Agrarian Academic Journal
doi: 10.32406/v7n6/2024/82-97/agrariacad
In vitro study of antioxidant and anti-inflammatory activities of Matricaria pubescens extracts. Estudo in vitro de atividades antioxidantes e antiinflamatórias de extratos de Matricaria pubescens.
Hafsa Boubekeur
1, Abderrahim Benslama1*, Hadjer Habouche1, Somia Mimoune1, Sarra Saadia Hellal2
1- Department of Microbiology and Biochemistry, Faculty of Sciences; University of M’Sila, University Pole, Road Bourdj Bou Arreiridj, M’Sila 28000, Algeria. Email: abderrahim.benslama@univ-msila.dz
2- Department of Medicine, Faculty of Medicine, University of Algiers, Ziania, Algeria.
Abstract
Our study is based on the evaluation of the biological activities of the aerial part of Matricaria pubescens, a medicinal plant of the traditional pharmacopoeia of Oued souf region. Two methods of extracting Soxhlet and maceration ethanolic. Phytochemical tests have revealed the presence of active ingredients, especially polyphenols and flavonoids. The Folin Ciocalteu method and AlCl3 aluminum trichloride, respectively determine total polyphenols and flavonoids. The results obtained indicate that the Soxhlet extract has the highest content of 59.25±0.27 µg gallic acid equivalent / mg of extract and 12.106 ±1.07 µg equivalent quercetin / mg of extract. While the low content present by the maceration 41.365±1.01 µg equivalent of gallic acid / mg of extract and 5.380 ± 0.085 µg equivalent quercetin / mg of extract. The evaluation of the anti-free radical activities by the free radical scavenging method (DPPH) has shown strong anti-free radical activities by Soxhlet, which is lower than that of the BHT with IC50 of 99.861±2.364 and 16.415±2.461 µg/mL respectively, compared to the maceration with IC50 of 106.340±2.407 µg/mL. The anti-inflammatory activity of the extracts was explored in vitro by the protein denaturation inhibition the test extracts showed inhibitory activity similar to diclofenac sodium with a percentage inhibition 67.038±1.36; 58.038±2.551 and 72.435±1.022 µg/mL, respectively.
Keywords: Medicinal plant. Phytochemical screening. Polyphenols and flavonoids. Biological activities.
Resumo
Nosso estudo baseia-se na avaliação das atividades biológicas da parte aérea de Matricaria pubescens, planta medicinal da farmacopéia tradicional da região de Oued souf. Dois métodos de extração de Soxhlet e maceração etanólica. Testes fitoquímicos revelaram a presença de princípios ativos, principalmente polifenóis e flavonóides. O método de Folin Ciocalteu e o tricloreto de alumínio AlCl3, respectivamente, determinam polifenóis e flavonóides totais. Os resultados obtidos indicam que o extrato de Soxhlet apresenta o maior teor de 59.25±0.27 µg equivalente de ácido gálico/mg de extrato e 12.106±1.07 µg equivalente de quercetina/mg de extrato. Enquanto o baixo teor apresentado pela maceração 41.365±1.01 µg equivalente de ácido gálico/mg de extrato e 5.380±0.085 µg equivalente de quercetina/mg de extrato. A avaliação das atividades anti-radicais livres pelo método de eliminação de radicais livres (DPPH) mostrou fortes atividades anti-radicais livres por Soxhlet, que é inferior à do BHT com IC50 de 99.861±2.364 e 16.415±2.461 µg/mL respectivamente, comparado à maceração com IC50 de 106.340±2.407 µg/mL. A atividade anti-inflamatória dos extratos foi explorada in vitro pela inibição da desnaturação proteica. Os extratos de teste apresentaram atividade inibitória semelhante ao diclofenaco sódico com percentual de inibição de 67.038±1.36, 58.038±2.551 e 72.435±1.022 µg/mL, respectivamente.
Palavras-chave: Planta medicinal. Triagem fitoquímica. Polifenóis e flavonóides. Atividades biológicas.
