Towards the use of Cupressus sempervirens L. organic extracts as a source of antioxidant, antibacterial and antileishmanial biomolecules

Industrial Crops & Products 131 (2019) 194–202

Contents lists available at ScienceDirect

Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop

Towards the use of Cupressus sempervirens L. organic extracts as a source of antioxidant, antibacterial and antileishmanial biomolecules

T

Safa Rgueza, Rym Essidb, Papetti Adelec, Kamel Msaadaa, , Majdi Hammamia, Khaoula Mkadminia, Nadia Faresb, Olfa Tabbeneb, Salem Elkahouib, Desirèe Portellic, Riadh Ksouria, Ibtissem Hamrouni Sellamia ⁎

a

Laboratory of Aromatic and Medicinal Plants, Center of Biotechnology of Borj Cedria, BP 901, Hammam-Lif, 2050, Tunisia Laboratory of Bioactive Substances, Center of Biotechnology of Borj Cedria, BP 901, Hammam-Lif, 2050, Tunisia c Nutraceutical & Food Chemical-Toxicological Analysis Laboratory, Department of Drug Sciences, University of Pavia, Viale Taramelli, Italy b

ARTICLE INFO

ABSTRACT

Keywords: Cupressus sempervirens Organic extracts Antioxidant Antibacterial Antileishmanial HPLC-MSn

Cupressus sempervirens L. is largely used in traditional medicine as an antimicrobial agent. The present study investigated the antioxidant, antibacterial and antileishmanial activities of C. sempervirens organic extracts at different phenological stages. Antioxidant activity was determined by DPPH (2,2-diphenyl-1-picrylhydrazylradical) scavenging assay, ferric reducing power and total antioxidant capacity. The antibacterial activity was evaluated against five clinical strains by disk diffusion method and minimum inhibitory concentration (MIC). The antileishmanial activity was determined against promastigote and amastigote forms of Leishmania (L.) infantum and L. major. Results of antioxidant activity showed that methanolic extract from vegetative stage had the most important activity. The ethyl acetate extract of C. sempervirens from flowering stage was the most active against Bacillus cereus (ATCC 14579) and Staphylococcus aureus (ATCC 25923) with MIC of 100 μg/mL and 50 μg/mL, respectively. Interestingly, this extract exhibited high antileishmanial activity against promastigote form of L. infantum and L. major (IC50 = 1.47 and 2.8 μg/mL, respectively) and amastigote form (IC50 = 3.61 and 5.42 μg/mL, respectively). Furthermore, ethyl acetate extract showed low cytotoxicity on macrophage cells Raw264.7 with selectivity index of 34.15 and 17.93 for L. infantum and L. major, respectively. The identification by HPLC and HPLC-MSn of active extracts of C. sempervirens revealed that major compounds of methanolic extract from vegetative stage and ethyl acetate extract from flowering stage were cupressuflavone and amentoflavone. Based on these results, C. sempervirens extracts could be used as an alternative to chemical drugs for the treatment of oxidative stress and infectious diseases.

1. Introduction In recent years, medicinal plants are being screened more efficiently to be considered as a novel therapeutic alternative and may substitute chemical based food preservative. In fact, plants constitute an important source of many pharmaceutical drugs and biological activities notably antioxidant, antimicrobial and antiparasitic properties (Lobo et al., 2010; Mothana et al., 2014; Guendouze-Bouchefa et al., 2015). Cupressus (C.) sempervirens (cypress) is an ornamental tree belonging to the family of cupressaceae. It is native to the Mediterranean basin. However, this plant was distributed in North Africa, Asia, Southern Europe and Northern America (Chaudhary et al., 2012). The chemical analysis of C. sempervirens showed that this plant contained alkaloids

(0.7%), flavonoids (0.2%), tannins (0.31%), saponins (1.9%), phenols (0.067%) and many other molecules (Khabir et al., 1987; Al-Othman et al., 2012). Many pharmacological studies revealed that C. sempervirens exhibited an antimicrobial (Zhang et al., 2012), antiviral (Amouroux et al., 1998), insecticidal (Moussa et al., 2011), antioxidant (Asgary et al., 2013), anticancer (Verma et al., 2014) and many other biological effects (Koriem, 2009; Tumen et al., 2012). Asgary et al. (2013) highlighted the antiradical power of both methanolic and chloroformic extracts of C. sempervirens. Also, Shahid et al. (2013) have confirmed the antioxidant power of methanolic extract obtained from fresh leaves of C. sempervirens. On the other hand, it has been shown that C. sempervirens aqueous and chloroformic extracts were characterized by high antimicrobial activity against gram positive

Abbreviations: GAE, gallic acid equivalent; OEs, organic extracts ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (K. Msaada). https://doi.org/10.1016/j.indcrop.2019.01.056 Received 1 October 2018; Received in revised form 20 January 2019; Accepted 25 January 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

Industrial Crops & Products 131 (2019) 194–202

S. Rguez et al.

bacteria Bacillus subtillus, Proteus vulgaris and Staphylococus aureus (Boukhris et al., 2012). As for methanolic extract and at a lesser degree ethanolic extract of C. sempervirens, these two extracts were found to exhibit an antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeroginosa, Echerchia coli, Klebsiella pneumonia and Salmonella typhimurium (Zhang et al., 2012). Furthermore, two diterpenes: taxodione and 6-deoxytaxodione, isolated from the ethanolic extract of C. sempervirens, displayed high antileishmanial activity against Leishmania donovani promastigotes (Zhang et al., 2012). AlMusayeib et al. (2012) demonstrated that non relevant results of methanolic extract of C. sempervirens were found against L. infantum. This extract also exhibited a noticeable cytotoxic effect against MRC-5. Diseases resulting from oxidative stress, bacteria and protozoa infections were the most common public health problems (Tay et al., 2011; Ramdane et al., 2017). Traditionally, the control of these diseases was performed by the use of chemical drugs. However, for a long period, microorganisms could develop resistance against chemical agents. Moreover, these products were toxic and caused different problems to human health. Accordingly, considerable attention has been given to identify new alternative products derived from medicinal plants with antioxidant and antimicrobial properties (Duarte et al., 2016). The present study aimed in the first part to evaluate polyphenols, flavonoids and condensed tannins content in organic extracts (OEs) of C. sempervirens and to assess the antioxidant activity of different OEs through DPPH scavenging assay, ferric reducing antioxidant power and total antioxidant capacity. In the second part, an evaluation of the antiinfectious capacity through antibacterial and antileishmanial activities of the OEs of C. sempervirens collected at three phenological stages was performed and to identify the chemical composition of active extracts.

material (DW). An aliquot (0.125 mL) of a suitable sample was accordingly added to 0.5 mL of deionized water and 0.125 mL of concentrated Folin–Ciocalteu reagent were mixed and incubated at room temperature. After 1 min, 1.5 mL of 7% sodium carbonate (Na2CO3) solution was added. The final mixture was shaken thoroughly and then incubated for 90 min in the dark at room temperature. The absorbance of all samples was measured at 760 nm using a spectrophotometer. 2.3.2. Total flavonoid contents Total flavonoid contents were measured according to Dewanto et al. (2002). An aliquot of diluted sample or standard solution of (+)-catechin was added to a 75 μL of NaNO2 (5% w/v) solution and mixed for 6 min, before adding 0.15 mL AlCl3 (100 g/L). After 5 min of incubation, 0.5 mL of NaOH (4% w/v) was added. The final volume was adjusted to 2.5 mL with distilled water and thoroughly mixed. Absorbance of the mixture was determined at 510 nm against the same mixture, without the sample, as a blank. Total flavonoid content was expressed as mg quercitin equivalents (CE) per g DW, through the calibration curve of (+)-quercitin. The calibration curve range was 50–400 μg/mL ( r2=0.99). 2.3.3. Total condensed tannins The content of total condensed tannins was estimated according to vanillin assay described by Sun et al. (1998). Briefly, 3 mL of methanol vanillin solution (4% w/v) and 1.5 mL of concentrated H2SO4 were added to an aliquot of 50 μL of properly diluted sample. Absorbance was determined at 500 nm against the solvent of extraction as a blank. Total condensed tannins were expressed as mg catechin/g DW using the calibration curve of catechin with concentrations ranging from 50 to 400 μg/mL ( r2=0.99).

