Introduction
1.
Free radicals are often atoms or molecules or molecular fragments containing one or more unpaired electrons in their atomic or molecular orbitals, which give them significant reactivity. Indeed, a radical will always tend to fill its orbital by capturing an electron from other compounds to become stable. Thus, the new molecule formed becomes itself a free radical by losing its electron, triggering a cascade of chain reactions which finally damages the living cell (Mukherji et al., 1986).
The main free radicals involved in human pathophysiological processes are the superoxide radical, the hydroxyl radical and the peroxyl radical. Singlet oxygen and hydrogen peroxide are reactive oxygen species that are not free radicals but very reactive. Besides reactive oxygenated species (ROS), there are reactive nitrogenous species (RNS) whose major representative is nitric oxide (Huet and Duranteau, 2008;Bargagli et al., 2009). In low doses, ROSs are very useful for the organism and play important roles in various physiological mechanisms such as signal transduction, cell differentiation, apoptosis, immune defense against pathogens, destruction by apoptosis of tumor cells, regulation of capillary dilation, the functioning of some neurons, especially those of memory, fertilization of the ovum, regulation of genes and participate in the functioning of some enzymes (Koechlin-Ramonatxo, 2006;Roberts and Sindhu, 2009). Nevertheless, the excessive production of free radicals causes direct damage to biological molecules (lipids, proteins and DNA), but also secondary damages due to the cytotoxic and mutagenic character of the metabolites released in particular during the oxidation of lipids (Deby-Dupont et al., 2002). This damage results in oxidative stress which causes various diseases like Alzheimer's disease, Parkinson's disease, heart disease, diabetes mellitus, cancer (Halliwell and Gutteridge, 1990;Qazi and Molvi, 2018). In order to reduce the risk of chronic diseases and prevent their progression, it is necessary to enhance the body's natural antioxidant defenses or supplemented with food antioxidants. In this context, the search for new antioxidant compounds from natural sources has been increased. The alkaloids consist of the most abundant naturally occurring group of secondary metabolites in plants (Rehman and Khan, 2017). Alkaloids are naturally occurring chemical compounds containing basic nitrogen atoms and often have pharmacological effects. Many studies have demonstrated that alkaloids have biological activities, such as antioxidant, anti-microbial, anti-inflammatory, anti-cancer, and anti-virus (Yan et al., 2008). Moreover, the vast biological activities make many alkaloids prominent start line for chemical medicinal chemistry (Rehman and Khan, 2017).
Matricaria pubescens is a medicinal plant much appreciated by the inhabitants of the Saharan regions and used as a remedy against rheumatism, aches, coughs, allergies, eye diseases, dysmenorrhea, and gastro-intestinal disorders. In children, it is used against measles, ailments related to teething, fever and dermatosis (Maiza et al., 1995).
Various secondary metabolites like alkaloids, saponins, terpenoids, phytosterols (Djellouli et al., 2013), flavonoids and tannins (Metrouh-Amir et al., 2015) have been isolated from M. pubescens. Alkaloid compounds exert a wide range of biological effects. The study carried out byMetrouh-Amir and Amir, (2018) found that the alkaloids of M. pubescens exerted antinociceptive and anti-inflammatory activities.
The aim of this study was to determine the effect of extraction method and solvent on alkaloid contents and antioxidant activities of M. pubescens.
Materials and methods
2.
Plant material
The entire plant was harvested during flowering in March 2016 at Ouargla area (Algerian Septentrional Sahara). Authentication and identification were made by the botanist J.P. Lebrun (National Museum of Natural History, Paris). A specimen of the plant was deposited at Laboratoire de Botanique d'Alger, Algeria (voucher number 644).
Plant processing and extraction of total alkaloids
The entire plant was air dried and ground and fractions with diameters of less than 250 μm were used for the extraction.
The extraction of alkaloids from M. pubescens was carried out using several protocols at different pH with various solvents (extraction in an alkaline medium, in an acid medium and in a neutral medium), by maceration and Soxhlet (Bruneton, 2009).
