Antioxidant, antimicrobial and anti-acetylcholinesterase activities of organic extracts from aerial parts of three Tunisian plants and correlation with polyphenols and flavonoids contents
Abstract
In goal of searching new active compounds with important biological activities, a screening of several plants from salt-marsh region of Chebba-Tunisia had been realized. Three species had been selected: Calendula arvensis, Chenopodium murale and Nicotiana glauca. The organic extracts of different aerial parts of these plants (stems, leaves and flowers) displayed variable contents of total polyphenols (TPP) and total flavonoids (TF). Flowers acetone extract from N. glauca contained the higher quantity in TPP (264.8 ± 1.6 µg GA/mg), while stems dichloromethane extract exhibited the best TF content (49.8 ± 2.2 µg QE/mg). The important TPP and TF contents reflected a good antioxidant and antimicrobial activities. The best acetylcholinesterase inhibitory activity had been shown in the fractions obtained after extraction with low polarity solvents. Whence, a correlation of flavonoids contents with biological activities had been shown, while, there was no correlation with acetylcholinesterase inhibitory activity.
Introduction
For centuries, extracts from plants have been used in traditional medicine to solve various health problems (Montiel Ruiza et al., 2013). Now-a-days, plants still play an important role in terms of prevention and cure of several diseases. They are the base for the development of modern medicine (Ameyaw and Duker-Eshun, 2009), and they have provided a great number of drugs (Kintzios and Barberaki, 2004).
The medicinal value of plants lies in the bioactive constituents, such as alkaloids, tannins, coumarins, terpenoids, phenolic compounds, etc (Chethan et al., 2012), that have been known to bear valuable therapeutic activities (Raina et al., 2014). Several studies showed significant variations of plants secondary metabolites production between spices and even for the same spices under different environmental conditions (Selmar and Kleinwaëchter, 2013). Furthermore, in arid and semi-arid regions, plants are often subjected to severe environmental conditions (salinity, drought, extreme temperatures etc), that influence secondary metabolites biosynthesis, especially the rise of phenolics production and distribution (Jallali et al., 2012).
The aims of this work were firstly, the gain of more information about total polyphenols and flavonoids quantities, the study of the potential natural antioxidant, antimicrobial and acetylcholinesterase inhibitory effect of diverse crude extracts from various areal parts of the three plants species from salt-marsh region of Chebba-Tunisia; Calendula arvensis, Chenopodium murale and Nicotiana glauca belonging respectively to Asteraceae, Chenopodiaceae and Solanaceae families, secondly to carry on the relationships between phenolics and flavonoids quantities and the different biological activities.
Materials and Methods
Chemicals and reagents
The following chemicals and reagents were used: Cyclohexane (Riedel-deHaën), dichloromethane (Loba Chemie), ethyl acetate (Loba Chemie), acetone (Loba Chemie), acetonitrile (Loba Chemie), Folin-Ciocalteau reagent (Fluka), Na2CO3 (Merck), ascorbic acid (Bio Basic), butylated hydroxytoluene (BHT) (Supelco), gallic acid (Bio Basic), AlCl3 (Merck), quercetin (HWI Analitik), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Aldrich), DMSO (Scharlau), 2, 2'-azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) (Fluka), MK2S2O8 (Merck), beta-carotene (Sigma), linoleic acid (Acros Organics), Tween 40 (Scharlau), peptone (Accumix), Yeast extract (Accumix), NaCl (Suvchem), agar (Biokar), glucose (Accumix), electric eel AChE (Type-VI-S, EC 3.1.1.7, Sigma), acetylthiocholine iodide (ATCI) (Sigma), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) (Sigma), Tris (Vivantis) and galanthamine (Sigma).
Plants collection and extracts preparation
C. arvensis, N. glauca and C. murale were collected from the marsh area of Chebba (Mahdia, Tunisia, latitude 35.23° and longitude 11.11°), this region strong saline and had a semi-arid climate with scarce rainfall (Mabrouk et al., 2014; Saidi et al., 2009). The identification of the three plants species was performed according to the flora of Tunisia deposited in the Center of Biotechnology of Sfax.
The plants were washed and air-dried in the shade at room temperature. The areal parts were separated and grounded to fine powder using a grinder. Each material resulted was extracted by maceration in 5 solvents successively in increasing polarity (cyclohexane-dichloromethane-ethyl acetate-acetone-acetonitrile) for 24 hours with a mass to volume ratio of 1:10 (g/mL), at room temperature. The resulting crude extracts were evaporated under rotary evaporator to dryness at 37°C. The dried extracts were kept at 4°C in the dark until a further analysis.
Total polyphenols content
Total polyphenols content was determined according to Folin-Ciocalteau method (Waterman and Mole, 1994) with some modifications. 10 µL of diluted extract solution was shaken for 5 min with 50 µL of Folin-Ciocalteau reagent. Then 150 µL of 20% Na2CO3 was added and the mixture was shaken once again for 1 min. Finally, the solution was brought up to 790 µL by adding distilled water. After 2 hours, the absorbance at 760 nm was evaluated using a spectrophotometer (Thermo Scientific / Genesys 20, Germany). Gallic acid was used as a standard for the calibration curve. Total polyphenolics content (TPP) of the extracts was calculated according to the following equations: y = 0.012 x + 0.017 (r2 = 0.997), expressed as µg gallic acid equivalent per milligram of dry extract (µg GA/mg) using the linear equation based on the calibration curve.
Flavonoids content
The flavonoids content in extract was determined by spectrophotometric method (Quettier Deleu et al., 2000). Briefly, 1 mL of AlCl3 was added to 1 mL diluted extract solution and vortexed, and then incubated for 15 min in the dark. The absorbance at 430 nm was evaluated for the samples and the quercetin as standard for the calibration curve. Total flavonoids (TF) content of the extracts was calculated according to the following equations: y = 0.051 x + 0,003 (r2 = 0.999), and expressed in µg of quercetin equivalent per milligram of dry plant extract (µg QE/mg).