2. Material and methods

2.4. Antioxidant activities of extracts

2.1. Plant material

2.4.1. DPPH radical scavenging assay The donation capacity of the obtained extracts was measured by bleaching of the purple-colored solution of the DPPH radical according to the method of Hanato et al. (1988). A total of 1 mL of extract at different concentrations was added to 0.5 mL of DPPH at 0.2 mM. The mixture was shaken vigorously and kept at room temperature for 30 min. The absorbance of the resulting solution was then measured at 517 nm. The antiradical activity was expressed as IC50 (μg/mL); the concentration required to cause 50% of DPPH inhibition. The ability to scavenge the DPPH radical was calculated using the following equation:

Aerial parts of C. sempervirens were harvested from plants located in the region of Akouda (Latitude 35° 53′ 33. 54″ N / Longitude 10° 34′ 19. 58″ E) on the central coasts of Tunisia. The identity of the plant was confirmed by Prof. Abderrazzak Smaoui and a voucher specimen was deposited in the herbarium at the Biotechnological Centre of BorjCedria. Aerial parts (leaves, fruits and terminal branches) were harvested at three phenological stages: vegetative, flowering, and fruiting stages during the year 2013. Fresh samples were air dried at shade and ambient temperature (24 °C).

DPPH scavenging effect % = [(Ao–A1)/Ao] x 100

2.2. Extraction of phenolic compounds

Where Ao was the absorbance of the control at 30 min and A1 was the absorbance of the sample at 30 min. BHT was used as a positive control.

Phenolic compounds were extracted by maceration into pure solvents according to the method of Mau et al. (2001). Briefly, 1 g of dried and powdered sample was immersed into 10 mL of pure organic solvents with different polarity. For this, five solvents were added: diethyl ether, ethyl acetate, dichloromethane, methanol, and water. Suspensions were stirred for 24 h in darkness at 4 °C. Each mixture was then filtered through a Whatman filter paper (No. 4) and the macerates were evaporated to dryness under vacuum and stored at 4 °C for further analysis.

2.4.2. Ferric-Reducing antioxidant power The method of Oyaizu (1986) was used to assess the reducing power of different extracts. A total of 1 mL extracts at different concentrations was mixed with 2.5 mL of sodium phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide [K3Fe(CN)6]. The mixture was incubated in a water bath at 50 °C for 20 min. Then, 2.5 mL of 10% trichloroacetic acid was added to the mixture that was centrifuged at 650 g for 10 min. The supernatant (2.5 mL) was then mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride solution. The intensity of the blue-green color was measured at 700 nm. Results were expressed as EC50 (mg/mL) which was the extract concentration at which the absorbance was 0.5 for the reducing power and was calculated from the graph of absorbance at 700 nm against extract concentration. Ascorbic acid was used as positive control.

2.3. Phenolic composition of extracts 2.3.1. Total phenolic contents Total phenolic content was evaluated using the Folin-Ciocalteu reagent, following Singleton’s method slightly modified by Dewanto et al. (2002). A calibration curve of gallic acid (ranging from 50 to 400 mg/ mL) was prepared and the results, determined by the regression equation of the calibration curve (y=–0.67+62.94x, r2=0.99), were expressed as μg gallic acid equivalents (GAE) per g dry weight of raw

2.4.3. Total antioxidant capacity The total antioxidant capacity of all extracts was evaluated by the 195

Industrial Crops & Products 131 (2019) 194–202

S. Rguez et al.

method of the phosphomolybdenum according to Prieto et al. (1999). The test was based on the reduction of ions Mo (VI) to Mo (V) by the antioxidant molecules present in the plant extract. Following this reduction reaction, a green complex phosphate/Mo (V) was formed in acidic medium. The absorbance measured at 695 nm was then evaluated to measure the total antioxidant capacity of the extract and results were expressed in gallic acid equivalents (GAE). After that, 100 μL of the diluted extract was mixed with 1 mL of a reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM molybdate ammonium). After stirring, the mixture was kept in a water bath at a temperature of 90 °C for 90 min. The absorbance was measured at 695 nm with a UV/visible spectrophotometer, referring to a control without extract. The total antioxidant capacity of each sample was expressed as mg of GAE per g of dry matter.

50%. Negative and positive control corresponding to untreated and amphotericin B treated parasites respectively were added (Essid et al., 2015). All tests were performed in triplicate. 2.6.3. Antiamastigote assay Macrophage cells were infected using logarithmic growing phase promastigotes at a parasite macrophage ratio of 10:1 and incubated for 2 h at 37 °C in 5% CO2. Monolayers cells were washed with PBS to remove free promastigotes and further incubated for 4 h. Extracts were added at varying concentrations ranging from 0.78 to 200 μg/mL for an additional 48 h (Torres-Santos et al., 1999). Cultures were then fixed with absolute methanol, stained with 10% Giemsa and examined under light microscope. Intracellular amastigotes were counted in at least 100 macrophages for each sample. Results were expressed as percent of reduction in the infection rate (IR) following extract treatment versus untreated control (De Muylder et al., 2011): % IR = 100–[(infection rate of the treated culture/infection rate of the untreated culture) × 100]. The infection rates were calculated by multiplying the percentage of infected macrophages by the number of amastigotes per infected macrophages (Ramdane et al., 2017). The IC50 were determined using GraphPad non-linear regression equation and triplicate determinations were performed.

2.5. Antibacterial activity 2.5.1. Strains collection For antibacterial activity, all extracts were tested against Gram positive bacteria including Bacillus (B.) cereus (ATCC 14579), Listeria (L.) monocytogenes (ATCC 19115) and Staphylococcus (S.) aureus (ATCC 25923) and Gram negative bacteria such as Escherichia (E.) Coli (ATCC 35214) and Pseudomonas (P.) aeruginosa (ATCC 27853). Bacterial strains were obtained from the collection of the "Laboratory of Bioactive Substances, Biotechnology Center of Borj Cedria, Tunisia" and maintained in LB medium.

2.6.4. Cytotoxicity assay and selectivity index Cytotoxicity of the C. sempervirens extracts was evaluated on murine macrophagic cells (Raw264.7). Macrophages were maintained in RPMI1640 medium supplemented with 10% FBS, antibacterial and antifungal solution (Gibco, USA) and were incubated at 37 °C in a humidified 5% CO2 atmosphere. Macrophages viability was controlled microscopically by counting cells after staining with 0.1% trypan blue solution. Macrophages were initially seeded in 96-well tissue culture plate at 105 cells/well and allowed to adhere overnight. The medium was then replaced with a fresh one containing different concentrations of extract (from 0.48 μg/mL to 1 mg/mL). After 72 h of incubation at 37 °C, viability was estimated by the MTT test as described above and selectivity index (SI) was determined as the ratio IC50 macrophage/IC50 parasite (Ramdane et al., 2017).