Extraction in an alkaline medium by maceration
The principle of this extraction protocol is based on the method ofBruneton, (2009). Ten grams of the powder were mixed with 100 mL of alkalized chloroform at pH 9 with concentrated ammonium hydroxide. After 24 hours of maceration, the mixture was filtered. In order to extract the maximum amount of alkaloids, this phase was repeated three times. The recovered filtrate was partially concentrated then extracted with 100 mL of water acidified at pH 3 with HCl. This acid solution was adjusted to pH 9 with concentrated ammonium hydroxide, and extracted with chloroform. The organic phase recovered was dried with anhydrous sodium sulfate and then concentrated to dryness.
Extraction in an acid medium by maceration
The principle of extraction of alkaloids in an acid medium was inspired by the method ofYubin et al. (2014). An amount of 10 g of powder was mixed with 300 mL of methanol acidified to pH 4 with sulfuric acid. After 72 hours of maceration, the extract was filtered. In this case, the extraction was followed by vacuum distillation which eliminates the alcohol and leaves an acidic aqueous solution of alkaloid salts. The acidic aqueous solution recovered was made alkaline with concentrated ammonium hydroxide up to pH 9, and then extracted with chloroform until complete exhaustion. The extract obtained was dried with anhydrous sodium sulphate and evaporated to dryness.
Extraction in a neutral medium
The extraction of alkaloids in a neutral medium was carried out using two solvents, ethanol and methanol, by the Soxhlet apparatus and by maceration.
The extraction protocol with ethanol and methanol by Soxhlet was carried out according toSuau et al. (2002) method, with some modifications. Ten grams of the powder were mixed with 300 mL of alcohol (methanol or ethanol). After 8 h of extraction by Soxhlet apparatus, the mixture was filtered and evaporated. The residue was acidified with hydrochloric acid to pH 3. The acid extract was adjusted with concentrated ammonium hydroxide to pH 9, and then extracted with chloroform until complete exhaustion. The organic phase was dried with anhydrous sodium sulphate and evaporated to afford a crude extract of alkaloids.
The extraction protocol with ethanol by maceration is the next: The powdered sample (10 g) was extracted with 300 mL of ethanol by maceration for 24 h. The obtained extract was treated in the same manner as described for the Soxhlet method (see above).
Determination of plant extract yield
The percentage yield of the alkaloids was calculated as: % yield = W2 / W1 × 100. Where W2 is the weight of alkaloids crude extract and W1 is the weight of the initial dried powder.
The crude extract of alkaloids was dissolved in dimethyl sulfoxide (DMSO 1%) to evaluate antioxidant activity.
Determination of antioxidant activity
The antioxidant activity of the alkaloid extracts of M. pubescens was determined using different tests.
Ferric reducing power (FRP)
The ferric reducing power was evaluated according to the method ofDeepa et al. (2015). First, 2.5 mL of alkaloid extracts at various concentrations were mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1 g/100 mL). The mixture was incubated at 50 °C. After 20 min, 2.5 mL of trichloroacetic acid (10 g/100 mL) was added. Then, 2.5 mL of supernatant was mixed with 2.5 mL of distilled water and 0.5 mL of ferric chloride solution (0.1 g/100 mL). After 10 min, the absorbance was measured at 700 nm. The ferric reducing activity was expressed as gram ascorbic acid equivalent (AAE) per 100 g of dry weight (DW). Butylated hydroxyanisole (BHA), gallic acid and ascorbic acid were used as standards.
Total antioxidant capacity (TAC)
The total antioxidant capacity of the alkaloid extracts was determined by the phosphomolybdenum method ofPrieto et al. (1999). A volume of 0.3 mL of each concentration was mixed with 2 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The mixtures were incubated at 95 °C for 90 min. After cooling to room temperature, the absorbance was measured at 695 nm. The total antioxidant capacity was expressed as gram ascorbic acid equivalent (AAE) per 100 g of dry weight (DW). Ascorbic acid, gallic acid and trolox were used as positive controls.
DPPH radical scavenging activity
The DPPH (1, 1-diphenyl-2-picrylhydrazy) radical scavenging activity was performed according to the method of Jain et al. (2008.). 0.3 mL of alkaloid extracts was added to 2.7 mL of DPPH solution (60µ M). After 60 min of incubation, the absorbance was assessed at 517 nm. The percentage of DPPH radical scavenging activity was determined according to the formula: {[(Ac − At)/Ac] × 100}, where Ac is the absorbance of the control and At is the absorbance of the extract. The extract concentration providing 50% antioxidant activity (IC50 value (µg/mL)) was calculated from the graph of antioxidant activity percentages against alkaloid concentrations. BHA, gallic acid and ascorbic acid were used as positive controls.