Introduction
Inflammation is the body’s natural protective response to tissue damage caused by physical trauma, chemical agents or infection. It is characterized by pain, redness, heat, swelling and disruption of physiological functions. In most cases, this reaction is beneficial to the attacked host, but excessive activation can lead to significant alterations, including denaturation of certain proteins. These proteins, having lost their three-dimensional structure, can trigger the appearance of autoantigens, transforming an inflammatory reaction into an autoimmune one (CHANDRA et al., 2012). Treatment of inflammation is often based on non-steroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids.
On the other hand, the uncontrolled production of reactive oxygen and nitrogen species provokes or maintains inflammatory processes, leading to several diseases such as cancer, diabetes, asthma, premature aging, cardiovascular, neurodegenerative and inflammatory diseases. The neutralization of these species by antioxidants, especially of natural origin, can limit damage to biomolecules (DNA, proteins, lipids and sugars). Plants have always played an important role in human life. Every known civilization has used plants, whether wild or cultivated, for food, defense, clothing or medicine. A plant is said to be medicinal when at least one part of it possesses medicinal properties, as it contains active principles capable of preventing, alleviating or curing disease (SOFOWORA et al., 2013). Regardless of the parts and forms in which they are used, plants are extremely rich in complex chemical structures. The metabolism of plants contains thousands of different constituents whose therapeutic effect is obviously not linked to all the compounds, nor to their harmful or toxic effects (WANG et al., 2019). Algeria, by virtue of its geographical position, presents a wide range of bioclimatic stages, inducing a biodiversity of plants used as condiments, natural foods and for therapeutic purposes (IMENE et al., 2023).
Plants contain a large number of effective compounds that reflect the therapeutic potential of these plants, it is known that some plant drugs have a therapeutic capacity greater than that of manufactured medicines in treating some diseases, and the use of these drugs is devoid of the harmful side effects that accompany the use of manufactured medicines sometimes. And among the other factors that led to the increase in the use of medicinal plants and other natural products, the emergence of new diseases accompanied by severe complications for which no suitable treatment has yet been found (ANTAL, 2010; EKOR, 2014).
The secondary metabolite diversity of the medicinal plants explains their multiple pharmacological activities, and as a result, many species of this family are used in traditional medicine. Flavonoids, anthocyanins, tannins, phenols, and other plant constituents are potential antioxidants (BENSLAMA et al., 2021). Foods’s rich in antioxidants play an essential part in the disease’s prevention, such as diabetic, cancer, neurodegenerative, cardiovascular disorders, inflammation, and problems caused by cell and cutaneous aging (BENSLAMA et al., 2019).
A great deal of research worldwide has focused on the valorization of natural substances endowed with biological activities, in order to establish scientific rules for their use. This is the background to the present study, whose main aim is to investigate the phytochemical screening and to evaluate the anti-inflammatory and antioxidant properties of extracts of the medicinal plant Matricaria pubescens, commonly used in traditional medicine for its various virtues.
Materials and methods
Plant materials
The Matricaria pubescens plant was harvested during the flowering stage in April 2017 in the El Meghaier region (Oued souf wilaya), southeastern Algeria. The aerial parts (leafy stems and flowers) were cleaned and shade-dried in a dry, airy place. They were then ground to powder using an electric grinder and collected in clean paper bags until use.
Extracts preparation
Maceration extraction
The maceration hydroalcoholic extract of the aerial parts of the M. pubescens plant was prepared according to the method described by Vargas-Madriz et al. (2020). 20 g of ground plant material was macerated in 200 mL of an ethanol/water mixture (80/20) under magnetic stirring and at room temperature. This maceration is repeated 3 times in succession, with solvent replacement every 24 hours. The resulting hydroalcoholic macerate is double-filtered on filter paper. The filtrates were concentrated in a rotary evaporator (Büchi R 114) at a temperature of 45°C.
Soxhlet extraction
A 20 g sample of the aerial part of the M. pubescens plant is introduced into a cellulose cartridge and subjected to extraction with 500 mL of an ethanol/water mixture (80/20) in the Soxhlet, for 6 hours. Extraction is stopped when the liquid surrounding the cartridge becomes clear, this color indicating that the solvent no longer extracts anything from the solid. Processing time is different depending on the solvent, often more than 3 hours (after about six cycles of solvents in the extractor Soxhlet). The hydroalcoholic extracts are evaporated under vacuum at a temperature of 45°C (PINILLA et al., 2021).
