Phytochemical Proﬁling, Mineral Elements, and Biological Activities of Artemisia campestris L. Grown in Algeria

: Artemisia campestris L. is commonly used in folk medicine due to its antioxidant, antidiabetic, nutritional, and culinary properties. Our study assessed the total phenolics contents, antioxidant, and pharmacological activities of various organic extracts prepared from the aerial parts of Artemisia campestris , and its mineral elements and chemical proﬁle were analyzed. ICP-OES was used to analyze the mineral proﬁle and the LC-MS/MS analysis was used to characterize the phytochemical proﬁling. A series of antioxidant tests were carried out using DPPH, ABTS, beta-carotene, GOR, RP, CUPRAC, and O-Phenanthroline assays. In vitro potent inhibitory actions of A. campestris extracts were investigated to evaluate their anti-cholinesterase, anti-lipase and anti-diabetic activities. The photoprotective effect of the plant was measured by the sun protection factor. The most powerful inhibitor of α -amylase was AcPEE (IC 50 = 11.79 ± 0.14 µ g/mL), which also showed a signiﬁcant butyrylcholinesterase inhibitory effect (IC 50 = 93.50 ± 1.60 µ g/mL). At IC 50 = 23.16 ± 0.19 µ g/mL, AcEAE showed the most powerful inhibitory effects on acetylcholinesterase. A. campestris was found to have a strong photoprotective ability, absorbing UV radiations with SPF values ranging from 26.07 ± 0.22 to 40.76 ± 0.11. The results showed that A. campestris extract has strong antioxidant activity in all the test samples except for the carotene bleaching assay. The LC/MS-MS results showed that AcDE, AcEAE, and AcBE identiﬁed 11 compounds belonging to Polyphenols Compounds. Our result also showed that A. campestris contains a high concentration of essential minerals, including macro-and micro-elements with their values close to the FAO’s recommended concentration. A. campestris has the capacity to improve pharmaceutical formulations, health, and medical research, due to its compositions and potent biological properties


Introduction
Medicinal plants have long been regarded for their medicinal properties and nutritional importance, both of which help in treating various diseases and for drug development, according to a survey conducted by the World Health Organization [1]. In light of the antioxidant properties that they have, natural chemicals that plants produce have the

Extraction of Secondary Metabolites
The plant materials were macerated in a hydro-methanolic solution (80:20) (methanol/ Water) (v/v) at room temperature for 24 h with steady stirring. The process of extraction was repeated three times with the solvent being reconditioned. The resulting suspension was filtered through Whatman filter paper and condensed in a vacuum evaporator. The filtrate was evaporated at 40 • C using a rotary evaporator (BUCHI, R-100, flail, Switzerland). The residue was dissolved in water and extracted with a series of solvents with increasing polarity. The organic fractions were concentrated to obtain the following dry extracts; methanol (AcME: 0.23%), petroleum ether (AcPEE: 0.05%), dichloromethane (AcDE: 0.35%), ethyl acetate (AcEAE: 0.79%), butanol (AcBE: 1.45%), and aqueous (AcAE: 7.95%).

Inductively Coupled Plasma ICP-OES
The analysis of the mineral contents was performed using ICP-OES (Inductively coupled plasma-optical emission spectrometry) [20][21][22][23]. A 500 mg powdered sample of A. campestris was placed in a burning cup. The powder was placed in a flask (200 mL capacity) and mixed with 3 mL of HNO 3 , which has a concentration of 65% (Sigma Aldrich-Germany), along with 3 mL of hydrochloric acid at a 37% concentration (Sigma Aldrich-Germany). Subsequent agitation was performed for 90 min followed by heating the mixture to 105 • C to ensure that all of the powder was transformed into liquid. The sample was then diluted with 50 mL distilled water and transferred to a new tube, where it was allowed to settle and homogenize. Finally, the supernatant was subjected to an ICP-OES analysis to determine the minerals present in the sample; each sample was examined in triplicate.