ABTS radical scavenging activity
The ABTS (2, 2-Azino-bis (3-ethylbenzothiazloine-6-sulfonic acid) radical scavenging capacity was evaluated according to the method given byRe et al. (1999) with a little modification. In order to prepare the ABTS solution, an aliquot of 7 mM of ABTS was mixed with 2.45 mM of potassium persulfate and incubated 16 h at room temperature in the dark. The ABTS solution was diluted with ethanol until an absorbance of 0.70 ± 0.02 at 734 nm. 10 µL of alkaloids extract was added to 1 mL of ABTS solution. The absorbance was measured after 6 min at 734 nm. The percentage of ABTS radical scavenging activity of alkaloid extracts was determined from {[(Ac − At)/Ac] × 100}, where Ac is the absorbance of the control and At is the absorbance of the extract. The decrease in absorption at 734 nm was used for calculating the IC50 value (µg/mL). Trolox, BHA and gallic acid were used as positive controls.
β-Carotene bleaching (BCB) assay
The β-carotene bleaching inhibiting activity of M. pubescens alkaloids was evaluated using the β-carotene/linoleate model system (Moure et al., 2000). An amount of β-carotene (2 mg) was dissolved in 10 mL of chloroform. 1 mL of β-carotene solution was added to 20 μL of linoleic acid and 200 mg of Tween 40 emulsifier. After total evaporation of the chloroform, 100 mL of distilled water saturated with oxygen was added. 0.2 mL of different concentrations of the alkaloids was mixed with 4 mL of this emulsion. The absorbance was immediately measured at 470 nm against a blank, consisting of the emulsion without β-carotene, which was considered at t = 0 min. After 2h of incubation at 50 °C in water bath, the absorbance was evaluated at 470 nm (A120). The inhibition of β-carotene bleaching percentage is determined by the following formula: {[(At(120) – Ac(120))/(Ac(0) – Ac(120))] × 100}, Where At(120) is the absorbance of the sample at t = 120 min; AC(120) is the absorbance of the control at t = 120 min and AC(0) is the absorbance of the control at t = 0 min. The extract concentration providing 50% antioxidant activity (IC50 value (mg/mL)) was calculated from the graph of antioxidant activity percentage against alkaloid concentration. BHA, ascorbic acid and gallic acid were used as standard.
Hydroxyl radical scavenging activity
In this study the hydroxyl radical scavenging activity was assessed by two different tests:
Deoxyribose test
The hydroxyl free radical scavenging activity (HRSA) was evaluated according to the method ofHalliwell et al. (1987), with some modifications. 1 mL of alkaloid extract was mixed with 0.1 mL of EDTA(1mM), 0.01 mL of chloride ferric (10 mM), 0.1 mL of hydrogen peroxide (10mM), 0.36 mL of deoxyribose (10 mM), 0.33 mL of phosphate buffer (50 mM, pH 7.4) and 0.1 mL of ascorbic acid (1mM) in sequence. After 1 hour of incubation at 37 °C, 1 mL of the mixture was added to 1 mL of thiobarbituric acid (1%) followed by 1 mL of trichloroacetic acid (0.5%). The mixtures were incubated in a water bath at 100 °C. After 20 min, the absorbance was measured at 532. The results were expressed as percentage of deoxyribose oxidation inhibition as determined by the following formula: {[(Ac − At)/Ac] × 100}, where Ac is the absorbance of the control and At is the absorbance of the extract. IC50 value (mg/mL) calculated denote the concentration of the extract required to decrease the absorbance by 50% at 532 nm. BHA, gallic acid and ascorbic acid were used as positive controls.
