases and to split the extracts at the same time in order to check if there is presence of others anthocyanins non known in the literature and that one could not be check their presence by LC–MS. Acknowledgments We are very grateful to Pr. Boussaid Mohamed Head of Biotechnology Laboratory, National Institute of Applied Sciences and Technology (INSAT, Tunis) for identification of plant. References Albert-Baskar, A., Ignacimuthu, S., 2010. Chemopreventive effect of Cynodon dactylon (L) Pers. extract against DMH-induced colon carcinogenesis in experimental animals. Exp. Toxicol. Pathol. 62, 423–431. Arvouet-Grand, A., Vennat, B., Pourrat, A., Legret, P., 1994. Standardisation d’un extrait de propolis et identification des principaux constituants. J. Pharm. Belg. 49, 462–468. Auddy, B., Ferreira, M., Blasina, F., Lafon, L., Arredonodo, F., Dajas, F., Tripathi, P.C., Seal, T., Mukherjee, B., 2003. Screening of antioxidant activity of three Indian
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