Agrarian Academic Journal
doi: 10.32406/v7n2/2024/71-82/agrariacad
Exploring and evaluating the phenolic profile and antioxidant properties of the Algerian wild carrot Daucus carota subsp. maximus aqueous extract. Exploração e avaliação do perfil fenólico e das propriedades antioxidantes do extrato aquoso da cenoura selvagem argelina Daucus carota subsp. maximus.
Maroua Hadji
1*, Tahar Smaili1, Khadidja Dehimi2, Fethi Farouk Kebaili3,4, Toka Hadji5, Amal Lahouaou 6, Ilyas Yildiz7, Ramazan Erenler8
1*- Biodiversity and Biotechnological Techniques for Plant Resources Valorization Laboratory (BTB_VRV), Department of Natural and Life Sciences, Faculty of Sciences, University of M’sila, PO Box 166 Ichebilia, 28000, Algeria. Corresponding Author. E-mail: maroua.hadji@univ-msila.dz. E-mail: tahar.smaili@univ-msila.dz
2- Department of Microbiology and Biochemistry, Faculty of Sciences, University of M’sila, PO Box 166 Ichebilia, 28000, Algeria. E-mail: khadidja.dehimi@univ-msila.dz
3- Laboratory of Microbiological Engineering and Application, Department of Biochemistry and Molecular and Cellular Biology, Faculty of Nature and Life Sciences, University of Mentouri Brothers Constantine 1, P.O. Box, 325 Ain El Bey Way, Constantine, 25017, Algeria. E-mail: kff.fethi@gmail.com
4- The Regional Laboratory of Scientific Police, Ain El Bey City Constantine; the Sub-Directorate of Scientific and Technical Police; the General Directorate of National Security, Algeria.
5- Laboratory of the Development and Valorization of Plant Genetic Resources, Department of Biology and Plant Ecology, University of Mentouri Brothers Constantine 1, P.O. Box, 325 Ain El Bey Way, Constantine, 25017, Algeria. E-mail: tokahad@gmail.com
6- Laboratory of Plant Biotechnology and Ethnobotany, Department of Physical and Chemical Biology, University of Bejaia, Algeria. E-mail: amel.lahouaou@univ-msila.dz
7- Faculty of Health Sciences and Arts, Department of Molecular Biology and Genetics, Tokat Gaziosmanpaşa University, Tokat 60100, Türkiye. E-mail: Ilyas.yildiz@gop.edu.tr
8- Research Laboratory Application and Research Center (ALUM), Foundation of the Faculty of Health Sciences, Nutrition and Dietetics Department, Iğdır University, Iğdır, 76000, Türkiye. E-mail: ramazan.erenler@gop.edu.tr
Abstract
This study presents, for the first time, the phenolic profile of Daucus carota subsp. maximus aqueous extract and evaluates its antioxidant properties using various in vitro assays. The aqueous extract contained significant polyphenol and flavonoid contents (99.00 µg GAE/mg E and 43.06 µg QE/mg E, respectively). Additionally, LC-ESI-MS/MS analysis made it possible to identify and quantify 19 phenolic compounds, with trans-ferulic acid as the predominant compound (1.460 mg/g). Moreover, the aqueous extract exhibited antioxidant effects in all assays, with IC50 and A0.5 values less than 200 µg/mL except for Phenanthroline reducing power and SNP assays.
Keywords: Flora of M’sila. Phytochemical Characterization. Radical Scavengers. Polyphenols.
Resumo
Este estudo apresenta, pela primeira vez, o perfil fenólico do extrato aquoso de Daucus carota subsp. maximus e avalia suas propriedades antioxidantes usando vários ensaios in vitro. O extrato aquoso continha teores significativos de polifenóis e flavonoides (99,00 µg GAE/mg E e 43,06 µg QE/mg E, respectivamente). Além disso, a análise LC-ESI-MS/MS tornou possível identificar e quantificar 19 compostos fenólicos, com ácido trans-ferúlico como o composto predominante (1,460 mg/g). Além disso, o extrato aquoso exibiu efeitos antioxidantes em todos os ensaios, com valores de IC50 e A0,5 menores que 200 µg/mL, exceto para o poder redutor de fenantrolina e ensaios de SNP.
Palavras-chave: Flora de M’sila. Caracterização Fitoquímica. Eliminadores de Radicais. Polifenóis.
Introduction
In recent years, scientists have become progressively more interested in exploring natural sources of bioactive compounds. Plants have been shown to be the main natural source of these bioactive chemicals, which have an extensive number of properties such as antioxidant (XU et al., 2017), antibacterial (PACYGA et al., 2024), anti-inflammatory (GONFA et al., 2023), anti-allergenic (WU et al., 2023), and antimutagenic (AKRAM et al., 2020) activities that may be effective against a number of noncommunicable diseases, including cancer, heart disease, autoimmune, inflammatory, metabolic, and neurological disorders (DINCHEVA et al., 2023).
A widely known class of bioactive compounds derived from plants secondary metabolism is polyphenols. Chemically, these compounds are identified by hydroxyl groups and phenyl rings (SINGLA et al., 2019). Interest in plant materials abundant in phenolic compounds has grown recently due to their potent antioxidant properties, which may provide protection against long-term illnesses such cancer, neurological diseases, and cardiovascular problems (BERTELLI et al., 2021).
Plant-based polyphenols are highly valuable components of functional foods for human nutrition (BAYIR et al., 2019). They are responsible for the antioxidant effects that enhance the stability of fats and foods and extend the shelf life of specific food products. Additionally, phenolic compounds increase the protective efficacy of antioxidant vitamins and enzymes in reducing oxidative stress induced by an excess of reactive oxygen species. Furthermore, oral polyphenol supplementation strengthens the body’s antioxidant defense mechanism and protects from oxidative stress (BOJARCZUK; DZITKOWSKA-ZABIELSKA, 2022). Moreover, they are important components of signaling pathways in plants that influence development, lignification, and interactions with other soil organisms. Their involvement in interactions between plants and microbes, like those with rhizobium bacteria, as well as their impact on allelopathic processes, water consumption, and seed germination, have all proved their diverse environmental effects (BOJARCZUK; DZITKOWSKA-ZABIELSKA, 2022).