HOSC test
The HOSC (Hydroxyl radical scavenging capacity) method was tested according to the method ofMoore et al. (2006). In a 96-well microplate, 170 µL of fluorescein (9.28 × 10−8 M) and 40 µL of H2O2 (0.1990 M) were added to 30 µL of alkaloid extract. The reaction was started by adding 60 µL of FeCl3 solution (3.43 mM) at each well of the microplate. For 60 min at 37 °C, the fluorescence emitted by the reduced form of fluorescein was measured and recorded every 1 min. Emission and excitation wavelengths were 485±20 nm and 530±25 nm, respectively. Solution of sodium saline buffer (75 mM, pH 7.4) was used to prepare the fluorescein solution. Solutions of H2O2 and FeCl3 were prepared in Milli-Q water. The trolox has been used as control standard at different concentrations (5, 10, 15, 20 and 30 µmol/L). The HOSC values have been expressed using regression equation between the net area under the fluorescein decay curve and the trolox at different concentrations. The HORAC capacities were expressed as mmoL of trolox equivalents (TE) per 100 g of dry weight (DW).
Oxygen radical absorbance capacity (ORAC)
The ORAC assay was evaluated according to the method ofHuang et al. (2002), with some modifications. 25 µL of alkaloid extract was mixed with 150 µL of disodium fluorescein (2 × 10−7 mM). The mixture was incubated at 37 °C in the plate reader. After 10 min, 25 µL of AAPH (2, 2’-azobis- 2-methyl-propanimidamide, dihydrochloride) (153 mM) was added. During 40 min at 37 °C, the fluorescence emitted by the reduced form of fluorescein was recorded every 1 min at 530 ± 25 nm excitation and 485± 20 nm emission wavelengths. Trolox at different concentration (5, 10, 20, 30 and 40 µmol/L) was used as control standard. The solution of phosphate buffer saline (75 mM, pH 7.4) was used to prepare AAPH, fluorescein, and trolox solutions. The ORAC values have been expressed by applying the same equation used for the previous test. The ORAC capacities were expressed as mmoL of trolox equivalents (TE) per 100 g of dry weight (DW).
Statistical analysis
Statistical analysis of the results was carried out with one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. The comparison of these results was taken at the probability of P <0.05. All the data represent the average of three tests ± standard deviation. Nonlinear regression test was used to determine the IC50.The correlation between all parameters was defined by Pearson correlation coefficient (r).
Results and discussion
3.
Extract yields
A good extraction method must allow the complete extraction of the compounds of interest, avoiding any chemical modification. The extraction of bioactive compounds from plant materials using various solvent is an important step in obtaining phytochemical-rich product. The bioactive compounds from various plants have commonly been isolated by extraction methods such as Soxhlet extraction and maceration (Hayati et al., 2019).
In order to extract the highest alkaloid content from M. pubescens, extraction was carried out by applying two extraction techniques, maceration and Soxhlet extraction, using several solvents at different pH (alkalized chloroform, acidified methanol, neutral methanol and neutral ethanol). The results of this study show that the obtained extraction yields were affected by the extraction technique, nature and pH of solvent used (Figure 1). The highest extraction rates were obtained with ethanolic neutral (Soxhlet) and methanolic acidified (maceration) extracts with yields of 4.03±0.01% and 4.02±0.01%, respectively, while the lowest extraction rate was found in the alkalinized chloroform extract (maceration) with a value of 2.69±0.02%.
_83-95-f1.jpg)
Results with different letters are significantly different. All the data represent the average of three tests ± standard deviation.
Using Soxhlet for the extraction, the results obtained showed that ethanol allowed to extract the highest rate of alkaloids compared to methanol. Concerning the extraction by maceration, it is the methanol which allowed to extract the highest rate in alkaloids that ethanol.
The extraction yield of plant extracts is affected strongly by the solvent, due to the presence of different compounds with different chemical characteristics and polarities. The variation between the extract yields was due to the polarities of different compounds present in the plant (Labiad et al., 2017).
The extraction yield using ethanol as solvent in Soxhlet extraction (4.03±0.01%) was higher than that obtained using the same solvent for extraction by maceration (3.32±0.01%), which indicates that the extraction technique influences the extraction yield of alkaloids.Djilani et al. (2006) demonstrated that the extraction technique used affects the extraction rate of alkaloids from the plants Ruta graveolens and Hyoscyamus muticus, which is consistent with the results of the present study.