The yield of the extraction was calculated by the flowing formula:
Yield (%) = 100 mass of the extract after evaporation solvent (g)/ mass dried of the sample plant (g).
Phytochemical screening
Phytochemical analysis was carried out on the extract obtained by infusion, Soxhlet and maceration using chemical procedures to identify the various constituents as described by Sheikh et al. (2013). The revelation of certain chemical families of the plant was achieved through chemical detection tests such as: alkaloids (Mayer and Wagner test), phenolic compounds and tannins (ferric chloride reaction), flavonoids (cyanidine reaction), saponins (foam index), sterols and triterpenes (Liebermann Buchard reaction), coumarins (confirmation test). Qualitative analysis of the extract is based on staining and/or precipitation reactions.
Alkaloids
To ensure reliable results, two types of alkaloid test reagents were used in parallel Mayer and Wagner. For the first test, a 0.5 mL volume of Mayer reagent was brought into contact with 0.5 mL of each extract. For the second test, a volume of 0.5 mL of each extract was mixed with 0.5 mL of Wagner’s reagent. The formation of a white or brown precipitate in both tests indicate the presence of alkaloids.
Polyphenol
Polyphenol detection involves introducing 2 mL of aqueous extract into a test tube, followed by 02 drops of 2% FeCl3. The appearance of blue-black, green or dark black coloration indicates the presence of polyphenol.
Flavonoids
A few drops of concentrated hydrochloric acid and a few milligrams of magnesium turnings were added to 0.5 mL of extract. Pink-red or yellow coloration indicates the presence of flavonoids.
Tannins
Tannins are detected by adding 1 mL of water and 1 to 2 drops of 1% FeCl3 solution to 1 mL of each extract. The appearance of a dark green or blue-green coloration indicates the presence of tannins. The appearance of a dark green color indicates the presence of catechic tannins. The appearance of blue-green coloration indicates the presence of gallic tannins.
Coumarins
In a tube, 5 mL of extract, then 2 mL of hot water is added to the residue. The solution is divided between 2 test tubes. To the contents of one of the tubes, 0.5 mL of 25% NH4OH is added. Fluorescence is observed under UV light at 366 nm. Intense fluorescence in the tube to which ammonia has been added indicates the presence of coumarins.
Quinonic substances
Free or combined quinone substances are detected using Borntraegen’s reagent. Practically 2 mL of the infusate is evaporated to dryness. The residue is triturated without 5 mL of 1/5 hydrochloric acid, then placed in a water bath for 30 min.
After cooling, the quinones are extracted with 20 mL chloroform. A 0.5 mL volume of ammonia diluted to one-half is added to the chloroform solution. A red or violet coloration is a positive sign of the presence of quinones.
Reducing compounds
Detection involves introducing 2 mL of aqueous extract into a test tube, then adding 2 mL of Fehling’s liqueur. The mixture is then heated in a boiling water bath for 8 min. Obtaining a brick-red precipitate indicates the presence of reducing compounds.
Sterols and polyterpenes
Sterols and polyterpenes have been identified by the Liebermann-Buchard reaction. 1 mL of infusate is mixed hot with 1 mL of chloroform, in a test tube into which 0.5 mL of a concentrated sulfuric acid solution is slowly poured to form a layer. The appearance of a red ring indicates a positive reaction.
Saponins
The identification of saponins in a plant extract is based on the ability of an aqueous solution of the extract to foam after agitation. This property is the basis of the method used to assess the richness of an extract in saponosides: measurement of the foam index.
To detect saponins, we introduced 10 mL of aqueous extract into a test tube. The tube was shaken for a few seconds, then left to stand for 15 min. A persistent foam height greater than 1 cm indicates the presence of saponosides.
Determination of total polyphenols contents (TPC)
Determination of polyphenols was carried out using a mini-1240 UV visible double-beam spectrophotometer. The double-beam technique helped us to eliminate the absorbance of the blank and give the optical density of the sample directly. To ensure reliable results, the determination of each phenolic compound was carried out in three trials, after averaging the measured optical densities. Determination of total polyphenols using the Folin-Cicalteu reagent (BENSLAMA; HARRAR, 2016).