The Apiaceae (formerly Umbelliferae) family is commonly used in many fields, such as the pharmaceutical and flavor industries, due to its abundance of antioxidants, mainly phenolic compounds (THIVIYA et al., 2021). Carrots, or Daucus carota, are aromatic, celery-like plants in the Apiaceae family. Daucus carota L. has been classified into 11 subspecies, including wild subspecies like bocconei, hispanicus, commutatus, carota, major, maximus, gummiferi, maritimus, and fontanesii, and the domesticated varieties D. carota ssp. sativus and D. carota ssp. boissieri (ISMAIL et al., 2023).
Daucus carota L. grows in temperate climates and was originally endemic to Europe and southwest Asia. It is now widely dispersed throughout the Americas, Asia, Europe, and Africa (HOLM et al., 1979). Numerous published studies have focused on cultivated carrot species and demonstrated their antioxidant properties, validating them as a natural source of antioxidants (BOADI et al., 2021). However, the phenolic profile and antioxidant properties of most wild carrot subspecies, including the largest known wild carrot in Algeria, Daucus carota subsp. maximus, have not yet been discovered.
Therefore, as far as we are aware, this research is the first to focus on determining the total polyphenols, and flavonoids content in Algerian Daucus carota subsp. maximus aqueous extract, providing its detailed phenolic profile using the LC-ESI-MS/MS technique, and evaluating its antioxidant properties through various in vitro experiments.
Material and methods
Chemical reagents and spectral measurements
The necessary chemical reagents were bought from Sigma-Aldrich in Taufkirchen, Germany. These included the phenol reagent from Folin-Ciocalteu, Na2CO3, AlCl3, DPPH (2,2-diphenyl-1-picrylhydrazyl), potassium persulfate (K2S2O8), ABTS (2,2′-azinobis (3-ethylbenzoline-6-sulfonic acid) diammonium salt), ammonium acetate (ACNH4), copper chloride dihydrate (CuCl2•2H2O), dipotassium phosphate (K₂HPO₄), monopotassium phosphate (KH2PO4), potassium ferricyanide (K3Fe(CN)6), trichloroacetic acid (TCA) ferric chloride FeCl3, phenanthroline, silver, butylated hydroxyanisole, α-tocopherol, and Trolox. Additionally, Honeywell and Sigma-Aldrich provided the methanol and dimethylsulfoxide (DMSO) solvents that were used. On the other hand, a Multiskan SkyHigh Microplate spectrophotometer from Thermo Scientific was used to measure the absorbance.
Plant material collection and identification
The aerial parts of the Algerian wild Daucus carota subsp. maximus was gathered in June 2021 during its blooming phase from M’sila region, which is located 248 km southeast of Algiers at 35° 42′ 7′′ N and 4° 32′ 50′′ E. Pr. Smaili T. from Faculty of Sciences, Department of Life and Nature Sciences, Mohamed Boudiaf University, M’sila, Algeria, botanically authenticated the plant using the Flora of Algeria (QUEZEL; SANTA, 1963) and the documented research of kazi tani et al. (2021).
Aqueous extract preparation
Before being crushed in a lab mill, the obtained plant aerial parts were cleaned and allowed to air dry away from the sun. Furthermore, the aqueous extract (Aq) was prepared following gnanaprakash et al. (2008) method with some updates. A solution of 50 g of powdered plant material and 500 mL of distilled water was prepared at 70°C for an hour with magnetic stirring. The mixture is then left for two days at room temperature in the dark, with occasional stirring. After filtration using whatman paper (125 mm), the filtrate is subjected to evaporating using a rotating evaporator to remove water, at 50oC temperature and 40 revolutions per minute (RPM). The resulting extract is dried in a laboratory oven and preserved in an amber vial at 4°C.
Quantification of the total phenolic compounds
Total polyphenol content (TPC)
Following Singleton and Rossi’s method (1965), the total polyphenol content in D. carota subsp. maximus Aq extract was determined by quantifying oxidized polyphenols using the Folin-Ciocalteu reagent. More precisely, in a 96-well microplate, 20 µL of Aq extract was added to 100 µL of the 10% Folin-Ciocalteu solution. After 4 minutes of incubation, 80 µL of a 7.5% sodium carbonate (Na2CO3) solution was added. After that, the mixture was incubated for two hours at ambient temperature and away from light. The total polyphenol content in Aq extract was expressed as µg of gallic acid equivalent per milligram of extract (µg GAE/mg E) after the absorbance was measured at 765 nm.
Total flavonoids content (TFC)
The Bahorun et al. (1996) method was used to quantify the total flavonoid content in the D. carota subsp. maximus Aq extract. After adding 100 µL of a 2% AlCl3 solution to 100 µL of extract, the mixture was incubated at room temperature for 15 minutes. The results were expressed as micrograms of quercetin equivalents per milligram of extract (µg QE/ mg E) after the absorbance was measured at 430 nm.
LC-ESI-MS/MS analysis
The LC-ESI-MS/MS technique was used to analyze the phenolic profile of D. carota subsp. maximus Aq extract in accordance with the Erenler et al. (2023) method. An Agilent 1260 Infinity II LC system with a tandem mass spectrometer was used to perform the LC-ESI-MS/MS analysis, and a reversed-phase Agilent Poroshell120 EC-C18 analytical column (100 mm × 3.0 mm, 2.7 μm) was used for the chromatographic separation.