2.5.2. Disc diffusion assay The antibacterial activity of C. sempervirens extracts were assessed using the disk diffusion method as described previously. Briefly, bacterial suspensions were adjusted to 108 CFU/mL and were uniformly inoculated on Muller-Hinton (MH) agar plates. Different concentrations of extract (ranging from 31.25 μg/mL to 1 mg/mL) were deposed on a filter paper disks then applied on the agar surface. Plates were incubated for 24 h at 37 °C. Tetracycline (30 μg/disk) was used as positive control. Clear inhibition zones around the disks indicated antibacterial activity. 2.5.3. Minimal inhibitory concentration For a diameter zone inhibition over than 10 mm, the minimal inhibitory concentration value (MIC) defined as the lowest concentration of the extract showing total bacterial inhibition, was calculated as described previously (Hassim et al., 2015). Briefly, two fold serial dilution of active extract were placed in 96-well microtiter plates. Bacterial suspension was added at 106 CFU/mL than plates were incubated overnight at 37 °C. All experiments were conducted in triplicate.

2.7. Identification and quantification of active extracts by HPLC-DAD The identification of phenolic compounds was done using HPLC system (consisting of a vacuum degasser, an autosampler, and a binary pump with a maximum pressure of 400 bar; Agilent 1260, Agilent technologies, Germany) equipped with a reversed phase C18 analytical column of 4.6 x 100 mm and 3.5 μm particle size (Zorbax Eclipse XDB C18). The DAD detector was set to a scanning range of 200–400 nm. Column temperature was maintained at 25 °C. The injected sample volume was 2 μL and the flow-rate of mobile phase was 0.4 mL/min. Mobile phase B was milli-Q water consisted of 0.1% formic acid and mobile phase A was methanol. The optimized gradient elution was illustrated as follows: 0–5 min, 10–20% A; 5–10 min, 20–30% A; 10–15 min, 30–50% A; 15–20 min, 50–70% A; 20–25 min, 70–90% A; 25–30 min, 90–50% A; 30–35 min, return to initial conditions. For the quantitative analysis, a calibration curve was obtained by plotting the peak area against different concentrations for each identified compounds at 280 nm: The obtained curves for all identified compounds showed a good linearity (with an average of r2=0.99): y=129.88+21.482x for amentoflavone; y = 44.775+13.488x for cuppressuflavone. The amount of each compound was expressed as mg/ g of residue.

2.6. Antileishmanial activity 2.6.1. Parasitic strains Leishmania major (LC04) and Leishmania infantum (LV24) strains were cultured in RPMI 1640 medium (Gibco-Invitrogen) supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/mL), streptomycin (100 μg/mL) and incubated at 27 °C in a humidified atmosphere with 5% CO2. 2.6.2. Antipromastigote assay Promastigotes at the stationary growth phase were seeded at 2 × 105 parasites per well in 100 μL growth medium. Two fold serial dilutions of tested compounds were added and incubated at 27 °C for 72 h. The parasitic viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) test. After 4 h of incubation, formazan crystals were solubilized with pure DMSO and estimated spectrophotometrically at 570 nm using a microplate reader. Antipromastigote activity was expressed as IC50 values; the concentration of the compound that inhibited the growth of promastigotes by

2.8. Identification of active extracts by RP-HPLC-DAD-ESI-MSn The chromatographic separation was carried out using a Gemini C18 (150.0 × 2.1 mm, i.d., 5 μm) with a Hypersil Gold C18 guard column (10.0 × 2.1 mm i.d. 5 mm, all from Phenomenex, Tor-rance, 196

Industrial Crops & Products 131 (2019) 194–202

S. Rguez et al.

CA). The mobile phase consisted of 0.1% formic acid aqueous solution (solvent A) and methanol (solvent B); flow rate 0.3 mL/min. Gradient elution: 0–5 min, 10–20% B; 5–10 min, 20–30% B; 10–15 min, 30–50% B; 15–20 min, 50–70% B; 20–28 min, 70–90% B, followed by column reconditioning for 10 min. LC–MS analyses were performed using a Thermo Finnigan Surveyor Plus HPLC apparatus equipped with a quaternary pump, a Surveyor Plus autosampler set at 5 μl injection volume, a degasser, a thermostat ted column oven set at 25.0 ± 0.5 °C, and a diode-array detector (DAD) set at 280 nm, and a vacuum degasser connected to an LCQ Advantage Max ion trap mass spectrometer (all from Thermo Fisher Scientific, Waltham, MA) through an electrospray ionization (ESI) source. The ion trap operated in data-dependent, full scan (100–1500 m/z), zoom scan, and MSn mode to obtain fragment ion m/z with collision energy of 35% and an isolation width of 3 m/z. The parameters of the ion mode ESI source had previously been optimized at a ionization voltage of 5.0 kV, a capillary temperature of 300 °C, a capillarity voltage of 33 V; a sheath gas flow rate of 50 arbitrary units (AU), and an auxiliary gas flow rate of 20 AU. The Thermo Fisher Scientific Excal-ibur 2.0 software was used for data acquisition and processing. No marked variations attributable to the nature of the detected fragments or the relative intensities were observed in 3 independent assays performed to analyse the sample.

Table 1 Total polyphenols flavonoids and condensed tannins quantities in organic extracts of C. sempervirens harvested at three phenological stages. Solvents H2O MEOH EA DCM EE H2O MEOH EA DCM EE H2O MEOH EA DCM EE

Vg

Fl

Total polyphenols (μg GAE/g DMW) 79.71Aa ± 2.43 45.38Ac ± 1.69 45.36Bb ± 0.63 43.29Ac ± 0.61 19.25Bc ± 0,65 24.94Ca ± 1.00 11.74Da ± 0,60 5.02Dc ± 0,22 10.51Cb ± 0.18 12.63Da ± 0.44 Total flavonoids (μg EQ/g DMW) 33.66Ea ± 1.69 34.33Ca ± 1.69 550.26Aa ± 36.21 375.86Ab ± 3.63 44.93Ba ± 1.06 45.20Ba ± 3.70 23.66Eb ± 0.13 36.40Ca ± 1.19 35.80Da ± 1.76 32.60Db ± 0.22 Condensed tannins (μg EC/g DMW) 5.27Ba ± 0,26 2.11Bb ± 0,11 11.84Aa ± 0.18 3.56Ab ± 0,15 1.15Ec ± 0.07 3.25Da ± 0.14 4.18Ca ± 0.07 1.84Cb ± 0.03 3.12Da ± 0.10 1.33Dc ± 0.03

Fr 49.27Ab ± 0.39 47.27Aa ± 0.22 21.12Bb ± 0.20 6.35Cb ± 0.21 6.92Cc ± 0.53 22.86Cb ± 0.91 573.02Aa ± 14.33 36.20Bb ± 1.79 18.60Dc ± 0.39 22.00Cc ± 1.13 2.27BCb ± 0.05 3.49Ab ± 0.10 2.18Cb ± 0.16 1.76Db ± 0.04 2.35Bb ± 0.05

H2O: water; MEOH: methanol; DCM: dichloromethane; EA: ethyl acetate; EE; diethyl ether; Vg: vegetative stage; Fl: flowering stage; Fr: fructification stage; GAE: gallic acid equivalents; DMW: dry matter weight. All measurements were done in triplicate and results were expressed as means ± SD. Variation between solvents for each studied phenological stage was significantly different at p < 0.05 according to Duncan’s test (capital letters); Variation between phenological stages for each studied solvent was significantly different at p < 0.05 according to Duncan’s test (small letters).