Soxhlet extraction allows the sample to come into repeated contact with hot solvent, promoting the dissolution of the desired compounds. Extraction time and temperature are important parameters for optimizing extraction conditions (Galvàn D’Alessandro, 2013). This could explain the higher yield of alkaloids extracted by Soxhlet comparing to that extracted by maceration using the same extraction solvent. The differences observed between the alkaloid contents obtained by applying several extraction protocols could be explained by the presence of alkaloids of different nature and thus of solubility in the different extracts of M. pubescens.
Authors have reported lower alkaloid contents than those found in this study. Contents ranging from 0.024 to 0.126 g/100 g, were extracted from two plants of the same family; Artemisia judaica and Artemisia herba alba (Elsharkawy and Shiboob, 2017). Different alkaloid fractions of Fumaria officinalis plant have given alkaloid contents ranging from 0.18 to 0.63 g/100 g (Khamtache-Abderrahim et al., 2016).
Antioxidant activities
Antioxidant compounds act through several mechanisms of action, including inhibition of free radical generation, enhancement of scavenging capacity against free radicals and reducing capacity. Thus, no test can accurately reflect the antioxidant effect of the compounds in a complex system. Therefore, to determine the antioxidant activity of compounds it is necessary to use the minimum two different methods (Antolovich et al., 2002). So, in the present study, the antioxidant activity of the alkaloid extracts of M. pubescens has been evaluated by various tests which determine the reducing activity of ferric and molybdate ions and the scavenging activity against DPPH, ABTS, hydroxyl and peroxyl radicals.
The extracts used to determine the antioxidant activity of M. pubescens are those which gave the best contents in alkaloids, and which are acidified methanol and alkalized chloroform extracts obtained by maceration and neutral ethanol extract obtained by Soxhlet.
Ascorbic acid, gallic acid, trolox and butylated hydroxyanisole (BHA) were standards used as reference antioxidants.
Ferric reducing power (FRP)
The reducing properties of plant extracts are generally due to the presence of reducing compounds, which exert an antioxidant effect by giving a hydrogen atom which leads to the breaking of the radical chain. These reducing compounds also have the ability to react with some peroxide precursors, thus preventing peroxide formation (Govindan and Muthukrishnan, 2013).
The results of this work showed that all alkaloid extracts of M. pubescens reduced ferric ions with significantly different values (p<0.05), depending on the tested alkaloid concentration, extraction technique, pH and the used solvent (Figure 2).
_83-95-f2.jpg)
For all tested concentrations ranging from 5 to 25 mg/mL, statistical analysis of the results revealed that the strongest ferric reducing power was exerted by acid methanolic extract (maceration) with values ranging from 0.12 to 0.96 g AAE/100 g (DW). While the alkaline chloroform extract (maceration) gave the lowest reducing power, with values between 0.10 and 0.39 g AAE/100 g (DW). This study revealed that by increasing the concentration of alkaloids, the ferric reducing capacity of the extracts tested increases significantly (p <0.05).
Results with different letters are significantly different. All the data represent the average of three tests ± standard deviation
Ascorbic acid, gallic acid and BHA have been shown to have greater reductive capacities of ferric than those obtained with alkaloid extracts of M. pubescens, at different concentrations, with values between 2.04 and 5.09 g AAE/100 g, 0.71 and 1.88 g AAE/100 g, and between 1.20 and 3.62 g AAE/100 g, respectively.
Total antioxidant capacity (TAC)
The capacities of alkaloid extracts of M. pubescens to reduce molybdate were significantly different (p <0.05), depending on the extraction protocol and the concentration of alkaloids tested. For alkaloid concentrations ranging from 5 to 25 mg/mL, the best total antioxidant capacity was obtained with the neutral ethanolic extract (Soxhlet), with contents between 0.32 and 0.88 g AAE/100 g (DW), while the lowest reducing activity of molybdate was determined with the chloroformic alkaline extract (maceration) with values ranging from 0.14 to 0.31 g AAE/100g (DW) (Figure 3). The statistical study did not reveal any significant difference (p<0.05) between the total antioxidant capacities of the alkaloid extracts prepared by alkaline chloroform (maceration) and acid methanol (maceration) at concentrations ranging from 5 to 15 mg/mL, and they exhibited the weakest capabilities.