Antioxidant assays
All antioxidant activities were expressed as EC50 (the concentration that causes 50% of effect). The free radical scavenging activity of the three plants extracts had been assessed by 2.2-diphenyl-1-picrylhydrazyl (DPPH) and 2.2'- azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS+) radicals assays. Gallic acid (GA), ascorbic acid (AA) and butylated hydroxytoluene (BHT) were used as controls, they are well known antioxidant compounds.
DPPH radical scavenging activity (Video clip)
DPPH is one of the compounds that possess a proton free radical, when DPPH encounters proton radical scavengers its purple color fades rapidly. This assay determines the scavenging of stable radical species according to the method of Kirby and Schmidt, (1997) with slight modifications. Briefly, extracts were dissolved in 10% DMSO and diluted with ultrapure water at different concentrations (500, 250, 125, 60.25, 30.125 µg/mL). Then, 500 µL of a 4% (w/v) solution of DPPH radical in methanol was mixed with 500 µL of samples. The mixture was incubated for 30 min in the dark at room temperature. The scavenging capacity was determined spectrophotometrically by monitoring the decrease in absorbance at 517 nm against a blank. The percentage of antiradical activity (%ArA) had been calculated as follows:
%ArA = [(absorbance of control - absorbance of test sample) / absorbance of control] x 100
ABTS+ radical scavenging activity
The potential of extracts to scavenge free radicals was also assessed by their ability to quench ABTS+ antiradical activity done by using the ABTS+ free radical discoloration assay developed by Re et al. (1999) with some modifications. Briefly, the preformed radical monocation of ABTS+ was generated by reacting 2.2' azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) solution (7 mM) with 2.45 mM K2S2O8. The mixture was allowed to stand for 15 hours in the dark at room temperature. The solution was diluted with ethanol to obtain the absorbance of 0.7 ± 0.2 units at 734 nm. Samples were separately dissolved in ethanol to yield the following concentrations (500, 250, 125, 60.25, 30.125, 15.625 µg/mL). In order to measure the antioxidant activity of extracts and standards, 20 µL of each sample at various concentrations was added to 180 µL of ABTS+ and vortexed for 30 sec, and then incubated for 6 min in dark at room temperature. The absorbance was measured spectrophotometrically at 734 nm. The percentage of scavenging activity of ABTS+ (% SA) was calculated by the following formula:
% SA = [(absorbance of control - absorbance of test sample) /absorbance of control] x 100
β-Carotene–linoleic acid bleaching assay
The extracts antioxidant activity was determined according to the beta-carotene bleaching method described by Pratt (1980): 0.5 mg of beta-carotene was dissolved in 1 mL chloroform, and then 25 µL of linoleic acid and 200 µL of Tween 40 were added. The chloroform was then evaporated under rotary evaporator, and the residue was dissolved in 100 mL of H2O. Extracts were diluted at different concentrations (500, 250, 125, 60.25, 30.125 µg/mL), and for 500 µL of each sample, 2.5 mL of beta-carotene-linoleic acid solution was added. The absorbance was read at 470 nm firstly at zero time and after 120 min of incubation in dark at 50°C. The antioxidant activity in beta-carotene bleaching model in percentage (AA%) was calculated using the following equation:
AA% = [1- (A0S-A120S)/ (A0C-A120C)] x 100
where A0S and A0C are absorbance of the sample and the control respectively measured at zero time, and A120S and A120C are absorbance of the sample and the control respectively measured after 120 min
Antimicrobial activities
Microorganisms and growth conditions
Bacteria and Candida species were obtained from International Culture Collections (ATCC) and local culture collection of Laboratory of Microorganisms and Biomolecules of the Centre of Biotechnology of Sfax - Tunisia. They included Gram-positive bacteria: Staphylococcus aureus ATCC 6538, Micrococcus luteus LB14110 and Listeria monocytogenes ATCC 19117 and Gram-negative bacteria: Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 49189 and Enterobacter aerogenes ATCC 13048 and two Candida strains: Candida tropicalis R2 CIP 203 and C. albicans ATCC 10231.
The bacterial cultures were performed in Luria-Bertani (LB) agar medium composed of (g/L): peptone 10; yeast extract 5; NaCl 5 and agar 20 at pH 7.2, then the bacterial strains were incubated at 37°C, except M. luteus and P. aeruginosa which are incubated at 30°C. The Candida species were cultured on Sabouraud agar medium composed of (g/L): peptone 10; glucose 10 and agar 20 at pH=5.6 and incubated at 28°C. Working bacterial cultures were prepared by inoculating a loopful of each test bacteria in 3 mL of LB broth. C. tropicalis was cultured in YP10 medium composed of (g/L): yeast extract 10; peptone 10; glucose 100 and 15 mL of 2 g/L adenine solution, while C. albicans was cultured in YEPD medium composed of (g/L): yeast extract 10; peptone 20 and dextrose 20 at pH 5.6. All strains were incubated at adequate temperature for 12 hours. For the test, final inoculum concentrations of 106 CFU/mL were used.
Agar diffusion method
In this research, the antimicrobial activities of each crude extract were assessed by diameters of inhibition areas (halo) in mm, by means of agar-well diffusion assay according to Güven et al. (2006) with some modifications. Fifteen milliliters of the molten agar (45°C) were poured into sterile petri dishes (~ 90 mm). Working cell suspensions were prepared and 100 uL was evenly spreaded onto the surface of the agar plates of LB agar medium for bacteria and sabouraud agar medium for Candida. Once the plates had been aseptically dried, 5 mm wells were punched into the agar with a sterile pasteur pipette. The extracts were dissolved in dimethylsulfoxide (DMSO)/water (1/9; v/v) to a final concentration of 100 mg/mL and then filtered through 0.22 μm pore-size black polycarbonate filters (Millipore). Thus, 100 uL were placed into the wells and the plates were incubated at adequate temperature overnight for bacterial strains and 72 hours for Candida strains.