This assay is based on quantification of the total concentration of hydroxyl groups present in the extract. The Folin-Ciocalteau reagent consists of an acidic yellow solution containing a polymeric complex of ions (heteropolyacids). In an alkaline environment, Folin-Ciocalteau reagent oxidizes phenols to phenolate ions and partially reduces its heteropolyacids, resulting in the formation of a blue complex.
To 0.2 mL of extract (prepared in distilled water with the appropriate dilutions) is added 0.8 mL of Na2CO3 solution (75 mg/mL distilled water), after shaking, 1 mL of Folin Ciocalteu solution (diluted tenfold in distilled water) is added to the whole, after 2 h incubation at laboratory temperature, absorbance is read at 765 nm against a blank without extract. The level of total polyphenols in our extracts was calculated from a linear calibration curve (y= ax + b), established with precise concentrations of gallic acid (0-250µg/mL), as a reference standard, under the same conditions as the sample. Results are expressed in micrograms of gallic acid equivalent per milligram of extract of powdered aerial part (µg GAE /mg).
Determination of total flavonoids contents (TFC)
The aluminum trichloride (AlCl3) method was used to determine the flavonoid content of various M. Pubescens extracts (BENSLAMA; HARRAR, 2016). 1 mL of sample (dissolved in methanol) with appropriate dilutions was added to an equal volume of AlCl3 solution (2% in methanol). The mixture was shaken vigorously, then incubated in the dark at room temperature for 10 min. Absorbance was measured at 430 nm using a UV spectrophotometer (UV mini-1240).
Flavonoids were quantified according to a linear calibration curve (y = a x + b) using a standard quercetin at different concentrations (0-150 μg/mL) under the same conditions as the sample. Results are expressed in micrograms of quercetin equivalent per milligram of extract (μg QE/mg).
Antioxidant activity using the DPPH free radical
The antioxidant test was carried out using the DPPH method (POPOVICI et al., 2009). 50 μl of each methanolic solution of the extracts at different concentrations (from 0.0625 to 2.5 mg/mL) are added to 1.95 mL of the DPPH methanolic solution (0.025g/l). At the same time, a negative control is prepared by mixing 50μl of methanol with 1.95 mL of the DPPH methanolic solution. Absorbance is read against a blank prepared for each concentration at 517nm after 30 min incubation in the dark at room temperature. The positive control is represented by a solution of a standard antioxidant, ascorbic acid, whose absorbance was measured under the same conditions as the samples, and for each concentration the test was repeated 3 times. Results are expressed as percent inhibition (I%).
I%= [(Abs control – Abs test)/ Abs control] x 100
The values of the IC 50 have summer determined graphically by their regression linear.
In vitro anti-inflammatory activity
The albumin denaturation assay was performed by adopting the method described by Kumari et al. (2015). A reaction vessel for each mixture was prepared consisting of 200 ul egg albumin, 1400 ul phosphate-buffered saline and 1000 ul test extract (at different concentrations: (100, 300,1000 mg/mL). Distilled water was used instead of extract as a negative control. The mixtures were then incubated at 37°C for 15 minutes and heated to 70°C for 5 minutes. After cooling, absorbance was measured at 660 nm (OSMAN et al., 2016).
Diclofenac sodium was used as a positive control under the same operating conditions. The experiment was performed in triplicate. Percentage inhibition of protein denaturation was calculated as follows.
Results
Extraction
In order to evaluate the best extraction technique for total polyphenols and flavonoids, we used two extraction methods for the aerial parts of the Matricaria pubescens plant: cold maceration of plant powder and hot Soxhlet with a hydroalcoholic solvent (ethanol/water). Extraction is the main stage in the recovery and isolation of bioactive phytochemicals. It is influenced by the extraction process used, the particle size of the sample, and the presence of interfering substances (STALIKAS, 2007). The ethanolic extracts (EtOH(S); EtOH(M)) obtained have a viscous brown and dark green appearance. The combined use of water and ethanol can facilitate the extraction of substances soluble in water and/or ethanol.
In fact, the use of dry material for polyphenol extraction is recommended, as flavonoids can undergo enzymatic degradation when plant material is fresh or undried (MARSTON; HOSTETTMANN, 2006). Drying in the dark prevents chemical transformations such as isomerization and degradation caused by UV radiation from sunlight (JONES; KINGHORN, 2012).