Liquid Chromatography-Mass Spectrometry (LC-MS-MS)
A qualitative analysis of constituents present in different fractions of A. campestris was performed using UPLC-ESI-MS-MS Shimadzu 8040 Ultra-High sensitivity with UFMS technology equipped with a binary bump Nexera XR LC-20AD. Separation was achieved with an Ultra-force C18 column (I.D. 2.5 mm × 100 mm, 1.8 µm particle size; Restek) at 25 • C oven temperature. The chromatographic separation was carried out using a mixture of 30% (water, 0.1% formic acid) as mobile phase A and 70% methanol as mobile phase B. The duration of each gradient elution was as follows: B; 0.10 min 5% B; 10 min 15% B; 30 min 95% B; 45 min 15% B; 50 min 5% B; 60 min 100%. The flow rate was established at 0.3 mL/min, while the injection volume was 6 µL, passed through a Millex-LCR (PTFE) filter with 0.22 µm pore sizes. The separation was performed at room temperature, while the run lasted for 60 min.
The ESI conditions employed in the MS/MS are as follows: 230 KPs CID gas; −6.00 Kv conversion dynode; 350 • C interface temperature; 250 • C DL temperature; 3.00 L/min nebulizing gas flow, 400 • C heat block; and 15.00 L/min drying gas flow. The ion trap mass spectrometer was used in both negative and positive ions in the MRM mode (multiple reaction monitoring). Accurate identification was made according to their typical fragments by comparing the mass spectra with the published literature.

Assessment of Total Phenolics, Flavonoids Compounds 2.5.1. Total Phenolics Content
Spectrophotometrically, the phenolic content of the extracts was determined in a microplate using the Folin-Ciocalteu method with some modifications [24]. First, 100 µL of Folin-Ciocalteu reagent (1:10) was mixed with 20 µL of each extract or varying concentrations of the gallic acid (standard); 75 µL of sodium carbonate (7.5%) was then added to the mixture. The mixture was kept at ambient temperature for 2 h, and kept in darkness while it was incubated. A 96-well microplate reader (Perkin Elmer EnSpire, Singapore) was used to record the reading of the absorbance at 765 nm. The total phenol levels were defined as µg GAE/mg extract, using a calibration graph of gallic acid.

Total Flavonoids Content
The flavonoid content of the extracts was determined using a slight modification method of the microplate assay reported by Topcu et al. [25]. The method depends on forming a complex between Al 3+ and the flavonoids. A total of 50 µL of the test sample was placed in a 96-well microplate containing 130 µL (MeOH). This was followed by the addition of 10 µL of 1 M potassium acetate and 10 µL of 10% aluminium nitrate. The mixture was incubated at room temperature for 40 min. The absorbance of the sample was measured at 415 nm using quercetin as a positive control. The data were reported in µg quercetin equivalent per mg of extract (µg QE/mg) depending on the quercetin calibration curve.
2.6. Antioxidant Activity 2.6.1. DPPH Scavenging Activity The ability of the different extracts of A. campestris to inhibit the DPPH free radical using the 2,2-diphenyl-1-picrylhydrazyl free radical was investigated using the procedure described by Blois [26]. A DPPH solution (0.1 M) newly prepared in methanol was used in combination with 40 µL of plant extract at various concentrations for the reaction. After incubation of the mixture in the dark for 30 min, the absorbances were measured at 517 nm using a microplate reader (Perkin Elmer EnSpire, Singapore). The synthetic antioxidants, BHA and BHT, were used as standard. Percentage (%) DPPH free radical scavenging activity was calculated using the Formula (1): % Inhibition = (Abs 517 blank − Abs 517 sample) Abs 517 blank × 100 (1) Abs 517 blank: absorbance of control reaction. Abs 517 sample: absorbance of test sample. The percentage inhibition curve at various concentrations was used in order to calculate the IC 50 value, which corresponds to the extract concentration and is expressed as (µg/mL).

ABTS Scavenging Activity
According to the procedure described by Re et al. [27], the extracts of A. campestris were conducted in order to determine their ability to scavenge free radicals in the presence of ABTS (2,2 -azinobis (3-ethylbenzothiazoline)-6-sulfonic. After reacting a 7 mM aqueous solution of ABTS with 2.45 mM of potassium persulfate (K 2 S 2 O 8 ) for 16 h, while storing the mixture in the dark at room temperature, the cation ABTS + was generated. After dilution, the ABTS solution had an absorbance of 0.700 ± 0.020. In a 96-well microplate reader (PerkinElmer Multi-mode Plate Reader EnSpire, USA), 40 µL of each sample prepared in methanol at different concentrations was mixed with 160 µL of the ABTS mixture and stored in the dark for 10 min. The absorbance was measured at a wavelength of 734 nm. The antioxidant standards were BHA and BHT, respectively. Formula (1) was used to calculate the inhibition percentage.