Acetylcholinesterase inhibitory potential
AChE inhibitory activity was measured by slightly modified spectrophotometric method of Ellman et al. (1961). Electric eel AChE was used, while acetylthiocholine iodide (ATCI) was employed as substrate of the reaction. 5.5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was used for the measurement of the anti-acetylcholinesterase activity. Briefly, in this method, 100 µL of Tris buffer at 50 mM (pH 8.0), 30 µL of sample or standard and 5 µL of AChE enzyme (0.5 U/mL) were added in a 96 well microplate and incubated for 10 min at 25°C. Then, 142 µL of DTNB (3 mM) and 23 µL of substrate (75 mM) were added. Hydrolysis of ATCI was monitored by the formation of the yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB with thiocholines, catalyzed by enzymes at 405 nm utilizing a 96 well micro plates reader (Thermo Scientific/Varioskan Flash, Germany). The kinetic reaction has been followed until the AChE activity decreased, and then the reaction has been stopped. Percentage of inhibition of AChE was determined by comparison of rates reaction of samples relative to control (10% DMSO in Tris buffer) using the following formula:
% AChEI = 1- (δA sample / δA control) x 100
Where δA sample: Sample absorbance at zero time - Sample absorbance at the end of reaction, and δA control: Control absorbance at zero time - Control absorbance at the end of reaction.
Galanthamine, the antiacetylcholinesterase alkaloid-type of drug obtained from the bulbs of snowdrop (Galanthus sp.), was used as standard.
Firstly, all extracts have been tested at 100 µg/mL of concentration. Then, dose-dependent AChE inhibitory activity (of extracts that display an activity with 100 µg/mL of concentration) was further studied and the results are shown in the IC50 values.
Statistical analyses
All tests are assayed in triplicate and expressed as the average ± standard deviation of the measurements. The statistical program SPSS version 21.00 for Windows (SPSS Inc., Chicago, IL) was used to analyse data. Variance was analysed by one-way ANOVA and Duncan's multiple range tests were calculated for the significant data at p<0.05. Correlation between the phenolic and the flavonoids contents and biological activities in plant extracts were analysed by Pearson correlation coefficient.
Results
Total polyphenols and flavonoids contents
TPP contents of the extracts from aerial parts of the three plants were tabulated in Table I. Overall, the values appear to vary between 16.2 ± 0.4 and 264.8 ± 1.1 µg GA/mg. Concerning TPP contents in C. arvensis extracts, the values are ranged from 26.8 ± 0.2 µg GA/mg (Flowers cyclohexane extract) to 188.3 ± 0.2 µg GA/mg (leaves dichloromethane extract). For C. murale, polyphenols lowest concentration has been shown with stems acetonitrile extract (16.2 ± 0.4 µg GA/mg), while flowers acetonitrile extract showed the highest concentration (215.3 ± 0.3 µg GA/mg). N. glauca extracts presented a polyphenols contents ranged from 20.8 ± 0.2 µg GA/mg for the stems cyclohexane extract to 264.8 ± 1.1 µg GA/mg for the flowers acetonitrile extract.
TF contents of the extracts are shown in Table I. Globally, the values varied between zero to 49.8 ± 0.0 ug QE/mg. For C. arvensis values were ranged from 0 ug QE/mg (stems cyclohexane extract), to 43.0 ± 0.0 ug QE/mg (flowers acetonitrile extract). Concerning C. murale the lowest flavonoids content had been obtained in the flowers cyclohexane extract (0.4 ± 0.0 ug QE/mg) and the highest content had been shown in the leaves dichloromethane extract (49.2 ± 0.1 ug QE/mg). While, N. glauca had the lowest content of flavonoids in the stems dichloromethane extract (5.4 ± 0.3 ug QE/mg) and the highest one in the flowers dichloromethane extract (49.8 ± 0.0 ug QE/mg).