For the sample preparation, a mixture of 1 mL methanol and 1 mL n-hexane was used to dissolve 50 mg of the extract. After that, the resulting solution was centrifuged for 10 minutes at 4°C at 9,000 rpm. Subsequently, the methanol phase was isolated and diluted (1:9) in distilled water. Finally, the resulting sample was filtred with a 0.22 µm filter, and subjected to LC-ESI-MS/MS analysis at a flow rate of 0.5 mL/min and an injection volume of 5.12 µL. The experiment was performed for a total of 30 minutes. The eluent A consisted of formic acid (0.1%) and ammonium formate (5.0 mM) in water, while the eluent B consisted of formic acid (0.1%) and ammonium formate (5.0 mM) in methanol. The adjusted gradient program for the B mobile phase consisted of 25% from 1 to 3 minutes, 50% from 4 to 12 minutes, 90% from 13 to 21 minutes, and 3% from 22 to 25 minutes. In addition, the temperature in the column was 40°C. The capillary voltage was set to 4000 V, the nebulizing gas (N2) flow rate was 11 L/min, the pressure was kept at 15 psi and gas temperature was 300 °C.
Determination of antioxidant properties
The antioxidant properties of D. carota subsp. maximus Aq extract were evaluated using various in vitro assays based on radicals scavenging, and reducing power activity. Butylated hydroxyanisole (BHA), α-tocopherol (TCP) and Trolox (TX), were used as standards for comparison.
DPPH radical scavenging assay
DPPH scavenging activity of D. carota subsp. maximus Aq extract was assessed spectrophotometrically through the method of BLOIS (1958). 160 µL of 60 µM DPPH solution was added to 40 µL of the sample at different concentrations. The absorbance was measured at 517 nm after 30 min of incubation away from light. The findings were presented as 50% inhibitory concentration (IC50 in µg/mL).
ABTS radical scavenging assay
The ABTS scavenging activity was measured according to Re et al. (1999) method. 160 µL of ABTS•+ solution was added to 40 µL of the sample at different concentrations. After 10 min of incubation in the dark, the absorbance was determined at 734 nm and the results were expressed as 50% inhibitory concentration (IC50 in µg/mL).
Cupric reducing capacity assay (CUPRAC)
Cupric ions reducing capacity were determined using the Apak et al. (2004) method. 40 µL of the sample at different concentrations was mixed with 60 µL of ammonium acetate buffer, 50 µL of neocuproine solution, and 50 µL of copper (II) chloride. After an hour of incubation, the absorbance was measured at 450 nm. The results are expressed as A0.5 (in µg/mL), indicating the concentration at which the absorbance is 0.50.
Potassium ferricyanide reducing power assay
The Oyaizu (1986) method was used with some modifications to evaluate the potassium ferricyanide reducing power activity. 10 µL of the sample at various concentrations was mixed with 40 μL of phosphate buffer (pH 6.6) and 50 μL of potassium ferricyanide (1%) K3Fe (CN)6 in a volume of 10 μL. After 20 min incubation period at 50°C, 50 μL of 10% trichloroacetic acid (TCA), 40 μL of distilled water, and 10 μL of 0.1% ferric chloride (FeCl3) were added. The absorbance was measured at 700 nm and the results were presented as A0.5 (in µg/mL), indicating the concentration at which the absorbance is 0.50.
Phenanthroline assay
Phenanthroline activity was determined using Szydlowska-Cserniaka et al. (2008) method. 10 µL of the sample at different concentrations was added to 50 µL of FeCl3 (0.2%), 30 µL of phenanthroline (0.5%), and 110 µL of MeOH. After 20 min of incubation away from light, the absorbance measured at 510 nm. The results were expressed as A0.5 (in µg/mL), in which A0.5 is the concentration where the absorbance is 0.50.
Silver nanoparticles assay (SNP)
The reducing activity of silver ions (Ag⁺) was determined using the method of Özyürek et al. (2012). 50 µL of distilled water and 130 µL of SNP solution (1.0 mM) were added to 20 µL of the sample at different concentrations. The absorbance at 423 nm was measured after 30 minutes of incubation at 25°C.
Statistical analysis
The findings are presented as the mean ± standard deviation of three measurements. The IC50 and A0.50 values were determined using linear regression analysis. The XLSTAT 2016 V1.0 (v18) software was used to carry out the statistical analysis. One-way analysis of variance (ANOVA) was used to assess group differences, with Tukey’s honestly significant difference (HSD) test for comparisons between groups (p-value < 0.001).
Results and discussion
Extraction yield, total polyphenols and flavonoids contents
Fruits and vegetables are a great source of phenolic compounds, which are abundant antioxidants in the human diet and are very beneficial due to their significant antioxidant properties (ZHANG et al., 2022). Furthermore, phenolic compounds, important natural sources of antioxidants, can also be found in high amounts in wild plants.
The extraction yield, total polyphenols (TPC), and total flavonoid content (TFC) were determined for D. carota subsp. maximus Aq extract. The results of TPC and TFC are expressed as micrograms of gallic acid equivalent (GAE) per mg of extract (E) and micrograms of quercetin equivalent (QE) per mg of extract (E), respectively. Furthermore, the extraction method yielded a total extraction rate of 16.37 ± 1.43%, with maximum TPC and TFC contents of 99.00 ± 0.71 µg GAE/mg E and 43.06 ± 0.77 µg QE/mg E, respectively. Our findings are higher than those of Arkoub-Djermoune et al. (2020), who reported the TPC and TFC in Algerian cultivated Daucus carota L. aqueous extracts.
LC-ESI-MS/MS profiling of phenolic compounds in D. carota subsp. maximus aqueous extract
The phenolic composition of D. carota subsp. maximus Aq extract was determined using the LC-ESI-MS/MS technique, which allows for a detailed exploration of its profile, identifying and quantifying the various phenolic compounds present in the extract. Figure 1 displays the phenolic composition of the aqueous extract of D. carota subsp. maximus by LC-ESI-MS/MS analysis.