β-Carotene-Linoleic Acid Bleaching Activity
The capacity of A. campestris extracts to inhibit β-carotene by the model system (βcarotene/linoleic acid) was determined using the method described by Marco [28]. The method consists of preparing β-carotene/linoleic acid emulsion by dissolving 0.5 mg of beta-carotene in 1 mL of chloroform. This mixture was transferred to a flask containing 20 µL linoleic acid and 200 µL Tween 40. The chloroform was removed using a vacuum evaporator, and 50 µL of hydrogen peroxide was added, followed by vigorously agitation. At 470 nm, the absorbance of the mixture was adjusted to between 0.8 and 0.9. 160 µL; the emulsion was mixed with 40 µL of the plant extracts or synthetic antioxidants (BHA and BHA) at various concentrations. Then, the mixture places in each well of the 96well microplate. After incubating the microplate at 50 • C, the absorbance was measured at a wavelength of 470 nm at different times ranging from t = 0 min to t = 120 min, with each having a 30 min interval. The following equation was used to express the percentage inhibition: I (%): percentage of inhibition AH 0 : Absorbance of β-carotene in extract at t = 0. AC 0 : Absorbance of β-carotene in negative control at t = 0. AH t : Absorbance of β-carotene in extract at 120 min. AC t : Absorbance of β-carotene in negative control at 120 min.

Galvinoxyl (GOR) Scavenging Activity
The Galvinoxyl free radical (GOR) antioxidant test was performed using the Shi et al. method [29]. For this experiment, 40 µL of different extract concentrations in methanol was mixed with 160 µL of Galvinoxyl methanolic solution at 0.1 mM. Using a spectrophotometer at 428 nm, the absorbance of the resulting solution was measured after 120 min of incubation at room temperature in darkness. The standards were BHT and BHA. Galvinoxyl's methanolic solution was employed as a control for this test. The inhibition percentage I (%) was expressed as follows:

. Reducing Power Activity
The capacity of the samples to reduce Fe 3+ contained in the complex K 3 Fe(CN) 6 to Fe 2+ was assessed by reducing iron ions. The reducing power was evaluated using the approach outlined by Oyaizu [30]. In brief, 10 µL of each A. campestris extract or standard at various concentrations was mixed with 40 µL of phosphate buffer solution (0.2 M, pH 6.6) and 50 µL 1% potassium ferricyanide. Afterwards, the mixture was incubated for 20 min at 50 • C. Then, 50 µL of 10% trichloroacetic acid, 40 µL of distilled water, and 10 µL of a 0.1% ferric chloride solution were added. A microplate reader was used to record the 700 nm absorbance reading (Perkin Elmer, EnSpire, Singapore). BHA and ascorbic acid were used as reference points in the assay.

Cupric Reducing Antioxidant Capacity (CUPRAC) Activity
In the CUPRAC procedure, the reduction in copper ions Cu 2+ is the basis for measurement [31]. Various concentrations of the sample (40 µL) were added to 50 µL each of 10 Mm CuCl 2 and 7.5 mM neocuprine; 60 µL CH 3 COONH 4 buffer (1 M, pH = 7). The resulting mixture was incubated at room temperature for 1 h under a dark environment. The absorbance was measured at 450 nm using the microplate reader (Perkin Elmer Enspire, Singapore). BHA and BHT were used as the antioxidant standards. The concentration, given an absorbance of 0.5 (A 0 . 5 ), was calculated from the absorbance curve at different concentrations.

O-Phenanthroline Chelating Activity
The O-phenanthroline chelating activities of the extracts were determined using the method of Szydłowska-Czerniak et al. [32]. We added 50 µL FeCl 3 (0.2%), 30 µL O-Phenanthroline methanol solution (0.5%), and 110 M methanol each to 10 µL of various concentrations of the extract and incubated them at room temperature for 20 min before the analysis. An orange-red solution absorbance was detected at 510 nm using a 96-well microplate reader. BHT and BHA were used as the standards.

Cholinesterase Inhibitory Activity
The inhibitory effect of the aerial parts of A. campestris on butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) was evaluated by employing the method of Ellman [33]; S-Butyrylthiocholine iodide (BuCI) and acetylcholine iodide (ACI) were used as substrates for the reaction. A solution of 150 µL of 100 mM sodium phosphate buffer (pH 8.0) with 10 µL of the sample at different concentrations and 20 µL AChE (5.32 × 10 −3 U) or BChE (6.85 × 10 −3 U) was mixed in a 96-well microplate. The solution was incubated at 25 • C for 15 min. A 10 µL of 0.5 mM, DTNB [5,5-Dithio-bis(2-nitrobenzoic acid)] was added along with 10 µL of acetylthiocholine iodide (0.71 mM) or 10 µL of butyrylthiocholine chloride (0.2 mM), at a wavelength of 412 nm. The absorbance of AChE or BChE enzymes was recorded every 5 min for 15 min. The inhibitory activity was measured by comparing the reaction rates of samples relative to the blank samples using the following formula: E: activity of the enzyme without sample; S: activity of the enzyme with the sample. Galantamine was used as a reference compound (positive control).