Table I: Antioxidant and acetylcholinesterase inhibitor activities of extracts
| Plant | C. arvensis | C. arvensis | C. arvensis | C. murale | C. murale | C. murale | N. glauca | N. glauca | N. glauca | GA | AA | BHT | Galanthamine | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Extract | Stems | Leaves | Flowers | Stems | Leaves | Flowers | Stems | Leaves | Flowers | 3.6 ± 0.1 | 4.2 ± 0.1 | 28.6 ± 0.3 | - | |
| ±EC 50% DPPH | Cyclohexane | 360.1 ± 1.1Be | 160.3 ± 0.5Cc | >500Ad | >500Cd | 92.5 ± 1.3Aa | 451.4 ± 1.2Be | >500Ae | >500Ae | >500Ac | 3.6 ± 0.1 | 4.2 ± 0.1 | 28.6 ± 0.3 | - |
| Dichloromethane | 358.2 ± 0.3Bd | 104.4 ± 2.7Aa | >500Cd | >500Cd | 270.1 ± 0.3Bd | 124.6 ± 3.1Ac | 97.3 ± 2.2Bc | 83.3 ± 0.7Ac | >500Cc | 3.6 ± 0.1 | 4.2 ± 0.1 | 28.6 ± 0.3 | - | |
| Ethyl acetate | 197.4 ± 0.5Bb | 110.4 ± 0.8Ab | 430.2 ± 1.4Cc | 350.2 ± 0.6Cb | 248.4 ± 0.9Bc | 85.2 ± 0.1Ab | 80.4 ± 2.1Bb | 47.1 ± 0.6Aa | >500Cc | 3.6 ± 0.1 | 4.2 ± 0.1 | 28.6 ± 0.3 | - | |
| Acetone | 213.2 ± 0.2Bc | >500Ce | 185.0 ± 0.7Ab | 304.6 ± 2.1Ca | 179.3 ± 0.5Bb | 82.7 ± 1.3Aa | 70.2 ± 0.3Aa | 83.5 ± 2.5Ad | 190 ± 3.2Bb | 3.6 ± 0.1 | 4.2 ± 0.1 | 28.6 ± 0.3 | - | |
| Acetonitrile | 179.4 ± 0.7Ba | 302.1 ± 0.6Cd | 168.2 ± 0.2Aa | 470.2 ± 0.1Cc | 275.8 ± 0.1Be | 269.0 ± 0.4Ab | 152.1 ± 0.2Cd | 79.3 ± 1.9Bb | 41.4 ± 1.6Aa | 3.6 ± 0.1 | 4.2 ± 0.1 | 28.6 ± 0.3 | - | |
| ±EC 50% ABTS | Cyclohexane | 69.1 ± 0.5Ce | 25.7 ± 0.6Aa | 35.4 ± 0.3Ba | 100.5 ± 0.6Be | 57.3 ± 1.5Ad | 95.0 ± 0.6Ce | 94.3 ± 1.2Ce | 22.5 ± 2.4Ac | 40.7 ± 0.3Bb | 1.9 ± 0.5 | 5.4 ± 0.1 | 17.3 ± 0.4 | - |
| Dichloromethane | 10.8 ± 0.2Aa | 31.7 ± 0.4Bb | 85.6 ± 0.1Ce | 82.4 ± 2Cb | 23.8 ± 0.6Ab | 38.2 ± 0.5Bc | 23.5 ± 0.5Ba | 12.7 ± 0.9Aa | 79.1 ± 0.6Cc | 1.9 ± 0.5 | 5.4 ± 0.1 | 17.3 ± 0.4 | - | |
| Ethyl acetate | 43.8 ± 0.3Cd | 32.5 ± 0.2Ac | 43.2 ± 0.3Bb | 91.4 ± 1.1Cc | 70.2 ± 0.7Be | 46.9 ± 2.1Ad | 39.7 ± 2.8Cc | 16.8 ± 1.5Ab | 34.2 ± 2.6Bd | 1.9 ± 0.5 | 5.4 ± 0.1 | 17.3 ± 0.4 | - | |
| Acetone | 28.2 ± 0.4Ab | 40.1 ± 0.1Bd | 49.8 ± 0.2Cc | 79.2 ± 1.5Ca | 42.2 ± 2.6Bc | 30.1 ± 0.6Aa | 29.7 ± 1.4Bb | 69.6 ± 0.9Ce | 13.7 ± 0.9Aa | 1.9 ± 0.5 | 5.4 ± 0.1 | 17.3 ± 0.4 | - | |
| Acetonitrile | 42.8 ± 1.1Ac | 49.7 ± 0.3Be | 52.5 ± 0.5Cd | 98.6 ± 1.3Cd | 11.7 ± 1.4Aa | 31.3 ± 0.9Bb | 51 ± 0.6Cd | 38.2 ± 2.9Bd | 13.2 ± 0.4Aa | 1.9 ± 0.5 | 5.4 ± 0.1 | 17.3 ± 0.4 | - | |
| ±EC 50% β-caroten | Cyclohexane | 47.3 ± 0.9Aa | 102 ± 1.3Bb | 495.3 ± 3.1Cd | 246.1 ± 4.1Ab | 359.0 ± 0.9Bb | 399.2 ± 4.1Cd | >500Ce | 410.2 ± 2.7Be | 357.5 ± 1.2Ad | - | - | 72.7 ± 0.3 | - |
| Dichloromethane | 90.2 ± 1.2Ac | 96.1 ± 2.2Bb | 158.8 ± 3.2Ca | 408.7 ± 1.2Cc | 257.1 ± 4.1Bc | 73.9 ± 1.5Ab | 62.5 ± 2.4Aa | 89.0 ± 1.6Bb | 400 ± 0.0Ce | - | - | 72.7 ± 0.3 | - | |
| Ethyl acetate | 52.1 ± 0.2Ab | 54.9 ± 0.4Ba | 183.3 ± 1.2Cb | 160.1 ± 0.4Ca | 38.5 ± 1.2Aa | 50.0 ± 0.9Ba | 250.8 ± 3.1Bd | 40.6 ± 2.1Aa | 308.7 ± 1.0Cc | - | - | 72.7 ± 0.3 | - | |
| Acetone | 95 ± 0.0Ad | 300.3 ± 0.5Bc | ˃500Ce | 470.6 ± 0.3Cd | 293.2 ± 1.0Bd | 265.8 ± 3.3Ac | 140 ± 3.3Ab | 374.4 ± 0.8Cd | 252.3 ± 2.2Bb | - | - | 72.7 ± 0.3 | - | |
| Acetonitrile | 470.2 ± 2.1Ae | ˃500Cd | 482.0 ± 1.2Bc | ˃500Be | ˃500Be | 430.1 ± 2.3Ae | 207.0 ± 1.4Ac | 289.6 ± 1.1Bc | 216.5 ± 0.3Aa | - | - | 72.7 ± 0.3 | - | |
| %AChEI* | Cyclohexane | 41.3 ± 1Cb | 20.2 ± 0.0Bb | 0 ± 0.0Aa | 0 ± 0.0Aa | 0 ± 0.0Aa | 53.08 ± 0.0Bc | 0 ± 0.0Aa | 83.33 ± 0.0Bb | 0 ± 0.0Aa | - | - | - | - |
| Dichloromethane | 0 ± 0.0Aa | 0 ± 0.0Aa | 47.8 ± 0.1Bb | 100 ± 0.0Cb | 0 ± 0.0Aa | 46.27 ± 0.3Bb | 100 ± 0.0Bb | 100 ± 0.0Bc | 0 ± 0.0Aa | - | - | - | - | |
| Ethyl acetate | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0Aa | 100 ± 0.0Bb | 0 ± 0.0Aa | 0 ± 0.0Aa | 0 ± 0.0Aa | 68.63 ± 0.2Bb | - | - | - | - | |
| Acetone | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | - | - | - | - | |
| Acetonitrile | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | 0 ± 0.0a | - | - | - | - | |
| IC 50% AChEI** | Cyclohexane | >100 | >100 | - | - | - | 95.7 ± 1.3 | - | 12.5 ± 0.01 | - | - | - | - | 0.1 ± 0.03 |
| Dichloromethane | - | - | >100 | 40,9 ± 0.5 | - | >100 | 6.2 ± 0.5 | 2.5 ± 0.7 | - | - | - | - | 0.1 ± 0.03 | |