2.9. Statistical analysis Data of polyphenols content and antioxidant activity were analyzed by one way ANOVA/MANOVA using SPSS software version 20. Mean values were compared using the Duncan multiple range test at P ≤ 0.05. The results of antibacterial and antileishmanial activities were expressed as mean ( ± Standard Deviation, SD) and statistically analyzed using the Student’s t-test by MS-Excel software, Differences were considered significant at P < 0.05. IC50 was calculated by Graph Pad Prism non-linear regression equation.

the extracts (Djeridane et al., 2006). Previous studies were interested to the influence of the solvent polarity on the chemical composition of the extract. Tian et al. (2009) demonstrated that the level of polyphenols detected in Galla chinensis L. was highest in the order of ethyl acetate, water, ethanol and ether extracts. The total phenolic contents of different extracts from leaves of tetrastigma were found to be highest in methanol extract followed by ethyl acetate, chloroform, hexane and butanolic extracts (Hossain et al., 2013). In this study, in addition to the extraction solvents, phenological stages largely affected levels of polyphenols, flavonoids and tannins. These results confirmed the study of Ben-Farhat et al. (2014) who showed that different parameters such as the geographic origin (locality and habitat), harvesting time and phenological stages significantly affected the extraction yield and the composition.

3. Results and discussion 3.1. Phenolic contents of different extracts Cupressus sempervirens was considered as one of the most important Mediterranean plant known by several medicinal and aromatic properties (Rawat et al., 2010). The content of total polyphenols, flavonoids and condensed tannins was presented in Table 1. Results showed that level of total polyphenols varied significantly (p < 0.05) according to both phenological stage and the solvent used for the extraction. Thus, the maximum level of total polyphenols was detected in the aqueous extract of vegetative stage (79.71 μg GAE/g DMW) followed by the methanolic extract of fructification stage with amounts of 47.27 μg GAE/g DMW. The maximum level of flavonoids was detected in the methanolic extract of fructification stage (573.02 μg EQ/g DMW). The highest level of condensed tannins was attributed to the methanolic extract of vegetative stage (11.81 μg EC/g DMW). This finding is in complete accordance with those reported by Romani et al. (2002) who demonstrated that the total polyphenols content in the ethanolic extract of Cupressaceae leaves contained 4 mg/g MF of total flavonoids, 0.5 mg/g MS flavonoids glycosides and trace of biflavonoïdes derivatives. In this study, aqueous aerial parts extracts showed a high phenolic content, however, high sugar levels may react with Folin-ciocalteu method in over estimation of the total phenolic content (Ramdane et al., 2017). This result suggested that methanolic extract could be considered as the best solvent to extract polyphenols from C. sempervirens. Moreover, most of the phenolic compounds were soluble in polar or moderately polar solvents such as methanol and ethyl acetate (Kefi et al., 2018). Eventually, the different composition of OEs could be attributed to the polarity of different solvents used which have a big influence on phytochemical nature of the components present within

3.2. Antioxidant activities of different extracts The antioxidant activity of C. sempervirens extracts with different solvents at three phenological stages was resumed in Table 2. The methanolic extract of C. sempervirens harvested at the vegetative stage showed a radical scavenging activity measured by DPPH test more important than extracts obtained from the other solvents (IC50 = 6.96 μg/mL). According to the present results, it seems that the activity of methanolic extracts from different phenological stages was stronger than that of the synthetic antioxidant BHT (IC50 = 16 μg/mL). For all phenological stages, this activity was followed by those of aqueous, dichloromethanolic, ethyl acetate and finally diethyl ether extracts. The reductive capacity of iron was also significantly affected by the phenological stages and the solvent of extraction. Indeed, the methanolic extract of vegetative stage had the most important activity (EC50 equal to 18.93 μg/mL) followed by that of aqueous extract of vegetative stage (EC50 equal to 30.93 μg/mL). Results showed that these extracts presented high reducing activity as compared to those of ascorbic acid used as positive control (EC50 equal to 34.96 μg/mL). The reducing capacity was found according to this order: methanolic or aqueous extract > ethyl acetate extract > diethyl ether extract > 197

Industrial Crops & Products 131 (2019) 194–202

S. Rguez et al.

studies. In fact, the antioxidant activity of Annona squamosa extracts showed that the activity for DPPH and nitric oxide scavenging tests was highest in this order: chloroform extract > methanolic extract > hexanoique extract. However, for ABTS scavenging test the order was methanolic extract > chloroformic extract > hexanoic extract (Bheemagani et al., 2017). The highest content of total phenolic compounds of Echium arenarium was obtained in the ethyl acetate extract which showed also the best DPPH scavenging activity (Kefi et al., 2018). The ethyl acetate fraction of Asteriscus graveolens L. exhibited high antioxidant potential by DPPH, reduction capacity and total antioxidant capacity tests. This activity was higher than those of methanolic, butanolic and ethanolic fractions (Ramdane et al., 2017). Similar results were reported by Ibrahim et al. (2007) showing high antioxidant activity of C. sempervirens due to the presence of several compounds like quercetin, rutin, caffeic acid, and p-coumaric acid.

Table 2 Antioxidant activity of organic extracts of C. sempervirens harvested at three phenological stages. Solvents H2O MEOH EA DCM EE BHT H2O MEOH EA DCM EE Ascorbic acid H2O MEOH EA DCM EE

Vg

Fl

DPPH (IC 50 μg/mL) 33.94Ba ± 0.57 35.40Bc ± 0.61 6.96Aa ± 0.07 8,36Ab ± 0.16 566.25Da ± 0.72 633.17Db ± 1.04 385.86Cb ± 7.07 378.97Cb ± 11.83 666.07Ea ± 1.15 752.62Eb ± 0.76 16 Reducing power (EC 50 μg/mL) 30.93Ba ± 0.62 34.22Ab ± 2.91 18.93Aa ± 0.45 58.13Bb ± 2.06 120.10Cb ± 5.04 150.02Cc ± 2.91 227.27Ea ± 0.00 250.00Eb ± 0.00 147.39Da ± 9.83 176.80Db ± 10.62 34,96 Total antioxidant capacity (μg GAE/g DMW) 420.20Ba ± 6.78 325.66Bc ± 2.60 752.34Aa ± 28.96 538.42Ac ± 16.05 121.01Ca ± 0.50 91.81Cc ± 0.30 38.73Ec ± 0.52 45.3Ea ± 0.92 80.66Da ± 0.52 70.55Dc ± 0.73

Fr 34.44Bb ± 0.31 9.36Aa ± 0. 708.54Dc ± 2.36 650.77Ca ± 40.19 899.82Ec ± 3.8 34.9Aab ± 1.55 64.93Bc ± 3.83 68.19Ca ± 1.59 263.15Ec ± 0.00 172.96Dab ± 13.52