α-Amylase Inhibitory Activity
The alpha-amylase inhibitory activity was investigated according to Zengin et al. [34], using the iodine/potassium iodide method, with slight modifications. The reaction mixture was prepared in a 96-well microplate by adding 25 µL of the sample at various concentrations with amylase solution in 1 U of sodium phosphate buffer (pH = 6.9 with 6 Mm NaCl). After incubating the resultant solution at 37 • C for 10 min, the reaction was initiated by adding 50 µL of 1% starch solution. A control was simultaneously prepared without the enzyme solution. Re-incubation for 20 min at 37 • C was performed, followed by the addition of 25 µL 1 M HCl and 100 µL of iodine-potassium iodide solution to stop the reaction. The absorbance was measured at 630 nm, and the % inhibition of α-amylase was estimated as follows: Abs s = Absorbance (Extract, Starch, Enzyme, IKI, HCl); Abs b = Absorbance (Extract, sodium phosphate buffer, IKI); Abs e = Absorbance (solvent vol Extract, Enzyme, Starch, HCl, IKI); Abs c = Absorbance (solvent vol Extract, sodium phosphate buffer, Starch, HCl, IKI).

Pancreatic Lipase Inhibitory Activity
The assay for evaluating the inhibitory potential against pancreatic lipase was performed using the method of Souza et al. [35], with minor modifications; where P-nitrophenyl butyrate (p-NPB) was utilized as a substrate to assess the reaction. An aliquot containing 100 µL of enzyme solution (1 ppm in Tris-HCl (50 mM, pH 8.0) was mixed to obtain 50 µL of each sample at various concentrations. The resultant solution was then incubated at 37 • C for 20 min. Following that, the reaction was initiated by adding 50 µL of p-NPP (p-Nitrophenol Palmitate) after 120 min of incubation at 37 • C. Orlistat was used as a positive control. A 96-well microplate reader measured the solution's absorbance at 630 nm (Perkin Elmer, EnSpire) at t = 0 min and t = 120 min. In order to calculate the percentage of pancreatic lipase inhibition, we used the following equations [36]: where: Abs A : the activity in the absence of an inhibitor; Abs a : the negative control in the absence of an inhibitor; Abs B : the activity in the presence of an inhibitor; Abs b : the negative control in the presence of an inhibitor.

Photoprotective Activity
As described by Mansur et al. [37], the sun protection factor of A. campestris extracts was determined in vitro to investigate its ability to protect against UV damage (SPF).
First, the samples were diluted to a 2000 ppm concentration in absolute methanol. The absorbance was then measured at seven wavelengths, each with a 5 nm gap, ranging from 290 to 320 nm. All measurements were carried out in triplicate, and the following equation was used to calculate the SPF: CF: correction factor (=10); EE: erythemal effect spectrum; I: solar intensity spectrum; Abs: absorbance of sunscreen product, EE × I: is a constant calculated by Sayre et al. and is displayed in Table 1 [38].

Statistical Data Analysis
All tests were carried out in triplicate. The obtained results were statistically analyzed using SPSS descriptive statistics (IBM SPSS Statistics, version 21 PL, IBM, United States) and one-way ANOVA (GraphPad Prism 5 program). To answer the question of how individual features differ from each other, an additional test was obtained, called the multiple comparison test (the so-called post-hoc); the Tukey test was used, and the differences in values were considered at p < 0.05. Table 2 shows the main concentrations of mineral elements found in A. campestris. The results obtained were arranged in values as the mean and standard error (±SD), measured on the basis of triplicate analyses. Sixteen elements (Ca, Fe, Mg, Fe, Na, Co, Cd, Cr, Li, Mo, Ni, Pb, Sr, Ti, Zn and Cu) were found in our samples as reported. The coefficient of variation is a unitless measure (most often expressed as a percentage) and this property makes it possible to compare the diversity of statistical features with each other, regardless of the scale of the units. It should be noted that similar comparisons were not made using absolute measures of dispersion. It can also be used to figure out the arithmetic mean. The magnesium content in the aerial parts of A. campestris turned out to be the most stable (V = 0.32%), while molybdenum showed the greatest variability (V = 23.45%) ( Table 2).