The use of powder improves extraction because the contact surface between sample and solvent is greater, and penetration into cells not destroyed after grinding is easier. The best extraction yields were recorded for maceration, with an average of 24.38% compared with 15.160% for Soxhlet (Table 1).
The yields calculated by Metrouh-Amir et al. (2015) and Bouziane et al. (2016) from a hydroalcoholic extract of Matricaria pubescens harvested from Ouargla are 23.22% and 34.68%, respectively. These yields are higher than those we found before. This yield variability depends on several parameters such as: solvent, pH, temperature, extraction time and method, as well as the place and time of sample harvesting.
Table 1 – Extraction yield, TPC and TFC of M. pubescens extracts.
Extract |
Yield (%) |
TPC |
TFC |
Maceration |
24.38 |
59.25 ± 0.27 |
12.106± 1.07 |
Soxhlet extraction |
15.160 |
41.365 ± 1.01 |
5.380 ± 0.085 |
The results obtained show that among the different extracts, the aqueous decoction extract represents the highest yield (40.48%) relative to the total weight of the plant, followed by the aqueous maceration extract (9.733%), the Soxhlet ethanolic extract (5.7294%), and the ethanolic maceration extract (4.563%).
Phytochemical screening
After obtaining the extractables (ethanol/water), we performed a phytochemical screening of the extract to qualitatively identify the different families of compounds present. Depending on the family of molecules sought, the test was carried out either directly with the extract or after infusion. The results of this screening give an idea of the likely biological activities. These colorimetric tests are based on the interaction of certain functions with the reagents used.
The results revealed the presence of polyphenols, flavonoids, catechic tannins, saponins, polytherpenes, coumarins and alkaloids (Table 2).
The polyphenol test produced an intense blackish-blue coloration, indicating the high polyphenol content of the aerial part of the plant. The flavonoid test was positive, with the appearance of a yellow coloration. The appearance of an intense dark green color indicates the presence of catechic tannins. The formation of foam after stirring the infusate and its persistence after 15 min of rest with a height of 2 cm show the relative richness of the aerial part in saponins. Tests for gall tannins and reducing compounds were negative. The presence of terpenes and coumarins was also demonstrated, but they were less abundant than the other chemical families revealed. The reaction for quinonic substances was negative. This is in line with the results of Ahmed et al. (2021).
The results of this typically qualitative primary phytochemical characterization show that the aerial parts of M. pubescens possess an appreciable amount of polyphenols, tannin and flavonoids.
The work of Djellouli et al. (2013) on the aerial parts of M. pubescens harvested from the south-western region of Algeria, showed the presence of the same families of compounds, with the exception of alkaloids and terpenes, which were more abundant compared to ours. In addition, he noted the presence of cardenolides, steroids. Our results concur with those of Djellouli et al. (2013) and Metrouh-Amir et al. (2015), who noted the absence of gallic tannins and the presence of catechic tannins trapenoids, steroid.
The results of the phytochemical composition of various plant extracts by chemical screening are shown in Table 2.
Table 2 – Phytochemical screening of M. Pubescens extracts.
Compounds |
Identification reagents |
Part used |
Indicator |
Result |
Polyphenols |
FeCl3 (2%) |
Extract |
Color blueblackish orgreen or black dark |
+++ |
Flavonoids |
Hydrochloricacid, magnesiummagnesium |
Extract |
The colorpink-red or yellow |
++ |
Alkaloid |
Mayer |
Infusate |
Precipitatewhite orbrown |
+ |
Tannins |
Solution ofFeCl3 diluted at1% |
Extract |
dark green(catechictannins) |
++ |
Coumarin |
NH4OH à 25%. |
Extract |
Fluoresces |
+ |
Quinonic substances |
Borntraegen reagent |
Infusate |
red coloror violet |
– |
Reducing compounds |
Fehling’sliqueur |
Extract |
Precipitatered |
– |
Sterols and Polyterpenes |
Liebermann reagent |
Extract |
BrickRing |
+ |
Saponoside |
Foamingfoam |
Infusate |
Red Moss persistent |
++ |
+++: a strongly positive reaction; ++: a moderately positive reaction; +: a weakly positive reaction; -: a negative reaction.