3.3. Antibacterial activity

382.82Bb ± 3.66 595.87Ab ± 13.29 100.13Cb ± 1.08 41.00Eb ± 0.18 78.76Db ± 0.57

Different extracts of C. sempervirens collected at different phenological stages (vegetative, flowering and fructification) were tested against bacterial strains (Fig. 1; Table 3). Results showed that diethyl ether and ethyl acetate extracts from flowering stage were active against B. cereus (ATCC 14579). The ethyl acetate extract was also effective against S. aureus (ATCC 25923). The inhibition zone diameter of B. cereus was 12 and 14 mm for diethyl ether and ethyl acetate extracts from flowering stage, respectively. The inhibition zone diameter of S. aureus for ethyl acetate extract from flowering stage was 16 mm. The MIC values of the most active extract, ethyl acetate extract, were 50 and 100 μg/mL for S. aureus ATCC 25923 and B. cereus ATCC 14579, respectively. The MIC values of tetracycline were reported in Table 3. According to Askun et al. (2013), plant extracts were considered active against bacteria for MIC values below 100 μg/mL, moderately active for MIC ranging from 100 to 625 μg/mL and inactive for MIC below 800 μg/mL. According to these criteria, the ethyl acetate extract of C. sempervirens had a high antibacterial activity against Staphylococcus aureus and moderately active against Bacillus cereus. Ethyl acetate extract from flowering stage showed the best antibacterial activity. This indicated that the active compounds which inhibited the growth of tested bacteria may be dissolved better in ethyl acetate solvent more than in ethyl ether, dichloromethane, water or methanol. Interestingly, these compounds were also highly concentrated in the methanolic extract but probably inhibited by other compounds. The contrast between the high numbers of secondary metabolites classes found in this extract reinforced the idea that the detection of the classes of phytochemicals in plants was not a guarantee for a good antibacterial property (Noumedem et al., 2013).

H2O: water; MEOH: methanol; DCM: dichloromethane; EA: ethyl acetate; EE; diethyl ether; Vg: vegetative stage; Fl: flowering stage; Fr: fructification stage; EC50: Effective concentration at 50; IC50: Inhibition concentration at 50%; DMW: dry matter weight All measurements were done in triplicate and results were expressed as means ± SD. Variation between solvents for each studied phenological stage was significantly different at p < 0.05 according to Duncan’s test (capital letters); Variation between phenological stages for each studied solvent was significantly different at p < 0.05 according to Duncan’s test (small letter).

dichloromethanolic extract. Finally, the screening of the total antioxidant capacity of OEs of C. sempervirens was evaluated by the method of reduction of molybdate. The results of this test (Table 2) showed that the studied extracts had different antioxidant capacities depending with the phenological stages and also with the extraction solvents. In this way, the methanolic extract of vegetative stage had the highest total antioxidant capacity equivalent to 752.34 μg EAG / g. Contrary to methanolic and aqueous extracts, the dichloromethanolic, ethyl acetate and diethyl ether extracts of all phenological stages showed the lowest total antioxidant capacity. The activity of reducing of molybdate of the different extracts could be classified in this order: methanolic extract > aqueous extract > ethyl acetate extract > diethyl ether extract > dichloromethanolic extract. Similar findings were reported by previous

Fig. 1. Effect of C. sempervirens organic extracts on B. cereus (A) and S. aureus (B). Arrows corresponded at inhibition diameters (14 mm and 16 mm) of ethyl acetate extract from flowering stage (1 mg/mL) of respectively B. cereus (A) and S. aureus (B). 198

Industrial Crops & Products 131 (2019) 194–202

S. Rguez et al.

Table 3 Antibacterial activity of organic extracts of C. sempervirens harvested at three phenological stages and using discs diffusion methods. Inhibition zone diameter (mm)

MIC (μg/mL)

Gram positive bacteria

Stages

H2O

MEOH

DCM

EE

EA

Tetracycline

EA extract

Bacillus Cereus (ATCC 14579)

Vg Fl Fr Vg Fl Fr Vg Fl Fr

NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA

NA NA NA NA NA NA NA NA NA

NA 12 NA NA NA NA NA NA NA

8.00 14.00 8.00 NA NA NA 10.00 16.00 10.00

125 ± 1.33 – – 31.25 ± 0.60 – – 31.25 ± 0.60 – –

– 100.00 ± 0.16 – – – – – 50.00 ± 0.08 –

Vg Fl Fr Vg Fl Fr

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

NA NA NA NA NA NA

125 ± 0.60 – – 250 ± 0.60 – –

– – – – – –

Listeria monocytogenes (ATCC 19115) Staphylococcus aureus (ATCC 25923) Gram negative bacteria Escherchia coli (ATCC 35214) Pseudomonas aeruginosa (ATCC 27853)

H2O: water; MEOH: methanol; DCM: dichloromethane; EA: ethyl acetate; EE; diethyl ether; Vg: vegetative stage; Fl: flowering stage; Fr: fructification stage. NA: not active from maximal concentration. MIC: minimum inhibitory concentration.

In addition, flavonoids are very common and prevalent secondary plant metabolites and were well known to inhibit a large number fungus and bacteria. In fact, they play an important role in plant resistance or defence against a big range of microorganisms (Cuyckens and Claeys, 2004). Many authors studied the efficiency of C. sempervirens extract as an antimicrobial agent. Zhang et al. (2012) demonstrated the potent antibacterial activity of ethanolic extract of C. sempervirens collected from Oxford against Staphylococcus aureus MRSA with IC50 of 12.48 μg/mL. In addition, 6-deoxytaxodione and taxodione isolated from C. sempervirens showed potent antibacterial activity against the same strains (IC50 of 0.8 and 0.85 μg/mL, respectively). Similar works were focalized on the valorization of OEs especially ethyl acetate as source of antibacterial molecules. Kefi et al. (2018) demonstrated that ethyl acetate extract of Echium arenarium L. exhibited the highest antimicrobial activity against Gram positive bacteria (Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis and Bacillus cereus). The ethyl acetate fraction of Myrtus nivellei showed also an important activity against Bacillus cereus and Listeria monocytogenes (Ramdane et al., 2017). The ethyl acetate fraction of Asteriscus graveolens L exhibited a high activity against S. aureus and B. cereus (MIC = 0.625 μg/mL) (Ramdane et al., 2017).

activity of extracts obtained from C. sempervirens. Al-Musayeib et al. (2012) demonstrated that non relevant results of methanolic extract of C. sempervirens were found against L. infantum (IC50 = 20 μg/mL). This extract also exhibited a noticeable cytotoxic effect against MRC-5 with IC50 values of 10.7 μg/mL. However, 6-deoxytaxodione and taxodione isolated from C. sempervirens by thin layer chromatography displayed a potent antileishmanial activity against Leishmania donovani promastigotes with IC50 equal to 0.077 μg/mL. This activity was more important than that of antileishmanial drugs pentamidine and amphotericin B (IC50 equal to 1.62 and 0.11 μg/mL respectively) (Zhang et al., 2012). In the same way, other authors demonstrated the efficiency of ethyl acetate solvent to extract bioactive compounds for antileishmanial activity. Kefi et al. (2018) demonstrated that ethyl acetate fraction of Echium arenarium exhibited a significant antileishmanial activity against amastigotes and promastigotes of L. major and L. infantum. The cytotoxicity studies of this extract revealed low toxicity against Raw 264.7 macrophage cell line (IC50 equal to 145.8 μg/mL; SI < 10). Similarly, Ramdane et al (2017) showed that the antileishmanial activity of ethyl acetate extract of Asteriscus graveolens L. exhibited the highest inhibition of promastigotes form of L. major and L. infantum with good selectivity index (SI > 10). On the same way, ethyl acetate fraction of Myrtus nivellei L. exerted an antileishmanial activity against promastigotes of L. major and L. infantum with IC50 equal to 224.1 and 190.43 μg/mL (Ramdane et al., 2017). Many authors studied mechanisms of action of active compounds of medicinal plants against leishmanial strains. These compounds may be phagocytized by the macrophages, and acting directly on the intracellular stages by alterating mitochondria of parasites (Grzanna et al., 2005). These compounds also may not reach the parasitophorus vacuole and metabolically converted into different products by macrophages or may simply be non-selectively cytotoxic. Finally, active compounds can act indirectly by activating macrophage microbiocidal mechanisms such as the production of nitrique oxide, which was considered as the most important macrophagal leishmaniacidal mechanisms (Sereno et al., 1998).