_83-95-f3.jpg)
Total antioxidant capacity of the extracts of the studied plant is significantly (p<0.05) proportional to the concentration of alkaloids for all tested extracts.
At concentrations ranging from 5 to 25 mg/mL, ascorbic acid, gallic acid and trolox tested in this activity showed a molybdate reducing capacity with values ranging from 1.53 to 2.82 g AAE/100 g, 1.36 to 1.82 g AAE/100 g and from 0.36 to 0.57 g AAE/100 g. These standards have shown greater molybdate reducing capacities than those obtained with alkaloid extracts of M. pubescens at different concentrations, except trolox.
Results with different letters are significantly different. All the data represent the average of three tests ± standard deviation.
DPPH radical scavenging activity
The evaluation of the DPPH radical scavenger activity revealed that the IC50 values of the various alkaloid extracts of M. pubescens obtained showed significant differences (p˂0.05), depending on the extraction technique, nature and pH of solvent (Table 1). The neutral ethanolic extract (Soxhlet) exerted the largest DPPH radical scavenging effect with an IC50 of 314 μg/mL, while the alkaline chloroform extract (maceration) exerted the weakest scavenger effect with an IC50 of 744.29 µg/mL.
BHA showed the highest DPPH radical scavenger activity than that exerted by all alkaloid extracts of the studied plant. However, alkaloid extracts obtained by neutral ethanol (Soxhlet) and acidified methanol (maceration) showed a significant (p˂0.05) DPPH radical scavenging activity superior to those exerted by gallic acid and ascorbic acid.
ABTS radical scavenging activity
The results of ABTS scavenger activity of M. pubescens extracts and of standards are shown in table number 1. Statistical analysis revealed the existence of a significant difference (p <0.05) between the tested alkaloid extracts according to the technique, pH and solvent. Acidified methanolic (maceration) and neutral ethanolic (Soxhlet) extracts exerted the most important ABTS radical scavenger effect, with IC50 values of 370.83 and 395.81 µg/mL, respectively. While alkaline chloroform extract (maceration) showed the lowest scavenger activity with an IC50 of 605.41 µg/mL.
The present work revealed that the alkaloid extracts obtained from M. pubescens exhibited lower ABTS radical scavenger activity than that expressed by trolox, BHA and gallic acid.
β-carotene bleaching inhibiting (BCB) activity
The potential of the plant to inhibit lipid peroxidation was evaluated using β-carotene/linoleic acid bleaching test. β-carotene bleaching assay is considered among the oldest and most commonly applied methods of evaluating antioxidant activity of substances or extracts (Andrzej and Olszowy, 2010). In this test, the oxidation of linoleic acid in an aqueous emulsion system generates peroxyl radicals. The break of 𝜋-conjugation by addition reaction of peroxyl radical to a C = C double bond of β-carotene, causes discoloration of the yellowish colour of the solution of β-carotene. The presence of antioxidants reduces the extent of destruction of β-carotene by neutralizing free radicals derived from linoleic acid and therefore helps to prevent the oxidation and bleaching of β-carotene (Ueno et al., 2014).
The results of β-carotene bleaching inhibition by alkaloid extracts of M. pubescens presented significant differences (p<0.05) according to the extraction technique, the pH and the nature of the solvent used (Figure 4). The acidic methanolic extract (maceration) revealed the most potent β-carotene bleaching inhibiting activity (IC50 = 4.07 mg/mL), while the alkaline chloroform extract (maceration) exhibited the lowest inhibiting activity (IC50 = 14.90 mg/mL).
_83-95-f4.jpg)
This work has shown that gallic acid, ascorbic acid and BHA have shown the lowest β-carotene oxidation inhibiting activities than those exerted by the alkaloids of M. pubescens. However, trolox and neutral ethanolic extract exhibited the most important inhibitory activity compared to all other tested extracts.
Results with different letters are significantly different. All the data represent the average of three tests ± standard deviation.
Hydroxyl radical scavenging activity (HRSA) determined by deoxyribose test
In this test, the ability to trap the hydroxyl radical by plant extracts is based on Fenton reaction by measuring the generation of the OH• radical and its effect on the oxidation and degradation of biological molecules such as DNA deoxyribose. At low pH, hydroxyl radicals attack deoxyribose forming products that, upon heating with thiobarbituric acid (TBA), yield a pink chromogen. The presence of antioxidants protects deoxyribose and reduces chromogen formation (Halliwell et al., 1987).