According to table 1, 19 compounds were identified in the Aq extract based on their retention time, high-resolution mass, and MS fragmentation patterns. Moreover, the identified compounds were also quantified using tandem mass spectrometry (MS/MS). Based on the same table, a total concentration of 4.729 mg/g was estimated for all the identified compounds, with trans-ferulic acid and scutellarin being the most abundant compounds (1.460 and 1.155 mg/g, respectively). Phenolic compounds identified in the Aq extract were as follows: trans-ferulic acid, scutellarin, gallic acid, vanillic acid, syringic acid, sinapic acid, salicylic acid, p-coumaric acid, chlorogenic acid, kaempferol, isoquercitrin, caffeic acid, hydroxybenzaldeyde, quercetin, vanillin, hesperidin, kaempferol-3-glucoside, fisetin, catechin. The major identified phenolic acids in D. carota subsp. maximus Aq extract were detected in cultivated carrots with similar concentrations (HUI et al., 2015).
Figure 1 – Phenolic composition of Daucus carota subsp. maximus aqueous extract.
Trans-ferulic acid, the predominant compound, is an organic compound found in many foods that humans consume. It has numerous pharmacological properties such as anti-inflammatory, antibacterial, antifungal, and antioxidant (REZAEIROSHAN et al., 2021). Chlorogenic acid and vanillin are nutritional phenolic substances, renowned for their anti-inflammatory and antioxidant effects (KUMAR et al., 2022; OLATUNDE, et al., 2023). Hydroxybenzaldehyde, caffeic acid, vanillic acid, p-coumaric acid, salicylic acid, isoquercitrin, kaempferol-3-glucoside, fisetin, kaempferol and quercetin are integral components in many fruits, vegetables, grains, and herbs, contributing to the health promoting properties of these foods. Additionally, they exhibit a multitude of biological properties, such as antioxidant and anti-inflammatory, antimicrobial and anticancer effects (KUMAR et al., 2022; LIM et al., 2008; ALDABA-MURUATO et al., 2021; ROSHEEN et al., 2023; STOMPOR-GORĄCY; MACHACZKA, 2021; MBIKAY; CHRÉTIEN, 2022; TAIWO et al., 2019). Furthermore, catechin is a flavonoid found abundantly in tea leaves, and is renowned for its antioxidant properties. It exhibits antimicrobial, anti-allergenic, and anti-inflammatory effects, alongside potential antiviral and anticancer activities (KUMAR et al., 2022). Also, many previous studies confirmed the anti-inflammatory characteristics of gallic acid, syringic acid, sinapic acid, and scutellarin (LUO et al., 2020; KUMAR et al., 2022; JANG et al., 2023). Besides, Hesperidin is a flavonoid that belongs to the subclass of flavanones, and is found in citrus fruits. It possesses anti-inflammatory, antioxidant, antitumor, and antibacterial properties (WDOWIAK et al., 2022).
Table 1 – Phenolic compounds identified and quantified with LC-ESI-MS/MS in D. carota subsp. maximus Aq extract.
n ̊ |
Compound |
RT (min) |
Concentration (mg/g) |
Ion source |
Ion transitions |
Ion mod |
R2 |
LOQ(ug/mL) |
LOD(ug/mL) |
Linearity range (ug/mL) |
||
1 |
Gallic acid |
3.769 |
0.703 |
ESI |
169.0 ->
|
Neg |
0.9986 |
18.5862 |
7.1674 |
31.25-500 |
||
2 |
Catechin |
6.849 |
0.001 |
ESI |
288.9 ->
|
Neg |
0.9946 |
7.5013 |
1.7055 |
343.750-5500 |
||
3 |
Chlorogenic acid |
7.326 |
0.036 |
ESI |
353.0 ->
|
Neg |
0.9981 |
25.9023 |
11.589 |
31.25-500 |
||
4 |
Hydroxybenzaldeyde |
7.618 |
0.008 |
ESI |
121.0 ->
|
Neg |
0.9993 |
12.8651 |
4.9742 |
15.625-250 |
||
5 |
Vanillic acid |
7.750 |
0.489 |
ESI |
167.0 ->
|
Neg |
0.9958 |
1424.2132 |
219.0421 |
1250-20000 |
||
6 |
Caffeic acid |
7.828 |
0.010 |
ESI |
178.9 ->
|
Neg |
0.9994 |
24,162 |
6.9205 |
31.25-500 |
||
7 |
Syringic acid |
8.375 |
0.475 |
ESI |
197.1 ->
|
Neg |
0.999 |
857.388 |
358.5 |
1250-20000 |
||
8 |
Vanillin |
8.506 |
0.005 |
ESI |
153.0 ->
|
Pos |
0.9949 |
40.5411 |
14.5885 |
62,5-1000 |
||
9 |
Salicylic acid |
9.503 |
0.134 |
ESI |
137.0 ->
|
Neg |
0.9981 |
82.9646 |
47.6695 |
112.5-1800 |
||
10 |
p-coumaric acid |
9.581 |
0.048 |
ESI |
163.0 ->
|
Neg |
0.9987 |
17.5416 |
3.5348 |
31.25-500 |
||
11 |
trans-ferulic acid |
10.088 |
1.460 |
ESI |
193.1 ->
|
Neg |
0.995 |
11.5276 |
6.1184 |
31.25-1000 |
||
12 |
Sinapic acid |
10.353 |
0.155 |
ESI |
223.1 ->
|
Neg |
0.9972 |
4.9652 |
1.9437 |
125-2000 |
||
13 |
Scutellarin |
11.179 |
1.155 |
ESI |
462.8 ->
|
Pos |
0.9978 |
4.0013 |
3.1346 |
9.375-300 |
||
14 |
Isoquercitrin |
11.461 |
0.013 |
ESI |
464.9 ->
|
Pos |
0.9982 |
11.268 |
9.9382 |
18.75-300 |
||
15 |
Hesperidin |
11.695 |
0.004 |
ESI |
611.0 ->
|
Pos |
0.9957 |
17.6753 |
4.1396 |
31.25-500 |
||
16 |
Kaempferol-3-glucoside |
12.827 |
0.003 |
ESI |
448.8 ->
|
Pos |
0.9997 |
4.5238 |
1.1609 |
7.8125-125 |
||
17 |
Fisetin |
13.420 |
0.002 |
ESI |
287.0 ->
|
Pos |
0.9954 |
44.3662 |
10.8961 |
15.625-250 |
||
18 |
Quercetin |
14.799 |
0.006 |
ESI |
300.8 ->
|
Neg |
0.9964 |
16.9127 |
4.6558 |
27.5-440 |
||
19 |
Kaempferol |
16.546 |
0.024 |
ESI |
284.9 ->
|
Neg |
0.9997 |
5.4004 |
1.8683 |
312.5-10000 |
||
Total quantification (mg/g) |
4.729 |
|||||||||||
RT: retention time, LOQ: Limit of quantitation, LOD: limit of detection, Neg: negative, Pos: positive.