3.4. Antileishmanial and cytotoxicity activity The antileishmanial activity of different C. sempervirens extracts was reported in Table 4. Results showed that the ethyl acetate, dichloromethane and diethyl ether extracts from flowering stage of C. sempervirens exhibited high antileishmanial activity. For flowering stage, the aqueous and methanolic extracts showed negligible antileishmanial activity against L. infantum and L. major. However, those obtained from vegetative and fructification stages exerted low antileishmanial activity. The most important activity was found in the ethyl acetate extract with an IC50 value of 1.47 μg/mL and 2.80 μg/mL for L. infantum and L. major, respectively (Table 4). Furthermore, it showed low cytotoxicity on macrophage cells Raw264.7 (LC50 = 50.21 μg/mL) with a high selective index (SI) equal to 34.15 and 17.93 for L. infantum and L. major, respectively. Interestingly, this extract inhibited the growth of intracellular amastigotes with IC50 = 3.61 and 5.42 μg/ml for L. infantum and L. major, respectively. Results showed in this study that inhibitory activity depended largely on the phenological stage and the solvent used for extraction of bioactive compounds. Restrained studies evaluated the antileishmanial

3.5. Identification and quantification of active extracts by HPLC and HPLCMSn Cupressus sempervirens methanolic extract from vegetative stage characterized by the best antioxidant activity and ethyl acetate extract from flowering stage characterized by its highest antibacterial and antileishmanial activities have been identified by LC-ESI-MS. The 199

Industrial Crops & Products 131 (2019) 194–202

S. Rguez et al.

Table 4 Antipromastigote activity against Leishmania species and cytotoxicity of organic extracts of C. sempervirens harvested at three phenological stages. Extracts

Vg

Fl

Fr

Amphotericin B

IC50

H2O MEOH DCM EA EE H2O MEOH DCM EA EE H2O MEOH DCM EA EE

LC50

SI

L. infantum

L. major

Raw 264.7

L. infantum

L. major

> 500.00 425.00 ± 2.35 198.76 ± 1.84 124.123 ± 1.64 225.14 ± 1.22 247.12 ± 1.57 37.12 ± 0.94 4.12 ± 0.32 1.47 ± 0.09 6.11 ± 0.16 > 500.00 395.12 ± 2.18 186.47 ± 1.57 122.34 ± 1.46 248.33 ± 1.93 0.48 ± 0.08

> 500.00 > 500.00 227.34 ± 1.67 187.21 ± 1.22 311.20 ± 2.17 340.70 ± 2.57 45.75 ± 1.12 5.37 ± 0.47 2.80 ± 0.11 7.22 ± 0.21 > 500.00 > 500.00 239.14 ± 1.77 179.12 ± 1.34 347.25 ± 2.13 1.06 ± 0.24

– – – – – > 500.00 213.45 ± 2.25 62.18 ± 0.97 50.21 ± 0.45 75.12 ± 1.33 – – – – – 10.76 ± 0.58

– – – – – – 5.75 15.09 34.15 12.29 – – – – – 22.41

– – – – – – 4.66 11.57 17.93 10.40 – – – – – 10.15

H2O: water; MEOH: methanol; DCM: dichloromethane; EA: ethyl acetate; EE; diethyl ether; Vg: vegetative stage; Fl: flowering stage; Fr: fructification stage. IC50: Inhibition concentration 50 (μg/mL). IC50: Lethal concentration 50 (μg/mL). SI: Selectivity index.

Fig. 2. Chromatographic profiles of ethyl acetate extract at flowering stage (a), methanolic extract at vegetative stage (b) registered at 280 nm. Compound 1: cupressuflavone; compound 2: amentoflavone; compound 3: methyl amentoflavone. Table 5 Quantification of major compounds of methanolic extracts of C. sempervirens. Compounds

Regression equation

R2

LOD

LOQ

Quantification

(μg/mL)

(μg/mL)

(mg/g DR) Vegetative stage

Cupressuflavone Amentoflavone

Y=44.757+13.488X Y=129.88+21.482X

0.998 0.989

0.5806 0.4089

1.9352 1.3643

chromatographic profiles registered at 280 nm were reported in Fig. 2. Two main peaks were present in the methanolic extract of vegetative stage and ethyl acetate extract of fructification stage. Compound 1 was tentatively identified as cupressuflavone due to its molecular mass of 538 Da (m/z 537, [M−H]−) and its fragmentation pattern which showed a base peak at m/z 375 corresponding to the loss of C9H6O3 (162 amu) from the B-ring of a flavonoid unit, as reported by Romani et al. (2002), and m/z 417.0713 ([M–C8H7O–2 H]ˉ) (46% base peak). In positive ionization mode also m/z 521 (32% base peak) corresponding to [M+H–H2O] + was present. Another isobaric compound was detected as amentoflavone (compound 2) (Table 5). Its identity was confirmed by the fragmentation pattern which was very similar to that registered for compound 1, differing only for the presence in positive

Ac

4.12 ± 0.03 0.36Dc ± 0.00

Flowering stage Ab

16.71 ± 0.02 2.25Bb ± 0.00

Fructification stage 25.04Aa ± 0.19 2.39Ba ± 0.02

ionization mode of m/z 387 (68% base peak) deriving from the C-ring cleavage of one of the flavonoid molecule at position 1/3 (Zhang et al., 2012; Wang et al., 2015; Yao et al., 2017). The presence of these two molecules was also confirmed by the selectivity and UV–vis profile (Zhang et al., 2012; Ibrahim et al., 2017). The third putatively identified molecule was a methyl amentoflavone (MM 552) whose fragmentation pattern registered in positive ionization mode (m/z 417 (76%), m/z 387(81%), and m/z 349(45%)) is quite close to that reported in literature (Zhang et al., 2012). The quantification by HPLC-DAD of major compounds cuppressuflavone and amentoflavone presented in the methanolic extract of C. sempervirens revealed that the highest levels of these two compounds was obtained with the extract of fructification stage with 25.04 mg/g 200

Industrial Crops & Products 131 (2019) 194–202

S. Rguez et al.

RS and 2.39 mg/g RS, respectively for the two compounds (Table 5). Cupressus sempervirens presented in this study an interestingly source of antioxidant molecules which act as free radical scavengers making this plant important to human health. This activity can be attributed to the presence of biflavonoids in cypress as previously reported by Romani et al. (2002). Biflavonoids were identified in several medicinal plants. In fact, they are the major bioactive ingredients in Selaginella tamariscina (Zhang et al., 2012) and doederleinii (Wang et al., 2015; Yao et al., 2017) and in Apocynum venetum L. leaves (Zhang et al., 2012). These compounds have potential antioxidant properties due to their ability to interact and scavenge free radicals via hydrogen or electron donation, which damage cell membranes and biological molecules (Rice-Evans and Miller, 1996). Moreover, several studies showed that alkaloides, flavonoids, chaconnes, triterpenoides, saponins and polyphenols are phytochemical compounds with antileishmanial effect (Sawadogo et al., 2012). In this study, C. sempervirens was characterized by antileishmanial properties and can represent a potential treatment for leishmaniasis. The phytochemical analyses showed that the ethyl acetate extract of this plant was characterized by a high level of flavonoids especially cuppressuflavone and amentoflavone that may be responsible for its antileishmanial activity. Sen et al. (2005) declared that flavonoids were known to possess an established protective effect against membrane lipoperoxidative damages. Flavonoids compounds can also inhibit DNA topoisomerases favoring the cleavenge of specific DNA site affecting the growth of leishmanial parasite (Braga et al., 2007).