Determination of total polyphenols and flavonoids
The choice of quantifying polyphenols among the various phytochemicals stems from the fact that polyphenols have very important biological activities. The same applies to flavonoids, which are considered the most important class of polyphenols.
Two calibration curves (Figures 1, 2) have been drawn for the determination of total polyphenols and flavonoids in Matricaria pubescens extracts, using standard solutions (gallic acid, quercetin) at different concentrations. Ethanol and water and their mixtures are the most widely used for good recovery of phenolic compounds from certain plants of the Asteraceae family (CAI et al., 2004) and for obtaining better antioxidant activity (BARROS et al., 2010). Total phenolic content of extracts was expressed as micrgrame gallic acid equivalent per milligram extract (µg GAE/mg E). After the addition of sodium carbonate monohydrate solution and Folin Ciocalteu reagent to M. pubescens extract, a blue color was obtained, which varied according to the concentration of the two extracts. A calibration curve was drawn with gallic acid at different concentrations (0-200 µg/mL); optical density measurements for each extract were carried out at 760 nm.
Figure 1 – Gallic acid calibration curve.
Figure 2 – Quercetin calibration curve.
Statistical analysis of the total polyphenol content obtained by the different extraction methods (Table 1), reveals a non-significant difference (p< 0.05) for the two ethanolic extracts, equivalent to 59.25±0.27 µgEAG /mg extract by Soxhlet compared to 41.365±1.01 µg EAG/mg extract by maceration. The study carried out by Metrouh-Amir et al. (2015), concerning the effect of extraction solvent on the phenolic compound content of M. pubescens, shows that the best polyphenol content is obtained using dilute organic solvents namely; aqueous methanol, aqueous ethanol and aqueous acetone. According to the results of Khacheba et al. (2014), the polyphenol content of methanolic extracts is 3.16 µg EAG/mg extract. However, Harbourne et al. (2009) found a value of 13 µgEAG/mg extract in a plant of the same genus. These values are well below our own. Several authors point out that there is a significant difference in phenolic content within the same species depending on its origin and habitat, as confirmed by Sánchez-Rodriguez et al. (2011). This variability is probably linked to severe environmental conditions such as soil type, salinity, low rainfall and water stress, which may induce the synthesis of phenolic compounds as a response to oxidative stress generated by the formation of reactive oxygen species. In studies carried out by Djeridane et al. (2006) on Algerian plants of the same family; Artemisia herba-hara, Artemisia campestris and Anthemisa arvensis, polyphenol contents lower than those found in M. pubescens were obtained using aqueous ethanol (70%) for extraction, although the contents obtained ranged from 10.31 to 30.23 µgEAG/ g extract.
Flavonoids are a widely distributed class of secondary metabolites in the plant kingdom. They constitute the most important polyphenolic class, comprising several thousand molecules in over 10 classes with more than 5,000 compounds (GÓMEZ-CARAVACA et al., 2006). In this work, flavonoids were determined by the aluminum trichloride method. After addition of AlCl3 and incubation, a yellowish color was obtained, the intensity of which was proportional to the concentration of both extracts, confirming the presence of flavonoids in the extract of the aerial part of M. pubescens.
Statistical analysis of the total flavonoid content obtained by the different extraction methods (Table 1), reveals a non-significant difference (p< 0.05) for the two ethanolic extracts, equivalent to12.106± 0.27 µg EQ/mg extract by Soxhlet compared with 5.380± 0.085 µg EQ/mg extract by maceration.
In view of the data in the literature, we note that total flavonoid content varies for the same plant depending on the region of origin, the solvent and the extraction method used. In this context, Khacheba et al. (2014) and Eddine et al. (2016) found that the aqueous and methanolic extract found by Soxhlet and by maceration of the aerial part of M. pubescens recolé from the El oued and Laghouat regions, contain different proportions of flavonoids and which are 9.76 and 1.04 mg EQ/g MS, respectively. Similarly, Bouziane et al. (2016) showed the remarkable richness of the hydro-acetone extract of the plant harvested from the Biskra region in flavonoids 526.3 mg EAG/g extract. These results are higher than those obtained with our extracts.