| Ethyl acetate | - | - | - | - | 31.7 ± 0.6 | - | - | - | 72.3 ± 0.06 | - | - | - | 0.1 ± 0.03 | |
| Acetone | - | - | - | - | - | - | - | - | - | - | - | - | 0.1 ± 0.03 | |
| Acetonitrile | - | - | - | - | - | - | - | - | - | - | - | - | 0.1 ± 0.03 | |
| AValues in the same row (for the same specie) followed by the same lower case letter are not significantly different between the plant parts, by Duncan’s multiple range test (p<0.05); aValues in the same column followed by the same lower case letter are not significantly different among extracting solvent, by Duncan’s multiple range test (p<0.05); ± Extract concentration that give 50% of antioxidant effect (EC 50%); *Percentage of acetylcholinesterase inhibitory activity (% AChEI) of the crude extracts at 100 ug/mL of concentration; **Acetylcholinesterase inhibitory concentration at 50% of activity (IC50 in ug/mL) of the active crude extracts | ||||||||||||||
Antioxidant power
The results of antioxidant activities are shown in Table II. As regard DPPH radical scavenging activity, EC50 values of the extracts are ranged from 41.4 ± 1.6 µg/mL (Flowers acetonitrile extract from N. glauca) to >500 µg/mL for several extracts. The EC50 values of GA, AA and BHT content were found to be 3.6 ± 0.0, 4.2 ± 0.1 and 28.6 ± 0.3 µg/mL respectively. Discoloration of ABTS+ reflects the capacity of antioxidant species to donate electrons or hydrogen atoms to inactivate this radical cation. In this finding, the EC50 of the different extracts in ABTS+ assay are widely variable, they are ranged between 10.8 ± 0.2 µg/mL (Stems dichloromethane extract from C. arvensis) to 100.5 ± 0.6 µg/mL (Stems cyclohexane extract from C. murale).
Table II: Total polyphenols and flavonoids content
| Plant | C. arvensis | C. murale | N. glauca | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Extract | Stems | Leaves | Flowers | Stems | Leaves | Flowers | Stems | Leaves | Flowers | |
| TPP* | Cyclohexane | 43.1 ± 0.1Ba | 59.2 ± 0.7Ca | 26.7 ± 0.2Aa | 16.2 ± 0.4Aa | 77.0 ± 1.2Cb | 39.3 ± 0.1Ba | 20.8 ± 0.2Aa | 98.0 ± 0.2Ce | 80.2 ± 0.1Ba |
| Dichloromethane | 59.0 ± 0.3Bb | 188.3 ± 0.2Ce | 43.8 ± 0.2Ab | 18.5 ± 0.1Ab | 182.3 ± 0.6Ce | 96.1 ± 0.2Bb | 92.0 ± 0.6Be | 54.8 ± 0.5Ac | 138.5 ± 0.2Cb | |
| Ethyl acetate | 96.6 ± 0.8Bc | 163.3 ± 0.8Cd | 68.2 ± 0.3Ac | 69.7 ± 0.2Bc | 49.1 ± 0.2Aa | 159.7 ± 0.1Cd | 91.2 ± 0.3Bd | 69.2 ± 0.2Ad | 196.1 ± 0.5Cc | |
| Acetone | 129.6 ± 0.3Bd | 149.2 ± 0.2Cb | 121.0.2Ad | 109.1 ± 0.5Be | 105.5 ± 0.4Ac | 146.0 ± 0.9Cc | 60.2 ± 0.4Bb | 49.1 ± 0.3Ab | 264.7 ± 1.6Cd | |
| Acetonitrile | 171.4 ± 0.3Ce | 157.5 ± 0.6Bc | 104.4 ± 0.4Ae | 33.0 ± 0.3Ac | 167.3 ± 1.3Bd | 215.3 ± 0.3Ce | 64.5 ± 0.2Ac | 47.5 ± 0.3Aa | 254.3 ± 0.8Bd | |
| TF** | Cyclohexane | 24.4 ± 0.1Cd | 0 ± 0Aa | 19.8 ± 0.3Ba | 4.9 ± 0.3Bb | 14.2 ± 2.4Ca | 0.4 ± 0.2Aa | 8.4 ± 0.2Aa | 28.0 ± 1.4Cd | 10.3 ± 0.2Bb |
| Dichloromethane | 3.8 ± 0.9Aa | 3.6 ± 0.3Ab | 35.1 ± 0.1Bb | 3.8 ± 1Aa | 49.2 ± 0.7Cd | 24.7 ± 0.6Bb | 49.8 ± 2.2Ce | 42.9 ± 0.2Bc | 5.4 ± 0.3Aa | |
| Ethyl acetate | 9.7 ± 0.5Ab | 41.7 ± 0.2Ce | 38.7 ± 0.1Bc | 19.4 ± 0.2Bc | 20.0 ± 0.1Cb | 12.5 ± 0.3Ad | 49.0 ± 0.7Bd | 49.3 ± 0.2Be | 20.2 ± 0.3Ac | |
| Acetone | 19.1 ± 0.2Ac | 25.7 ± 0.3Bc | 40.1 ± 0.2Cd | 19.4 ± 0.5Ac | 20.2 ± 0.1Bb | 26.4 ± 0.2Ce | 45.8 ± 2.4Bc | 9.2 ± 0.6Aa | 36.7 ± 0.1Cd | |
| Acetonitrile | 27.0 ± 0.2Ae | 30.6 ± 0.2Bd | 43.0 ± 0.0Cd | 20.6 ± 0.4Be | 48.8 ± 0.5Cc | 15.8 ± 0.4Ac | 38.6 ± 0.4Ab | 46.9 ± 0.2Bd | 39.8 ± 2.1Ad | |
| AValues in the same row (for the same specie) followed by the same lower case letter are not significantly different between the plant parts, by Duncan's multiple range test (p<0.05); aValues in the same column followed by the same lower case letter are not significantly different among extracting solvent, by Duncan's multiple range test (p<0.05); *TPP contents in ug equivalent of gallic acid to 1 mg of extract (ug GA/mg); **TF content in μg equivalent of quercetin to 1 mg of extract (ug QE/mg) | ||||||||||