Bheemagani, A.J., Premkumar, P., Anupalli, R.R., 2017. Evaluation of antioxidant and antimicrobial activity of Annona squamosa L. seed extracts. J. Cell. Tissue Res. 17 (2), 6109–6114. Boukhris, M., Regane, G., Yangui, T., Sayadi, S., Bouaziz, M., 2012. Chemical composition and biological potential of essential oil from Tunisian Cupressus sempervirens L. J. Arid. Land. Stud. 22 (1), 329–332. Braga, F.G., Bouzada, M.L., Fabri, R.L., de, O.M.M., Moreira, F.O., Scio, E., Coimbra, E.S., 2007. Antileishmanial and antifungal activity of plants used in traditional medicine in Brazil. J. Ethnopharmacol. 111, 396–402. Chaudhary, H.J., Shahid, W., Bano, A., Ullah, F., Munis, F., Fahad, S., Ahmad, I., 2012. In vitro analysis of Cupressus sempervirens L. plant extracts antibaterial activity. J. Med. Plant Res. 6 (2), 273–276. Cuyckens, F., Claeys, M., 2004. Mass spectrometry in the structural analysis of flavonoids. J. Mass Spectrom. 39, 1–15. De Muylder, Geraldine, Steven Chen, K.H.A., Arkin, K., Juan, M.R., Engel, C., McKerrow, J.H., 2011. Screen against leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit. PLoS Negl. Trop. Dis. 5 (7). Dewanto, V., Wu, X., Adom, K.K., Liu, R.H., 2002. Thermal processing enhances the nutritiona value of tomatoes by increasing total antioxidant activity. J. Agric. Food Chem 50, 3010–3014. Djeridane, A., Yousfi, M., Nadjemi, B., Boutassouna, D., Stocker, P., Vidal, N., 2006. Antioxidant activity of some Algerian medicinal plants extracts containing phenolic compounds. Food Chem. 97, 654–660. Duarte, M.C., Tavares, G.S., Valadares, D.G., Lage, D.P., Ribeiro, T.G., Lage, L.M., Rodrigues, M.R., Faraco, A.A., Soto, M., da Silva, E.S., Chávez Fumagalli, M.A., Tavares, C.A., Leite, J.P., Oliveira, J.S., Castilho, R.O., Coelho, E.A., 2016. Antileishmanial activity and mechanism of action from a purified fraction of Zingiber officinalis Roscoe against Leishmania amazonensis. Exp. Parasit. 166, 21–28. Essid, R., Rahali, F.Z., Msaada, K., Sghair, I., Hammami, M., Aida, B., Karim, A., Limam, F., 2015. Antileishmanial and cytotoxic potential of essential oils from medicinal plants in Northern Tunisia, 2015. Ind. Crops Prod. 77, 795–802. Grzanna, R., Lindmark, L., Frondoza, C.G., 2005. Ginger-an herbal medicinal product with broad 549 anti-inflammatory actions. J. Med. Food 8, 125–132. Guendouze-Bouchefa, N., Madani, K., Chibane, M., Boulekbache Makhlouf, L., Hauchard, D., Kiendrebeogo, M., Stévigny, C., Ndjolo Okusa, P., Duez, P., 2015. Phenolic compounds, antioxidant and antibacterial activities of three Ericaceae from Algeria. Ind. Crop Prod. 70, 459–466. Hanato, T., Kagawa, H., Yasuhara, T., Okuda, T., 1988. Two new flavonoides and other constituents in licorice root their relative astringency and radical scavenging effect. J. Chem. Pharm. Res. 36, 1090–1097. Hassim, N., Markom, M., Anuar, N., Dewi, K.H., Baharum, S.N., Noor, N.M., 2015. Antioxidant and antibacterial assays on Polygonum minus extracts: different extraction methods. J. Chem. Pharm. 10. Hossain, M.A., AL-Raqmi, K.A., AL-Mijizy, Z.H., Weli, A.M., Al-Riyami, Q., 2013. Study of total phenol, flavonoids contents and phytochemical screening of various leaves crude extracts of locally grown Thymus vulgaris. Asian Pac. J. Trop. Biomed. 3 (9), 705–710. Ibrahim, N.A., EL-Seed, H.R., Mohamed, M.M., 2007. Phytochemical investigation and hepatoprotective activity of Cupressus sempervirens L. leaves growing in Egypt. Nat. Prod. Res. 21 (10), 857–866. Ibrahim, E.A., Desoukey, S.Y., Hadad, G.M., Salam, R.A.A., Ibrahim, A.K., 2017. Analysis of cupressuflavone and amentoflavone from Cupressus sempervirens L. and its tissue cultured callus using HPLC-DAD method. Pharm. Pharmacol. Int. J. 5 (5). Kefi, S., Essid, R., Mkadmini, K., Kefi, A., Mahjoub Haddada, F., Tabbene, O., Limam, F., 2018. Phytochemical investigation and biological activities of Echium arenarium (Guss) extracts. Microb. Pathog. 118, 202–210. Khabir, M., Khatoon, F., Ansari, W.H., 1987. Flavonoids of Cupressus sempervirens and Cupressus cashmeriana. J. Nat. Prod. 50 (3), 511–512. Koriem, K.M.M., 2009. Lead toxicity and the protective role of Cupressus sempervirens seeds growing in Egypt. Rev. Lat. Am. Psicol. 37, 230–242. Lobo, V., Patil, A., Phatak, A., Chandra, N., 2010. Free radicals, antioxidants and functional foods: impact on human health. Pharmacogn. Rev. 4, 8. Mau, J.L., Chao, G.R., Wu, K.T., 2001. Antioxidant properties of methanolic extracts from several ear mushrooms. J. Agric. Food Chem. 49, 5461–5467. Mothana, R.A., Al-Musayeib, N.M., Al-Ajmi, M.F., Cos, P., Maes, L., 2014. Evaluation of the in vitro antiplasmodial, antileishmanial, and antitrypanosomal activity of medicinal plants used in Saudi and Yemeni traditional medicine. Evid. Complement. Alternat. Med. 2014, 905639. Moussa, A.M., Emam, A.M., Diab, Y.M., Mahmoud, M.E., Mahmoud, A., 2011. Evaluation of antioxidant potential of 124 Egyptian plants with emphasis on the action of Punica granatum leaf extract on rats. Food Res. Int. 18, 535–542. Noumedem, J., Mihasan, M., Lacmata, S., Stefan, M., Kuiate, J., Kuete, V., 2013. Antibacterial activities of the methanol extracts of ten Cameroonian vegetables against Gram-negative multidrug-resistant bacteria. BMC Complement. Altern. Med. 6882, 13–26. Oyaizu, M., 1986. Studies on products of the browning reaction: antioxidative activities of browning reaction. Jpn. J. Physiol. 44, 307–315. Prieto, P., Pineda, M., Aguilar, M., 1999. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Ann. Biochem. 269, 337–341. Ramdane, F., Essid, R., Mkadmini, K., Hammami, M., Fares, N., Mahammed, M.H., El Ouassise, D., Tabbene, O., Limam, F., Ould Hadj, M.D., 2017. Phytochemical composition and biological activities of Asteriscus graveolens (Forssk) extracts. Process Biochem. 56, 186–192. Rawat, P., Khan, M.F., Kumar, M., Tamar, M., Tamarak, A.K., Sivastava, A.K., Arya, K.R.,