This variability is also observed for species of the same genus, in fact Harbourne et al. (2009) showed the richness of Matricaria camomilla (24.5 µg EQ/ mg extract) in flavonoids compared to plants of the same family such as: Artimisia hera alba, Artimisia compastiris and Artimisia arvensis which contain between 7.5 and 13.1 µg ER/ g extract using aqueous ethanol (70%) as solvent (DJERIDANE et al., 2006).
Antioxidant activity
The DPPH method was chosen for its simplicity, rapidity and sensitivity, and is based on the reduction of a DPPH in the presence of an antioxidant that donates a hydrogen or an electron. The non-radical form is formed. This method also makes it possible to compare IC50s expressed in mg/mL with each other, and not just with a reference. IC50 defines the concentration of plant extract responsible for inhibiting 50% of DPPH radicals. DPPH radical inhibition was assessed for each extract of M. pubescens and the anti-free radical activity profile of each extract tested against the DPPH radical is shown in Figure 3.
Figure 3 – The anti-free radical activity profile of M. pubescens extracts against the DPPH radical.
The anti-free radical activity profiles obtained were tested by the DPPH method, a stable organic radical that reacts with the polyphenol by electron and hydrogen atom transfer. Antioxidants react with DPPH to neutralize the radical. The color of the reaction mixture changes from violet to yellow. The intensity of the discoloration measures the potential activity of antioxidant scavenging (VLADIMIR-KNEZEVIC et al., 2011). IC50 values were obtained from the graph plotted as a function of percentage DPPH scavenging inhibition and sample concentration, this value is required to reduce 50% DPPH radical. The majority of plant extracts show significantly lower anti-radical activity than the reference product (BHT). The results are shown in Figure 3.
Hydroethanol extracts obtained by both extraction methods (Soxhlet /maceration) of the aerial part of M. pubescens have a concentration-dependent free radical scavenging activity towards the DPPH radical. Indeed, the IC50 values obtained with these extracts are low, in the order of 99.861 and 106.340 µg/mL respectively, compared with the reference antioxidant BHT, which has an IC50 of 16.415 µg/mL (Table 3). The activity of both extracts is probably due to the presence of phenolic compounds, which are known to have the ability to trap radical species and reactive forms of oxygen (DEMENCIANO et al., 2020).
Table 3 – The IC50 values in DPPH antioxidant activity assay.
Extract/BHT |
IC 50 (mg/mL) |
EtOH(S) |
99.861±2.364 |
EtOH(M) |
106.340 ± 2.407 |
BHT |
16.415±2.461 |
Since the Soxhlet extract is richer in polyphenols than the maceration extract, its capacity to trap DPPH radicals is higher. Makhloufi et al. (2021) found that the ethanolic extract of Matricaria pubescens had DPPH radical scavenging effects of 81.45 µg/mL. According to studies by Bouziane et al. (2016), the plant’s hydroacetone and methanol extracts have an inhibition power equivalent to 43.95% and 75%, respectively.
The chemical compound 2,2-diphenyl-1-picrylhydrazyl was one of the first free radicals used to study the structure-antioxidant activity relationship of phenolic compounds. It has an unpaired electron on a nitrogen bridge atom (POPOVOCI et al., 2009). DPPH is a stable radical with a characteristic absorption at 517 nm in solution, giving it a violet hue. This color quickly disappears when DPPH is reduced by a free radical scavenger. Where (AH) n represents a compound capable of yielding a hydrogen to the DPPH radical. (violet) to transform it into a DPPH-H molecule. Correlations were analyzed by Pearson’s test. Several studies have reported on the relationship between phytochemicals and antioxidant activity. Some authors found a strong correlation between phenolic content and antioxidant activity (SELLES et al., 2012; BENSLAMA et al., 2021; HECHAICHI et al., 2023). The antioxidant activity of the various extracts tested could be attributed to their richness in molecules with high anti-free radical potential, such as polyphenols, flavonoids and tannins. This hypothesis is confirmed by several authors who attribute the anti-free radical activity of plant extracts to these molecules (BENSLAMA et al., 2017).