There was a correlation between degradation rate and the bleaching of b-carotene; where, the extract with the lowest beta-carotene degradation rate exhibits the highest antioxidant activity. In this work, extracts showed a large variation of their protective action against bleaching of beta-carotene, the EC50 values are ranged from 38 µg/mL (Ethyl acetate extract from C. murale leaves) to >500 µg/mL for several extracts. The EC50 value of BHT is 72.7 ± 0.3 µg/mL. Several extracts from these 3 plants exhibited better activities than BHT.
In this work, extracts showed a large variation of their protective action against bleaching of beta-carotene, the EC50 values are ranged from 38 µg/mL (Ethyl acetate extract from C. murale leaves) to >500 µg/mL for several extracts. The EC50 value of BHT is 72.7 ± 0.3 µg/mL. Several extracts from these 3 plants exhibit better activities than BHT.
These results revealed that considerable antiradical components were contained in the different plants parts. Differences in species and in solvents polarities and thus different extractability of the antioxidant components may explain the differences in antioxidant activity of the extracts from the three plants.
Antimicrobial activities
The results against both bacterial and Candida species are presented in Figure 1-3. For C. arvensis, leaves extracts showed the best activities (Figure 1B), especially in acetonitrile extract, which gave inhibition diameters of 20.4 ± 0.6, 15.0 ± 0.0 and 15.6 ± 0.2 mm respectively against Candida tropicalis, Pseudomonas aeruginosa and Enterobacter aerogenes. While, the leaves acetone and ethyl acetate extracts were the most active respectively against Salmonella typhimarium (27.1 ± 0.2 mm) and Candida albicans (20.0 ± 0.1 mm). Flowers extracts carried out good activities against some pathogens species (Figure 1C), indeed, ethyl acetate extract exhibited the best activity against L. monocytogenes (40.7 ± 1.1 mm), and acetone extract was the most active against M. luteus (34.9 ± 0.2 mm), while the best activity against S. aureus has been shown in acetonitrile extract (20.0 ± 0.0 mm). C. murale extracts seemed to be less active than the two others species extracts. Indeed, the best activities were shown for flowers acetonitrile extract (Figure 2C), especially for C. albicans (19.1 ± 0.1 mm), P. aeruginosa (30.2 ± 0.1 mm), S. typhimarium (25.0 ± 0.0 mm), L. monocytogenes (34.7 ± 0.6 mm), M. luteus (30.9 ± 1.1 mm) and S. aureus (22.1 ± 1.8 mm). While leaves acetonitrile extract (Figure 2B) exhibited the best activities against C. tropicalis (5.1 ± 0.3 mm) and E. aerogenes (33.93 ± 0.09 mm). N. glauca leaves crude extracts (Figure 3B) were the most active: Ethyl acetate extract presents the best activities against S. typhimurium (29.7 ± 1.3 mm), E. aerogenes (47.2 ± 0.7 mm), L. monocytogenes (50.1 ± 0.2 mm), M. luteus (54.0 ± 0.0 mm) and S. aureus (26.2 ± 1.0 mm). While, acetonitrile and ethyl acetate extracts had fairy the same activities against C. tropicalis (10.0 ± 0.0 and 10.1 ± 0.1 mm, respectively). Dichloromethane extract had the best activities against C. albicans (12.1 ± 0.5 mm) and P. aeruginosa (31.0 ± 0.2 mm).
Figure 1: C. arvensis extracts antimicrobial activities by agar diffusion test (inhibition zone in mm). (A) stems extracts activities, (B) leaves extracts activities, (C) flowers extracts activities
Figure 2: C. murale extracts antimicrobial activities by agar diffusion test (inhibition zone in mm). (A) stems extracts activities, (B) leaves extracts activities, (C) flowers extracts activities
Figure 3: N. glauca extracts antimicrobial activities by agar diffusion test (inhibition zone in mm). (A) stems extracts activities, (B) leaves extracts activities, (C) flowers extracts activities
Globally, by comparing the three species aerial parts extracts, N. glauca leaves ethyl acetate extract was the most active against the three Gram+ bacteria strains and against E. aerogenes, while, N. glauca leaves was the best against P. aeruginosa. Moreover, C. arvensis leaves ethyl acetate, acetone, and acetonitrile extracts were the most active respectively against C. albicans, S. typhimarium and C. tropicalis. In comparison with findings in other studies, we remark that several extracts from the three plants exhibit a good activity against the used microbial strains.