In vitro antioxidant activity
The antioxidant properties of D. carota subsp. maximus Aq extract were determined through radical scavenging and reducing power in vitro assays, which serve as significant indicators of its antioxidant potential. DPPH and ABTS assays were used to evaluate free radical scavenging potential, while CUPRAC, Phenanthroline, Potassium ferricyanide reducing power, and SNP assays were used to determine reducing power properties. A sample is considered more effective when its IC50 and A0.5 values are lower.
In all antioxidant assays, the Aq extract consistently exhibited antioxidant potential, but it was significantly lower than the standards used in each assay. The detailed results are presented in table 2, showing IC50 and A0.5 values for both the Aq extract and the standards in all assays. The Aq extract exhibited the highest radical scavenging activity in the ABTS assay with the lowest IC50 value (ABTS: 65.17±0.22, DPPH: 92.97±0.44 µg/mL). Furthermore, the capacity of the Aq extract in reducing potassium ferricyanide (Fe³⁺) to potassium ferrocyanide (Fe²⁺) in the potassium ferricyanide RP assay was higher than its capacity in reducing Cu²⁺ ions to Cu⁺ ions in the CUPRAC assay, with the lowest A0.5 in the potassium ferricyanide RP assay (Potassium ferricyanide RP: 128.11±0.69, CUPRAC: 135.76±0.76 µg/mL). Only in the Phenanthroline and SNP assays did the extract show A0.5 values higher than 200 µg/mL. Our results present higher DPPH scavenging activity compared to methanolic and hexanic extracts from aerial parts of cultivated carrot (AYENI et al., 2018).
Table 2 – In vitro antioxidant activity of D. carota subsp. maximus Aq extract.
DPPH |
ABTS |
CUPRAC |
Phenanthroline |
Potassium ferricyanide RP |
SNP |
|
(IC50 µg/mL) |
(IC50 µg/mL) |
(A0.5 µg/mL) |
(A0.5 µg/mL) |
(A0.5 µg/mL) |
(A0.5 µg/mL) |
|
Aq |
92.97±0.44(c) |
65.17±0.22(b) |
135.76±0.76 (b) |
> 200 |
128.11±0.69(c) |
> 200 |
BHA |
9.69 ±0,18(a) |
8.01 ±0.11(a) |
NT |
1.84±0.12(a) |
1.68±0.37(a) |
NT |
TCP |
6.75 ±0,06(b) |
NT |
17.00 ±0.37 (a) |
7.69±1.39(b) |
3.46±0.06(b) |
NT |
TX |
NT |
NT |
NT |
NT |
NT |
34.17±1.23 |
Means ± SD (n = 3) were used to express the findings. Tukey’s honestly significant difference (HSD) test was used to determine the significance between groups; different letters indicating significant differences at P<0.001. Aq: Aqueous extract, BHA: Butylhydroxyanisole, TCP: α-Tocopherol, Tx: Trolox, NT: not tested.
The scavenging and reducing activity exhibited by the Aq extract may be caused by the phenolic compounds present in the Aq extract, which are identified in the current study using LC-ESI-MS/MS. The phenolic compounds were already mentioned in the previous literature for their antioxidant potential (BERTELLI et al., 2021). The antioxidant activity of phenolic substances is primarily attributed to three major mechanisms: single electron transfer, metal chelating approaches, and reactive oxygen species scavenging by hydrogen atom transfer (BHUYAN; HANDIQUE, 2022).
Indeed, phenolic compounds antioxidant properties are primarily attributed to their specific functional groups and structural features. Among these are aromatic rings and hydroxyl groups, which enable them to give hydrogen atoms and electrons to neutralize free radicals and reduce oxidative damage (TSAO, 2010). The presence of phenoxy radicals and carboxylic and methoxylic acid moieties make trans-ferulic acid has the ability to scavenge radicals and produce a stable structures. (REZAEIROSHAN et al., 2021). Furthermore, a previous study demonstrated the antioxidant properties of scutellarin, including radical scavenging due to its multiple hydroxyl groups which enhances hydrogen donation (SPIEGEL et al., 2022). Other previous studies demonstrated that all other identified compounds in the Aq extract characterized by several functional groups, including hydroxyl groups, methoxy groups, and carboxylic acid groups contribute to their antioxidant properties (DI MAJO et al., 2011; CAROCHO; FERREIRA, 2013; NARÓG; SOBKOWIAK, 2023).
Conclusion
Wild plants have rich biodiversity and unique phytochemical profiles, which potentiates the discovery of novel sources of bioactive compounds in those that have not been researched yet. This paper determined the total polyphenols and flavonoids content, the phenolic profile, and in vitro antioxidant properties of the aqueous extract from one wild plant: Daucus carota subsp. maximus. The study demonstrates that the aqueous extracts from the Algerian wild carrot D. carota subsp. maximus aerial parts contains a substantial amount of antioxidant compounds as identified and quantified using LC-ESI-MS/MS, which can contribute to the antioxidant effect of the extract. Based on the findings, the current study suggest that wild plants can be a valuable source of antioxidants like cultivated plants. Furthermore, the aerial parts of wild D. carota subsp. maximus can provide a natural source of trans-ferulic acid isolated in high quantities. The latter is highly beneficial in various applications, especially those related to food products, supplements, and pharmaceuticals. Moreover, researching other biological properties will enhance our understanding of this plant’s potential.