Liquid Chromatography-Mass Spectrometry Analysis LC-MS-MS
After establishing the optimal UPLC-ESI-MS-MS conditions, the AcDE, AcEAE, and AcBE were analyzed utilizing the method's full scan with negative and positive ions mode. Figures 1 and 2 illustrate the UPLC-ESI-MS-MS and mass spectra of eleven chemicals identified from A. campestris extracts. Table 3 outlines retention time (Rt), m/z, and the formula of compounds proposed or deduced based on data reported in previously identified in Artemisia genus.      Table 4 summarizes the spectrophotometric results of the bioactive components in A. campestris extracts. Through employing a calibration curve (Phenol: y = 0.0034x + 0.1044, R2 = 0.9972; Flavonoid: y = 0.0048x, R2 = 0.997), the total phenol and flavonoid contents of the extract were, respectively, expressed in μg GAE/mg and μg QE/mg. The highest successive contents of AcEAE for phenolic compounds and flavonoid were 527.333 ± 0.61 μg GAE/mg and 203.4194 ± 0.14 μg QE/mg. In contrast, when compared to the other extracts, AcEAE demonstrated that it contained a high concentration of phenolic and flavonoid components. It was followed by the AcDE, which showed a high content of phenolic compounds and flavonoids at 203.607 ± 0.67 μg GAE/mg and 69.444 ± 0.147 μg QE/mg, respectively.   Table 4 summarizes the spectrophotometric results of the bioactive components in A. campestris extracts. Through employing a calibration curve (Phenol: y = 0.0034x + 0.1044, R2 = 0.9972; Flavonoid: y = 0.0048x, R2 = 0.997), the total phenol and flavonoid contents of the extract were, respectively, expressed in µg GAE/mg and µg QE/mg. The highest successive contents of AcEAE for phenolic compounds and flavonoid were 527.333 ± 0.61 µg GAE/mg and 203.4194 ± 0.14 µg QE/mg. In contrast, when compared to the other extracts, AcEAE demonstrated that it contained a high concentration of phenolic and flavonoid components. It was followed by the AcDE, which showed a high content of phenolic compounds and flavonoids at 203.607 ± 0.67 µg GAE/mg and 69.444 ± 0.147 µg QE/mg, respectively.

Antioxidant Activity
Due to the complexity of phytochemicals, it is expected that the antioxidant capabilities of plant extracts need to be investigated using more than one method. The tests of DPPH, ABTS, β-Carotene, Reducing Power, CUPRAC, GOR, and Phenanthroline were used to identify the antioxidant activities of A. campestris extracts. The results of these assays are listed in Table 5, which shows comprehensive results that are easier to consider, more basic to interpret, and correlate to other compounds. The indices of A 0 . 5 and IC 50 were used to measure the antioxidant activity of the extracts under consideration in this study, which were calculated using linear regression analysis. The A 0 . 5 and IC 50 levels have an inverse dependence with the activity's efficiency.

DPPH Scavenging Activity
In the case of the hydrogen proton transfer method, against stable radicals the DPPH assay measures the antioxidant capability of substances according to their ability to scavenge free radicals [47]. A. campestris extracts exhibited a significant capacity of scavenging the radical DPPH. Both standards and plant extracts decreased in the order of BHA > AcEAE > BHT > AcDE > AcAE > AcME > AcBE. According to the results presented in Table 5, AcEAE (IC 50 : 10.45 ± 0.19 µg/mL) showed strong antioxidant activity in the DPPH method, in which the IC 50 was relatively close to the IC 50 stated by the BHA (IC 50 : 5.73 ± 0.41 µg/mL) and more efficient compared with the standard (BHT) (IC 50 : 22.32 ± 0.19 µg/mL).

ABTS Scavenging Activity
The ABTS radicals represent another approach for assessing the antiradical activity of A. campestris extracts, which operate on the same principle as DPPH and evaluate electron transfer in the media; the ABTS + scavenging capacities among the various extracts were in the following order: BHT > BHA > AcEAE > AcDE > AcME > AcAE > AcBE. The result obtained from studying the highest concentration (IC 50 : 9.52 ± 0.12 µg/mL) ( Table 5). AcEAE showed an excellent high capacity to scavenge the radical ABTS + .