Acetylcholinesterase inhibition
The results of AChEI are tabulated in Table II. Extracts from C. arvensis displayed the weakest inhibitory activities, even the most active one (flowers dichloromethane extract) had an IC50 above 100 µg/mL. Extracts from C. murale exhibited moderate activity. The most potent one was the leaves ethyl acetate extract (31.7 ± 0.6 µg/mL). While, extract from N. glauca seemed to be most powerful AChE inhibitors, especially leaves dichloromethane extract which displayed the highest activity (IC50% = 2.5 ± 0.8 µg/mL). The % AChEI is remarkably influenced by the solvent type, indeed dichloromethane extract exhibited the best activities, followed by cyclohexane extracts then ethyl acetate extracts, while none of acetone or acetonitrile extracts has shown activity with a concentration less than 100 µg/mL. Thus, the best AChEI activity appears in extracts obtained by maceration in solvents with weak polarity.
Correlation
In this study, there was not a correlation between TPP content and TF content (R2 = 0.225), and for the antioxidant assays, only ABTS+ activity had correlation with TPP content (R2 = -0.525). Concerning antimicrobial activities, there was a weak correlation between TPP content and the activities against C. albicans, S. typhimarium, E. aerogenes and S. aureus (R2 were 0.337, 0.334, 0.322 and 0.320, respectively) p<0.1, the correlation was more important with M. luteus (R2 = 0.410) p<0.01. Whereas, a distinct correlation between the amounts of TF content with the antioxidant and antimicrobial activities was revealed. Although a trend can be found, relating the higher content of TF to the DPPH activity (R2 = -0.421) and ABTS+ activity (R2 = -0.460) p˂0.01, therefore, this good correlation suggests that flavonoids compounds, present in the extracts from the three plants play an important role as anti-radicals. While, TF and beta-carotene test were not correlated (R2 = -0.058). Furthermore, TF content and the antimicrobial activities were highly correlated (p<0.01) with all the pathogenic strains. As regards the AChEI activity, no correlation was noticed with any of the other parameters.
Discussion
Our results show that TPP and TF contents varied depending on solvent used for the extraction and plant part. Acetonitrile extracts seemed to be the richest in TF, and leaves extracts seemed to contain the major quantities of TPP and TF, except for N. glauca that exhibited the highest content in TPP in flowers extracts. Concerning the antiradical capacity assayed by the most commonly used test based on DPPH and ABTS+ radicals scavenging (Liu et al., 2014), results reveal flatulence antiradical potential, which statistically dependent on flavonoids content, while the results of beta-carotene-linoleic acid bleaching system had correlation neither with TPP content nor with TF content.
Then, the assessment of extracts antimicrobial activity had been performed and their positive relationship with TTP and TF contents had been likewise highlighted by studying their correlation.
In this study we have also assessed the acetylcholinesterase inhibitory power; results reveal that the active extracts were those obtained by extraction with low polarity solvents, and the higher activity had been shown with the cyclohexanic extract from N. glauca flowers.
In comparison with other studies for the same species, much more important quantities of TPP and TF have been remarked in our findings. Indeed, Ercetin et al. (2012) found a TPP content of 43 µg GA/mg approximately in flowers dichloromethane extract from C. arvensis, which is similar to our result (43.8 ± 0.1 µg GA/mg), however, for the same extract, they found a weak quantity of TF (5 ug QE/mg) while we found 35.1 ± 0.1 µg QE/mg. Other study showed that a methanolic extract of flowers from C. arvensis exhibited a TPP and TF contents of 14.5 ± 0.4 µg GA/mg and 5.3 ± 0.4 ug QE/mg, respectively (Cetkovic et al., 2004), and this quantities are much fewer than the contents found with all flowers extracts in our work. For N. glauca, Tabana et al. (2015) reported that an organic fraction from this plant contain 108.2 µg GA/mg of TPP and 20.8 ug QE/mg of TF, in our work we found a much higher quantities, for example, the TPP content in flowers acetone extract was 264.8 ± 1.6 µg GA/mg and the TF for the same extract was 36.7 ± 0.1 ug QE/mg. grown.
Concerning antioxidant activities, our findings seem to be better than results reported from other works on these three plants, indeed, Ercetin et al. (2012) findings showed that extracts from C. arvensis exhibits a weak DPPH anti-radical activity (EC50 >1000 µg/mL for all extracts), while in our work we found that extracts from this same plant present a potent activity. Furthermore, Tabana et al. (2015) indicated that an extract from N. glauca had been exhibit an anti-radical activities against DPPH and ABTS of 86.7 µg /mL and 67.6 µg /mL respectively, besides, we found a much better activities with several extracts from this same plant. For antimicrobial activities, it had been demonstrated that C. arvensis methanol and hexane extracts from flowers are able to inhibit some pathogen such as S. aureus, L. monocytogenes and P. aeruginosa, while the aqueous extract is unable to inhibit them (Abudunia et al., 2014), moreover the inhibitory capacity of these extracts against S. aureus and L. monocytogenes remain weaker than our results found with C. arvensis flowers extracts. Indeed in our work the largest zones of inhibition of the pathogens were respectively 20.0 ± 0.1 mm (Acetonitrile extract), 40.7 ± 1.1 mm (Ethyl acetate extract), while the best result against P. aeruginosa in our work was found in acetonitrile extract (Zone of inhibition reached 8.7 ± 0.6 mm of diameter) and it is less active than C. arvensis flowers methanolic extract (Abudunia et al., 2014). No data about antimicrobial activity from other areal parts of this plant. Concerning C. murale antimicrobial potential, Zain et al. (2012) found that methanolic extract of C. murale leaves is active against P. aeruginosa and C. albicans, but unable to inhibit S. typhimurium, M. luteus and S. aureus. Awadh Ali et al. (2001) found the lack of activity of the whole plant methanolic extract against S. aureus and also P. aeruginosa. In other study (Ahmad et al., 2003), they found that the whole crude methanolic extract is inactive against C. albicans, S. aureus and S. typhimurium, but exhibited a medium activity against P. aeruginosa (Zone of inhibition reached 16 mm). Nevertheless in our work, C. murale extracts had a good inhibitory activity against these pathogens, for example, leaves acetonitrile extract gave important activity with zones of inhibition that reached 10.2 ± 0.6 mm, 24.3 ± 0.4 mm, 17.0 ± 0.2 mm, 29.7 ± 0.6 mm and 15.9 ± 0.2 mm respectively against C. albicans, P. aeruginosa, S. typhimurium, M. luteus and S. aureus. As regards N. glauca, studies reported its cytotoxic activities against pathogens, indeed, Abdel Rahman et al. (2011) found that hexane and methanol extracts from N. glauca whole aerial part inhibit weakly different strains of S. aureus but don't inhibit L. monocytogenes, while in our case all extracts from each areal part (Stems, leaves and flowers) had strongly inhibiting both S. aureus and L. monocytogenes.