Conflicts of interest
There is no conflict of interest of the authors.
Authors’ contribution
Maroua Hadji – concepts, original idea, experimental studies and original writing; Tahar Smaili – supervision and manuscript review; Khadidja Dehimi – guidelines and manuscript review; Fethi Farouk Kebaili – guidelines, manuscript editing and review; Toka Hadji – literature search and statistical analysis; Amal Lahouaou – experimental studies; Yildiz Ilyas – data analysis; Erenler Ramazan – data analysis.
Financial support
There is no funding associated with this work.
Acknowledgements
The authors are grateful to HADJI Ikhlass (University of Biskra, Department of English Language and Literature) for proofreading and language editing, and to Pr. BENDIF Hamdi (University of M’sila, Department of Natural Life and Sciences) for the support.
References
AKRAM, M.; RIAZ, M.; WADOOD, A. W. C.; HAZRAT, A.; MUKHTIAR, M.; ZAKKI, S. A.; DANIYAL, M.; SHARIATI, M. A.; KHAN, F. S.; ZAINAB, R. Medicinal plants with anti-mutagenic potential. Biotechnology & Biotechnological Equipment, v. 34, n. 1, p. 309-318, 2020. https://doi.org/10.1080/13102818.2020.1749527
ALDABA‑MURUATO, L.; VENTURA‑JUÁREZ, J.; PEREZ‑HERNANDEZ, A.; HERNÁNDEZ‑MORALES, A.; MUÑOZ‑ORTEGA, M.; MARTÍNEZ‑HERNÁNDEZ, S.; ALVARADO‑SÁNCHEZ, B.; MACÍAS‑PÉREZ, J. Therapeutic perspectives of p‑coumaric acid: Anti‑necrotic, anti‑cholestatic and anti‑amoebic activities. World Academy of Sciences Journal, v. 3, n. 5, p. 1-8, 2021. https://doi.org/10.3892/wasj.2021.118
APAK, R.; GUCLU, K.; OZYUREK, M.; KARADEMIR, S. E. Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC Method. Journal of Agricultural and Food Chemistry, v. 52, n. 26, p. 7970-7981, 2004. https://doi.org/10.1021/jf048741x
ARKOUB-DJERMOUNE, L.; LOUAILECHE, H.; BENMEZIANE, F.; MADANI, K.; BOULEKBACHE-MAKHLOUF, L. Impact of refrigerated storage on the bioactive compounds and antioxidant capacity of two Algerian carrot varieties (Daucus carota L.). Acta Universitatis Sapientiae Alimentaria, v. 13, n. 1, p. 5-31, 2020. https://doi.org/10.2478/ausal-2020-0001
AYENI, E. A.; ABUBAKAR, A.; IBRAHIM, G.; ATINGA, V.; MUHAMMAD, Z. Phytochemical, nutraceutical and antioxidant studies of the aerial parts of Daucus carota L. (Apiaceae). Journal of Herbmed Pharmacololgy, v. 7, n. 2, p. 68-73, 2018. https://doi.org/10.15171/jhp.2018.12
BAHORUN, T.; GRESSIER, B.; TROTIN, F.; BRUNET, C.; DINE, T.; LUYCKX, M.; VASSEUR, J.; CAZIN, M.; CAZIN, J. C.; PINKAS, M. Oxygen species scavenging activity of phenolic extracts from hawthorn fresh plant organs and pharmaceutical preparations. Arzneimittelforschung, v. 46, n. 11, p. 1086-1089, 1996. https://pubmed.ncbi.nlm.nih.gov/8955870
BAYIR, A. G.; AKSOY, A. N.; KOÇYİĞİT, A. The importance of polyphenols as functional food in health. Bezmialem Science, v. 7, n. 2, p. 157-163, 2019. https://doi.org/10.14235/bas.galenos.2018.2486
BERTELLI, A.; BIAGI, M.; CORSINI, M.; BAINI, G.; CAPPELLUCCI, G.; MIRALDI, E. Polyphenols: from theory to practice. Foods, v. 10, n. 11, p, 1-15, 2021. https://doi.org/10.3390/foods10112595
BHUYAN, U.; HANDIQUE, J. G. Plant polyphenols as potent antioxidants: highlighting the mechanism of antioxidant activity and synthesis/development of some polyphenol conjugates. Studies in Natural Products Chemistry, p. 243-266, 2022. https://doi.org/10.1016/b978-0-323-91250-1.00006-9
BLOIS, M. S. Antioxidant determinations by the use of a stable free radical. Nature, v. 181, n. 4617, p. 1199-1200, 1958. https://doi.org/10.1038/1811199a0
BOADI, N. O.; BADU, M.; KORTEI, N. K.; SAAH, S. A.; ANNOR, B.; MENSAH, M. B.; OKYERE, H.; FIEBOR, A. Nutritional composition and antioxidant properties of three varieties of carrot (Daucus carota). Scientific African, v. 12, 2021. https://doi.org/10.1016/j.sciaf.2021.e00801
BOJARCZUK, A.; DZITKOWSKA-ZABIELSKA, M. Polyphenol supplementation and antioxidant status in athletes: a narrative review. Nutrients, v. 15, n. 1, p. 1- 34, 2022. https://doi.org/10.3390/nu15010158
CAROCHO, M.; FERREIRA, I. C. A review on antioxidants, prooxidants and related controversy: natural and synthetic compounds, screening and analysis methodologies and future perspectives. Food and Chemical Toxicology, v. 51, n. 1, p. 15-25, 2013. https://doi.org/10.1016/j.fct.2012.09.021
DI MAJO, D.; LA NEVE, L.; LA GUARDIA, M.; CASUCCIO, A.; GIAMMANCO, M. The influence of two different pH levels on the antioxidant properties of flavonols, flavan-3-ols, phenolic acids and aldehyde compounds analysed in synthetic wine and in a phosphate buffer. Journal of Food Composition and Analysis, v. 24, n. 2, p. 265-269, 2011. https://doi.org/10.1016/j.jfca.2010.09.013
DINCHEVA, I.; BADJAKOV, I.; GALUNSKA, B. New insights into the research of bioactive compounds from plant origins with nutraceutical and pharmaceutical potential. Plants, v. 12, n. 2, p. 1-4, 2023. https://doi.org/10.3390/plants12020258
ERENLER, R.; ATALAR, M. N.; YILDIZ, I.; GECER, E.; YILDIRIM, A.; DEMIRTAS, I.; ALMA, M. Quantitative analysis of bioactive compounds by LC-MS/MS from Inula graveolens. Bütünleyici Ve Anadolu Tıbbı Dergisi, v. 4, n. 3, p. 3-10, 2023. https://doi.org/10.53445/batd.1278048
GNANAPRAKASH, K.; MADHUSUDHANACHETTY, C.; RAMKANTH, S.; ALAGUSUNDARAM, M.; VS, T.; ANGALAPARAMESWARI, S.; TS, M. S. Aqueous extract of Flacourtia indica prevents carbon tetrachloride induced hepatotoxicity in rat. International Journal of Medical, Health, Biomedical, Bioengineering and Pharmaceutical Engineering, v. 4, p. 46-50, 2010.