In vitro anti-inflammatory activity
In our study, the anti-inflammatory action of the plant was assessed in vitro by the inhibition test of protein denaturation (egg albumin) induced by heat treatment. This test was performed as a preliminary assay to verify the presence of anti-inflammatory properties. The maximum percentages of inhibition of protein denaturation by the extract (EtOH(S) and EtOH(M)) and the positive control (diclofenac sodium) are shown in Figure 3. Different concentrations of hydroalcoholic extract (EtOH(S) and EtOH(M)) of M. pubescens plant gave non-significant percentages of inhibition of protein denaturation (p˂ 0.05) ranging from: 25.26% and 67.04% for EtOH(S) and 23%, 58.105% for EtOH(M).
Diclofenac was used as a standard to compare its anti-inflammatory activity with our extract, finding a percentage 72.43% close to the extract studied.
Autoantigen production in some arthritic diseases may be due to protein denaturation, membrane lysis and proteinase action (AL NOMAN et al., 2016). The anti-inflammatory activity of ethanolic extracts of M. pubescens may be due to one constituent or to the synergistic effect of several phytochemical constituents such as the flavonoids and tannins present. Some flavonoids possess potent inhibitory activity against a variety of enzymes such as protein kinase C, protein tyrosine kinase and phospholipase A2 (PARVIN et al., 2015). The phospholipase A2 enzyme is known to be responsible for the formation of inflammatory mediators such as prostaglandins and leukotrienes, which, by attracting leukocytes to the site of inflammation, cause tissue damage, probably through the release of free radicals. Phospholipase A2 hydrolyzes phospholipids in the cell membrane into arachidonic acid, which is very rapidly metabolized by cyclooxygenase into prostaglandins, which are major components responsible for pain inflammation (SHIRWAIKAR et al., 2011; RAMADEVI et al., 2014). According to the literature free radicals can harm surrounding tissues, initiating lipid peroxidation that leads to membrane destruction. Damaged tissue provokes an inflammatory response through the production of mediators. Free radical scavengers can be beneficial in the treatment of inflammatory disorders (SEN et al., 2010). Denaturation is a process in which proteins lose their tertiary and secondary structure through the application of an external stress or chemical compound such as a strong acid or base, a concentrated inorganic salt, an organic solvent or heat. Most proteins lose their biological function when denatured. Denaturation of tissue proteins is one of the causes of inflammatory diseases. The mechanism of denaturation probably involves electrostatic alteration of hydrogen and the disulfide bridge (MISHRA et al., 2011). Diclofenac sodium (an anti-inflammatory drug) and hydroalcoholic extract have showed a dose-dependent ability to inhibit thermally induced protein denaturation. These experimental results support the traditional use of this plant for the treatment of various ailments, particularly inflammation. To our knowledge, no results on the evaluation of in vitro anti-inflammatory activity by the protein denaturation inhibition assay have been reported by other authors on M. pubescens to be able to compare our results, but numerous studies have reported the anti-inflammatory and analgesic effect of Matricaria pubescens alkaloids and essential oils (METROUH-AMIR; AMIR, 2018; BOUTAGHANE et al., 2011).
Conclusion
The aim of this work was to adopt a scientific basis for the validation of certain biological properties attributed to this plant, chosen on the basis of its traditional use. The hydro-ethanol mixture proved to be the ideal solvent for extracting phenolic compounds, thanks to its ability to extract both polar and apolar molecules, which was reflected in the good yields obtained with this solvent. Phytochemical analysis showed that ethanolic extracts are rich in polyphenols and flavonoids. In addition, all extracts possess antioxidant activity, scavenging free radicals and protecting macromolecules against oxidation. Our different extracts showed effective inhibition of thermal denaturation of albumin, compared with diclofenac, and are therefore endowed with anti-inflammatory activity. The medicinal plant is a promising source of antioxidant and anti-inflammatory agents, which is explained by the nature of the compounds present in this plant. Our study showed that the plant has good antioxidant and anti-inflammatory activity, which justifies its use in complementary (traditional) medicine.
Conflict of interest
Authors declare that there is no conflict of interest.
Authors’ contribution
Hafsa Boubekeur and Abderrahim Benslama – original idea, direction, evaluation and original writing, data collection, corrections and text review; Nadjet Maarouf, Leila Ladjal, Sarra Saadia Hellal – guidelines, data collection, data analysis.
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Received on February 22, 2024
Returned for adjustments on January 7, 2025
Received with adjustments on January 11, 2025
Accepted on February 3, 2025
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