Hydroxyl radical is the most reactive oxygen species, and has the shortest half-life compared with other ROS. The highly reactive hydroxyl radical can cause oxidative damage to DNA, lipids, and proteins (Halliwell and Gutteridge, 1985). In this study, the results of the hydroxyl radical scavenger activity, determined by deoxyribose test, revealed that the IC50 values obtained show significant differences (p˂0.05), according to the technique, pH and solvent (Figure 5). The strongest hydroxyl radical scavenger activities were found using alkaline chloroform (maceration) and acidic methanol (maceration), with IC50 values of 1.11 and 1.24 mg/mL respectively. The neutral ethanolic extract (Soxhlet) exerted the weakest hydroxyl radical scavenger activity with an IC50 of 2.70 mg/mL.
_83-95-f5.jpg)
The extracts prepared by alkaline chloroform and acid methanol showed the highest hydroxyl radical inhibitory activities than those exerted by all the standards tested, except gallic acid.
Results with different letters are significantly different. All the data represent the average of three tests ± standard deviation.
HOSC and ORAC capacities
Analysis of the results showed that acidic methanol extract exhibited the highest antioxidant activities among the tested extracts, thus HOSC and ORAC capacities were determined only on this extract (Figure 6).
_83-95-f6.jpg)
All the data represent the average of three tests ± standard deviation.
In the present study, the hydroxyl radical scavenging capacity was evaluated using also the HOSC assay (hydroxyl radical scavenging capacity). Hydroxyl radical is generated under physiological pH using a Fenton-like reaction with a definite end point. The HOSC assay is the best method compared to the deoxyribose assay widely used, because the HOSC method generates more hydroxyl radicals (Karadag et al., 2009). This method combines both the percentage inhibition of oxidation reaction and the length over time of this inhibition in a single measurement. This study showed that the alkaloids extracted by acidic methanol exhibited a hydroxyl radical scavenging capacity (HOSC) with a value of 672.44 mmoL TE/100 g (DW).
The ORAC (Oxygen radical absorbance capacity) method uses biologically relevant free radicals, integrates both time and degree of antioxidant activity into one data value, and it is readily adaptable to a high-throughput assay system. This method is based on the inhibition of the peroxyl radical (ROO·) induced oxidation initiated by thermal decomposition of AAPH (2, 2’-azobis (2-amidino propane) dihydrochloride). The antioxidants react with the peroxyl radicals and delay the degradation of fluorescein (Prior et al., 2003). The alkaloids extracted by acidic methanol exerted an ORAC capacity, with a value of 164.31 mmoL TE/100 g (DW).
Analysis of the data of the antioxidant activities determined by the fluorimetric methods showed that the alkaloids of M. pubescens obtained by acidic methanol exhibited a HOSC activity higher than the ORAC activity. This indicates that the alkaloids of M. pubescens are more effective on the hydroxyl radical than on the peroxyl radical.
Analysis of the results reveals that the antioxidant activities of alkaloid extracts of M. pubescens vary depending on the solvent and the technique used for the extraction and according to the method applied for the determination of antioxidant activity. This variation could be due to the quantity and/or the nature of the antioxidant substances present in the extracts studied. The antioxidant activity of the extracts was affected by the extraction solvent.
From the overall results of this study, the alkaloid extracts of M. pubescens exerted a reducing activity of ferric and molybdate ions. They also showed significant radical scavenger activity against DPPH, ABTS, hydroxyl, and peroxyl radicals. These different antioxidant activities could be due to the structural diversity of the alkaloids present in extracts of M. pubescens and interactions between these compounds, which influence their mechanisms of action.