GONFA, Y. H.; TESSEMA, F. B.; BACHHETI, A.; RAI, N.; TADESSE, M. G.; SINGAB, A. N.; CHAUBEY, K. K.; BACHHETI, R. K. Anti-inflammatory activity of phytochemicals from medicinal plants and their nanoparticles: a review. Current Research in Biotechnology, v. 6, n. 6, p. 1-16, 2023. https://doi.org/10.1016/j.crbiot.2023.100152
HOLM, L.; PANCHO, J. V.; HERBERGER, J. P.; PLUCKNETT, D. L. A Geographical Atlas of World Weeds. John Wiley and Sons; Hoboken, NJ, USA, 1979, 391p.
HUI, Y. H., GHAZALA, S.; GRAHAM, D. M.; MURRELL, K. D.; NIP, W.-K. Handbook of Vegetable Preservation and Processing. 1st Edition. Boca Raton: CRV Press, 2003, 752p. https://doi.org/10.1201/9780203912911
ISMAIL, J.; SHEBABY, W. N.; DAHER, J.; BOULOS, J. C.; TALEB, R.; DAHER, C. F.; MROUEH, M. The wild Carrot (Daucus carota): a phytochemical and pharmacological review. Plants, v. 13, n. 1, 2023. https://doi.org/10.3390/plants13010093
JANG, S.; KIM, S.; SO, B. R.; KIM, Y.; KIM, C.; LEE, J. J.; JUNG, S. K. Sinapic acid alleviates inflammatory bowel disease (IBD) through localization of tight junction proteins by direct binding to TAK1 and improves intestinal microbiota. Frontiers in Pharmacology, v. 14, 2023. https://doi.org/10.3389/fphar.2023.1217111
KAZI TANI, C.; GRARD, P.; LE BOURGEOIS, T. AdvenAlg 1.0 Identification et connaissance des principales adventices d’Algérie méditerranéenne. Al Yasmina, Revue de Botanique. v. 2, n. 3, 2021, 184p. https://hal.inrae.fr/hal-03259469
KUMAR, A.; KHAN, F.; SAIKIA, D. Phenolic compounds and their biological and pharmaceutical activities. The Chemistry inside Spices & Herbs: Research and Development, p. 206-236, 2022. https://doi.org/10.2174/9789815039566122010010
LIM, E.; KANG, H.; JUNG, H.; KIM, K.; LIM, C.; PARK, E. Anti-inflammatory, anti-angiogenic and anti-nociceptive activities of 4-hydroxybenzaldehyde. Biomolecules & Therapeutics, v. 16, n. 3, p. 231-236, 2008. https://doi.org/10.4062/biomolther.2008.16.3.231
LUO, Z.; HU, Z.; BIAN, Y.; SU, W.; LI, X.; LI, S.; WU, J.; SHI, L.; SONG, Y.; ZHENG, G.; NI, W.; XUE, J. Scutellarin attenuates the IL-1Β-Induced inflammation in mouse chondrocytes and prevents osteoarthritic progression. Frontiers in Pharmacology, v. 11, 2020. https://doi.org/10.3389/fphar.2020.00107
MBIKAY, M.; CHRÉTIEN, M. Isoquercetin as an anti-covid-19 medication: a potential to realize. Frontiers in Pharmacology, v. 13, 2022. https://doi.org/10.3389/fphar.2022.830205
NARÓG, D., SOBKOWIAK, A. Electrochemistry of flavonoids. Molecules/Molecules Online/Molecules Annual, v. 28, n. 22, p. 1-27, 2023. https://doi.org/10.3390/molecules28227618
OLATUNDE, A.; MOHAMMED, A.; IBRAHIM, M.; TAJUDDEEN, N.; SHUAIBU, M. Vanillin: a food additive with multiple biological activities. European Journal of Medicinal Chemistry Reports, v. 5, p. 1-16, 2023. https://doi.org/10.1016/j.ejmcr.2022.100055
OYAIZU, M. Studies on products of browning reactions: antioxidative activities of browning reaction prepared from glucosamine. Japanese Journal of Nutrition, v. 44, n. 6, p. 307-315, 1986. https://doi.org/10.5264/eiyogakuzashi.44.307
ÖZYÜREK, M.; GÜNGÖR, N.; BAKI, S. Development of a silver nanoparticle-based method for the antioxidant capacity measurement of polyphenols, Analytical Chemistry, v. 84, n. 18, p. 8052-8059, 2012. https://doi.org/10.1021/ac301925bac301925b
PACYGA, K.; PACYGA, P.; TOPOLA, E.; VISCARDI, S.; DUDA-MADEJ, A. Bioactive compounds from plant origin as natural antimicrobial agents for the treatment of wound infections. International Journal of Molecular Sciences, v. 25, n. 4, p. 1-44, 2024. https://doi.org/10.3390/ijms25042100
QUEZEL, P.; SANTA, S. Nouvelle flore de l’Algérie et des régions désertiques méridionales. Tome II. Paris, France: National Centre for Scientific Research, v. 2, 1963.