Concerning AChEI activity, although the potent activities of organic extracts from C. arvensis flowers and leaves reported by Ercetin et al. (2012), they remain weak comparing with our findings. Literature didn't reported AChEI activity for C. murale and N. glauca.
Phenolics are very important plant constituents; they exhibit antioxidant activity by inactivating free radicals or preventing decomposition of hydroperoxides into free radicals. The intermediate compounds formed by the action of the phenolic antioxidants are relatively stable due to charge distribution throughout the aromatic ring system (Costamagna et al., 2013; Maisuthisakul et al., 2007). This large variation of TPP and TF contents in the different extracts from the three plants can be explained by the varying solubility of the phenolic compounds in the used solvents, and this due to the difference of solvent polarities (Marinova and Yanishlieva, 1997). Furthermore, the different parts of plants are known to contain varying quantities of bioactive compounds, indeed, most phenolic compounds and flavonoids were found in the leaf extract, due to photosynthesis in this organ (Andersen and Markham, 2006).
The high correlation between flavonoids content and anti-radical capacity is due to hydroxyl groups in flavonoids molecules that confer scavenging ability on them (Emeka et al., 2015). As regard beta-carotene bleaching system, linoleic acid produces hydroperoxides as free radicals during incubation at 50°C. Beta-carotene undergoes rapid discoloration in the absence of antioxidants, while, the presence of antioxidants in the extracts will minimize its oxidation by the hydroperoxides (Kchaou et al., 2016). This hydroperoxides formed in this system will be neutralized by antioxidants. Thus, the degradation rate of beta-carotene depends on the antioxidant capacity. In the present work, several extracts from the three plants had shown an excellent capacity to protect beta-carotene from discoloration, thus, there are able to neutralize hydroperoxides. It is important to develop new compounds that protect beta-carotene from degradation because of the strong biological activity and physiological important role of this pigment (Maisarah et al., 2014).
The antimicrobial ability of the extracts in this work can be attributed to levels of phenolics contents, especially flavonoids levels, which was making out by numerous studies (Ozcelik et al., 2011). Indeed, the effectiveness of flavonoids against several pathogenic bacteria and fungi had been carried out (Rauha et al., 2000; Sohn et al., 2004), and researches had make evidence that flavonoids are able to form complexes with extracellular soluble proteins which bind to the bacterial cell wall (Tsuchiya et al., 1996).
Inhibition of acetylcholinesterase (AChE), the key enzyme in the breakdown of acetylcholine, is considered as a promising strategy for the treatment of neurological disorders such as Alzheimer's disease, senile dementia, ataxia and myasthenia gravis (Mukherjeea et al., 2007). In our findings, extracts obtained by low polarity solvents like cyclohexane and dichloromethane exhibited the strongest AChEI activity which hadn't any correlation with TPP and TF contents. This may due to power of other compounds presents in the three plants such as alkaloids, a nonpolar compounds. Indeed, many studies make out the effectiveness of alkaloids like a strong AChE inhibitors (Ahmed et al., 2013; Murraya et al., 2013). Moreover, many Alzheimer's drugs are made from plants alkaloids or their derivatives (Ng et al., 2015; Dall'Acqua, 2013).
The great activity from N. glauca (in leaves dichloromethane extract) can be explained by the fact that this plant contains some compounds that inhibit the AChE, such as anabisine (Lisko et al., 2013). Indeed, it had been reported that the anabisine, an alkaloid, and a principal compound of N. glauca, reversibly inhibits AChE by binding a serine in the active site of acetylcholinesterase, inhibiting the enzyme completely (Dobren'kov et al., 1988; Karadsheh et al., 1991).
We can finally deduce that the important contents in phenolics, and the good biological profiles of C. arvensis, C. murale and N. glauca, are probably due to the hostile conditions of the marsh area, where these three plants had grown. Indeed the high level of salinity causes stress in the plants from which, they produce much more quantities of secondary metabolites to survive in this conditions (Chanwitheesuk et al., 2005), especially flavonoids to improve a good antioxidative response (Reginato et al., 2014; Rezazadeh et al., 2012).
Conclusion
Globally, these screening investigations indicate that extracts from different aerial parts with different solvent polarities (cyclohexane, dichloromethane, ethyl acetate, acetone and acetonitrile) of the three species C. arvensis, C. murale and N. glauca, exhibit different biological activities. Then, confirm the great potential of plants from Chebba marshy region for the production of bioactive compounds, under salinity stress condition, especially polyphenolics and flavonoids.
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