Numerous studies have reported that alkaloids extracted from plants have the ability to inhibit lipid peroxidation (Schmeda-Hirschmann et al., 2003; Khamtache-Abderrahim et al., 2016;Gan et al., 2017), to exert a radical scavenger activity against DPPH, ABTS (Liu et al., 2014;Gutiérrez et al., 2014;Dalimunthe et al., 2018). The reducing power of ferric and molydate exerted by alkaloids has also been reported by other authors (Khamtache-Abderrahim et al., 2016;Liu et al., 2014;Gan et al., 2017). Studies have shown the effectiveness of alkaloids in inhibiting other free radicals such as hydroxyl radical (Kiplimo et al., 2011;Gan et al., 2017;Darkwah et al., 2018) and peroxyl radical (Tiong et al., 2013;Tian et al., 2018). These data indicate that the alkaloids can exert ferric and molybdate ion reducing activities and can inhibit DPPH, ABTS, hydroxyl and peroxyl radicals. These results confirm the different antioxidant activities exerted by the alkaloid extracts of M. pubescens.
Correlation between alkaloids and antioxidant activities
The correlation coefficients between the alkaloid concentrations of M. pubescens and the antioxidant activities obtained by several methods have been determined and are shown inTables 2 and 3{ label needed for table-wrap[@id='t3'] }.
| Alkaloids | FRP | TAC | |
|---|---|---|---|
| Alkaloids | 1 | ||
| FRP | 0.80 *** | 1 | |
| TAC | -0.58*** | 0.44** | 1 |
| *p < 0.05, significant correlation. **p < 0.01, very significant correlation. ***p < 0.001, extremely significant correlation. | |||
{ label needed for table-wrap[@id='t3'] }
| DPPH | ABTS | HRSA | BCB | |
|---|---|---|---|---|
| DPPH | 1 | |||
| ABTS | 0.93 *** | 1 | ||
| HRSA | -0.6 | -0.42 | 1 | |
| BCB | -0.22 | 0.01 | 0.73** | 1 |
| *p < 0.05, significant correlation. **p < 0.01, very significant correlation. ***p < 0.001, extremely significant correlation. | ||||
The results of this work revealed the presence of a very highly significant correlation (p<0.001) between the alkaloid contents and the ferric and molybdate reducing activities. These results reflect the powerful contribution of M. pubescens alkaloids in these reducing activities. The study of the correlations between the reducing activities revealed a very significant correlation (p<0.01) between the ferric reducing power (FRP) and the molybdate reducing activity (TAC).
A very highly significant correlation (p<0.001) was established between the DPPH and ABTS scavenger activities. However, the correlation between the hydroxyl radical scavenger activity (deoxyribose test) (HRSA) and β-carotene bleaching inhibiting activity (BCB) is highly significant (p <0.01). These results indicate that the alkaloids of M. pubescens have a good capacity to trap: DPPH, ABTS, hydroxyl, and peroxyl radicals.
The study showed insignificant correlations between DPPH, ABTS and HRSA, BCB. These results show that the bioactive substances responsible for each activity are not the same.
Few studies have been done on establishing correlations between alkaloid levels and antioxidant activities.Gan et al. (2017) reported relationship between the ferric reducing capacity, the hydroxyl radical scavenger activity, the lipid peroxidation inhibition ability and alkaloids.
The antioxidant activity of extract depends on the antioxidant contents, the structure and interaction between these antioxidant compounds (Rice-Evans et al., 1997;Bourgou et al., 2008). The antioxidant activities of the individual compounds may depend on structural factors, such as the number of phenolic hydroxyl or methoxyl groups, flavone hydroxyl, keto groups, free carboxylic groups and other structural features (Patt et al., 1990). Due to their redox properties, antioxidants act as hydrogen donators, reducing agents, and singlet and triplet oxygen quenchers (Pietta, 2000).
Conclusion
4.
This present work is the first study to evaluate the effect of the extraction method and solvent on alkaloids content of M. pubescens and their antioxidant activity. No study has determined the antioxidant activity of alkaloids of the plant. Based on the above results, it can be concluded that the extraction technique, pH and nature of extraction solvent have a significant effect on the alkaloids content and the antioxidant activity. In addition, the results of the antioxidant activity determined by several tests have shown that the alkaloid extracts of M. pubescens exerted a reducing activity of ferric and molybdate ions and showed significant radical scavenger activity against DPPH, ABTS, hydroxyl, and peroxyl radicals. The richness in alkaloids and the various antioxidant activities of M. pubescens observed in this work could justify its traditional use. It would be interesting to characterize the alkaloids of this plant and to study their biological activities.