RE, R.; PELLEGRINI, N.; PROTEGGENTE, A.; PANNALA, A.; YANG, M.; RICE-EVANS, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, v. 26, n. 9-10, p. 1231-1237, 1999. https://doi.org/10.1016/s0891-5849(98)00315-3
REZAEIROSHAN, A.; SAEEDI, M.; MORTEZA-SEMNANI, K.; AKBARI, J.; HEDAYATIZADEH-OMRAN, A.; GOLI, H.; NOKHODCHI, A. Vesicular formation of trans-ferulic acid: an efficient approach to improve the radical scavenging and antimicrobial properties. Journal of Pharmaceutical Innovation, v. 17, n. 3, p. 652-661, 2021. https://doi.org/10.1007/s12247-021-09543-8
ROSHEEN, N.; SHARMA, S.; UTREJA, D. Salicylic acid: synthetic strategies and their biological activities. ChemistrySelect, v. 8, n. 13, p. 1-15, 2023. https://doi.org/10.1002/slct.202204614
SINGLA, R. K.; DUBEY, A. K.; GARG, A.; SHARMA, R. K.; FIORINO, M.; AMEEN, S. M.; HADDAD, M. A.; AL-HIARY, M. Natural polyphenols: chemical classification, definition of classes, subcategories, and structures. Journal of AOAC International, v. 102, n. 5, p. 1397-1400, 2019. https://doi.org/10.5740/jaoacint.19-0133
SINGLETON, V.; ROSSI, J. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture, v. 16, p. 144-158, 1965. https://doi.org/10.5344/ajev.1965.16.3.144
SPIEGEL, M.; MARINO, T.; PREJANÒ, M.; RUSSO, N. Primary and secondary antioxidant properties of scutellarin and scutellarein in water and lipid-like environments: a theoretical investigation. Journal of Molecular Liquids, v. 366, p. 1-9, 2022. https://doi.org/10.1016/j.molliq.2022.120343
STOMPOR-GORĄCY, M.; MACHACZKA, M. Recent advances in biological activity, new formulations and prodrugs of ferulic acid. International Journal of Molecular Sciences, v. 22, n. 23, p. 1-16, 2021. https://doi.org/10.3390/ijms222312889
SZYDLOWSKA-CZERNIAKA, A.; DIANOCZKI, C.; RECSEG, K.; KARLOVITS, G.; SZLYK, E. Determination of antioxidant capacities of vegetable oils by ferric-ion spectrophotometric methods. Talanta, v. 76, n. 4, p. 899-905, 2008. https://doi.org/10.1016/j.talanta.2008.04.055
TAIWO, F. O.; OYEDEJI, O.; OSUNDAHUNSI, M. T. Antimicrobial and antioxidant properties of kaempferol-3-O-glucoside and 1-(4-Hydroxyphenyl)-3-phenylpropan-1-one isolated from the leaves of Annona muricata (Linn.). Journal of Pharmaceutical Research International, v. 26, n. 3, p. 1-13, 2019. https://doi.org/10.9734/jpri/2019/v26i330138
THIVIYA, P.; GAMAGE, A.; PIUMALI, D.; MERAH, O.; MADHUJITH, T. Apiaceae as an important source of antioxidants and their applications. Cosmetics, v. 8, n. 4, p. 1-19, 2021. https://doi.org/10.3390/cosmetics8040111
TSAO, R. Chemistry and biochemistry of dietary polyphenols. Nutrients, v. 2, n. 12, p. 1231-1246, 2010. https://doi.org/10.3390/nu2121231
WDOWIAK, K.; WALKOWIAK, J.; PIETRZAK, R.; BAZAN-WOŹNIAK, A.; CIELECKA-PIONTEK, J. Bioavailability of hesperidin and its aglycone hesperetin-compounds found in citrus fruits as a parameter conditioning the pro-health potential (Neuroprotective and antidiabetic Activity) – mini-review. Nutrients, v. 14, n. 13, p. 1-23, 2022. https://doi.org/10.3390/nu14132647
WU, T.; LI, Z.; WU, Y.; YANG, X.; LI, L.; CHEN, S.; QI, B.; WANG, Y.; LI, C.; ZHAO, Y. Exploring plant polyphenols as anti-allergic functional products to manage the growing incidence of food allergy. Frontiers in Nutrition, v. 10, p. 1-9, 2023. https://doi.org/10.3389/fnut.2023.1102225
XU, D. P.; LI, Y.; MENG, X.; ZHOU, T.; ZHOU, Y.; ZHENG, J.; ZHANG, J. J.; LI, H. B. Natural antioxidants in foods and medicinal plants: extraction, assessment and resources. International Journal of Molecular Sciences, v. 18, n. 1, 2017. https://doi.org/10.3390/ijms18010096
ZHANG, H.; PU, J.; TANG, Y.; WANG, M.; TIAN, K.; WANG, Y.; LUO, X.; DENG, Q. Changes in Phenolic compounds and antioxidant activity during development of ‘Qiangcuili’ and ‘Cuihongli’ fruit. Foods, v. 11, n. 20, p. 1-17, 2022. https://doi.org/10.3390/foods11203198
Received on February 29, 2024
Returned for adjustments on May 8, 2024
Received with adjustments on May 11, 2024
Accepted on May 12, 2024
The post Exploring and evaluating the phenolic profile and antioxidant properties of the Algerian wild carrot Daucus carota subsp. maximus aqueous extract first appeared on Revista Agrária Acadêmica.