4. Conclusion In conclusion, C. sempervirens seemed to be an effective antioxidant agent used as natural alternative for chemicals to treat several diseases caused by oxidative stress. Ethyl acetate extract of cypress was also highly selective to cuppressuflavone and amentoflavone. These molecules seemed to be responsible for the anti-infectious activities of C. sempervirens. Furthermore, experiments are needed to isolate active compounds from cypress and to explore the synergistic combination of this plant with conventional drugs in order to enhance selective activity against leishmaniasis and bacterial infections. Moreover, in vivo studies could be conducted to confirm the anti-infectious potential of these compounds. Acknowledgment Authors would like to acknowledge Pr. Abderrazak Smaoui a botanist at the CBBC for taxonomic identification of plant material and the Laboratory of Bioactive Substances members in which an important part of this work was conducted. References Al-Musayeib, N.M., Mothana, R.A., Matheeussen, A., Cos, P., Maes, L., 2012. In vitro antiplasmodial, antileishmanial and antitrypanosomal activities of selected medicinal plants used in the traditional Arabian Peninsular region. BMC Complement. Altern. Med. 20, 12–49. Al-Othman, A.M., Hussain, I., Khan, H., Rehman, Ur.M., Abdeltawab, A.A., Ullah, R., Rohullah, N.S., Talha, M., 2012. Phytochemical analysis and biological activities of selected medicinal plants. J. Med. Plants. Res. 6 (23), 4005–4010. Amouroux, P., Jean, D., Lamaison, J., 1998. Antiviral activity in vitro of Cupressus sempervirens on two human retroviruses HIV and HTLV. Phytother. Res. 12 (5), 367–368. Asgary, S., Naderi, G.A., Ardekani, M.R.S., Sahebkar, A., Airin, A., Aslani, S., Kasher, T., Emami, S.A., 2013. Chemical analysis and biological activities of Cupressus sempervirens var. horizontalis essential oils. Pharma. Biol. 51 (2), 137–144. Askun, E.M., Tekwu, F., Satil, S., Modanlioglu, H., Aydeniz, A., 2013. Preliminary antimycobacterial study on selected Turkish plants (Lamiaceae) against Mycobacterium tuberculosis and search for some phenolic constituents. BMC Complement. Altern. Med. 13, 365. Ben-Farhat, M., Chaouch-Hamada, R., Sotomayor, J., Landoulsi, A., Jordán, M., 2014. Antioxidant potential of Salvia officinalis L. residues as affected by the harvesting time. Ind. Crops Prod. 54, 78–85.

201

Industrial Crops & Products 131 (2019) 194–202

S. Rguez et al. Maurya, R., 2010. Constituents from fruits of Cupressus sempervirens. Fitote 81 (3), 162–166. Rice-Evans, C.A., Miller, N.J., 1996. Paganga G Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20 (7), 933–956. Romani, A., Galardi, C., Pinelli, P., Mulinacci, N., 2002. HPLC quantification of flavonoids and biflavonoids in Cupressaceae leaves. Chromatographia 56, 469–474. Sawadogo, W.R., Le Douaron, A., Maciuk Bories, C., Loiseau, P.M., Figadère, B., Guissou, I.P., Nacoulma, O.G., 2012. In vitro antileishmanial and antitrypanosomal activities of five medicinal plants from Burkina Faso. Parasitol. Res. 110, 1779–1783. Sen, G., Mandal, S., Roy, S.S., Mukhopadhyay, S., Biswas, T., 2005. Therapeutic use of quercetine in the control of infection and anemia associated with visceral leishmaniasis. Free Radic. Biol. Med. 38, 1257–1264. Sereno, D., Cavaleyra, M., Zemzoumi, K., Maquaire, S., Ouaissi, A., Lemesre, J.L., 1998. Axenically grown amastigotes of Leishmania infantum used as an in vitro model to investigate the pentavalent antimony mode of action. Antimicrob. Agents Chemother. 42, 3097–3102. Shahid, W., Durrani, R., Iram, S., Durrani, M., Khan, F.A., 2013. Antibacterial activity in vitro of medicinal plants. Sky. J. Mic. Res. 1 (2), 5–21. Sun, B., Richardo-da-Silvia, J.M., Spranger, I., 1998. Critical factors of vanillin assay for catechins and proanthocyanidines. J. Agric. Food. Chem. 46, 4267–4274. Tay, B., Giday, M., Animt, A., Seid, J., 2011. Antibacterial activities of selected medicinal plants in traditional treatment of human wounds in Ethiopia. Asian Pac. J. Trop. Biomed. 1 (5), 370–375.

Tian, B.L., Baoping, J., Jinhua, Y., Guizhi, Z., Yang, C., Yangchao, L., 2009. Antioxidant and antimicrobial activities of consecutive extracts from Gallachinensis: the polarity affects the bioactivities Fang. Food Chem. 113, 173–179. Torres-Santos, D., Lima Moreira, M., Auxiliadora, C., Kaplan, M., Nazareth, M., 1999. Antimicrobial agents and chemotherapy. M. Bio. 43, 1234–1241. Tumen, I., Suntar, I., Keles, H., Akkol, E.K.A., 2012. Therapeutic approach for wound healing by using essential oils of Cupressus and Juniperus species growing in Turkey. J. Evid. Complementary Altern. Med. 7. Verma, V., Sharma, V., Singh, V., Kumar, R., Khan, M.F., Singh, A.K., Sharma, R., Arya, K.R., Maikhuri, J.P., Dalela, D., Maurya, R., Gupta, G., 2014. Labda-8 (17), 12,14trien-19-oic acid contained in fruits of Cupressus sempervirens suppresses benign prostatic hyperplasia in rat and in vitro human models through inhibition of androgen and STAT-3 signaling. Phytother. Res. 28 (8), 1196–1203. Wang, G., Yao, S., Zhang, X.-X., Song, H., 2015. Rapid screening and structural characterization of antioxidants from the extract of Selaginella doederleinii Hieron with DPPH-UPLC-Q-TOF/MS method. Int. J. Anal. Chem. 2, 1–9. Yao, H., Chen, B., Zhang, Y., Ou, H., Li, Y., Li, S., Shi, P., Lin, X., 2017. Analysis of the total biflavonoids extract from Selaginella doederleinii by HPLC-QTOF-MS and its in vitro and in vivo anticancer effects, 2017. Molecules 22, 325–341. Zhang, J., Rahman, A.A., Jain, S., Jacob, M.R., Khan, S.I., Tekwani, B.L., Ilias, M., 2012. Antimicrobial and antiparasitic abietane diterpenoids from Cupressus sempervirens. Res. Rep. Med. Chem. 2, 1–6.

202