Reducing Power Activity
The mechanism of reducing power assay is based on the transformation of Fe 3+ into Fe 2+ [48]. AcEAE was the most effective in reducing iron ions, with the lowest A 0 . 5 value (A 0 . 5 : 16.05 ± 0.16 µg/mL). In decreasing order, the effective concentrations at which the absorbance was 0.5 were as follows: BHA > Ascorbic acid > AcEAE > AcDE > AcME > AcAE.

Cholinesterase Inhibitory Activity
Additionally, the efficacy of each extract to inhibit enzyme activity was examined using the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) ( Table 6), with the aim of increasing patients' cognitive capacity and reestablishing cholinergic transmission [49]. AcEAE showed a significant inhibitory effect for ACHE; it was more effective than AcPEE, whilst AcDE showed the weakest inhibitory effectiveness (IC 50 > 200 µg/mL). Although a substantial inhibition was shown towards BChE where the AcPEE displayed the most heightened inhibitory effect, both AcBE and AcAE had no effect on AchE and BchE.

α-Amylase Inhibitory Activity
In order to evaluate the antidiabetic efficient of A. campestris extracts was determined by estimating the potential of the different extracts to inhibit the α-amylase enzyme. From the results obtained (Table 6), the reaction of Artemisa extracts offered the optimum response with a substantially lower IC 50 value than that of acarbose, except that the AcAEwas not active against α-amylase. The highest α-amylase inhibitory activity was found in AcPEE (IC 50 = 11.97 ± 0.14 µg/mL). This activity is three 333 times higher than acarbose (IC 50 = 3650.93 ± 10.70 µg/mL).  Table 6 shows the data on the Pancreatic Lipase inhibitory activity of extracts from A. campestris aerial parts. Anti-lipase activity was significantly inhibited by A. campestris extracts, with the AcPEE being the most effective compared to the reference (IC 50 = 0.06 ± 0.001 µg/mL). The inhibitory activity follows the decreasing order of orlistat > AcPEE > AcDE > AcEAE, respectively, while AcME, AcBE and AcAE did not affect pancreatic lipase.

Photoprotective Activity
The Sun Protection Factor (SPF) was determined as an indicator of the photoprotective efficiency A. campetris, as presented in Table 7. SPF values of the extracts of A. campestris ranged from 24.79 ± 0.07 to 40.76 ± 0.1. These findings revealed that all of the extracts had evidence of photoprotective activity. In addition, the extracts of AcME, AcDE, AcEAE, and AcBE were found to have high photoprotective effects with SPF 42.07 ± 0,17; 40.76 ± 0.11; 39.51 ± 0.09; 38 ± 0.05, respectively. The coefficients of variation of the SPF values of the photoprotective activity turned out to be very low (V = 0.13-1.00), which indicates a very high accuracy in their determination (Table 7).

Discussion
A wide range of illnesses can be treated or at least reduced by using plants and/or plant materials; in addition, plants are essential sources for food applications [50]. According to the WHO (World Health Organization), there is abundant proof that minerals have biochemical nutritional and structural activities that are important for overall physical and mental health [51]. The mineral composition of A. campestris has not been investigated in previous studies; hence, this is the first time such a study has been undertaken. The present purpose is to serve the lacuna in research by utilizing the ICP-OES method in order to provide empirical proof of the mineral make-up of A. campetris, along with the specific concentrations. The results of this study can be used as a database for the healthcare industry, and the mineral analysis reveals that A. campestris is a valuable Ca, Fe, Mg, and Na source. In human nutrition, these mineral components are essential. Calcium (Ca) is the most prevalent element in aerial parts of A. campetris, with a concentration of 10,538.1 ± 69.9 mg/Kg. Calcium is an important structural mineral that is commonly present in bones and teeth, and it regulates nerve and muscle function. It plays a critical function in enzyme activation [52]. A. campestris is an excellent source of calcium. A. campestris contains 1181.8 ± 82.37 mg/Kg of iron (Fe), which is the second most common element as shown in this study. Iron helps in the formation of haemoglobin in the transfer of oxygen; it is also a key component of some enzymes that performs biological oxidation in cellular respiration [52,53]. Magnesium and Sodium were both abundant with concentrations of 990.7 ± 3.14 mg/kg and 289.41 ± 1.319 mg/kg, respectively. Additionally, magnesium is a component of many enzyme systems and is also found in bones and teeth [54]. Sodium is the most abundant cation in extracellular fluids, and also plays a role in controlling the volume of plasma and the acid-base equilibrium of cells [55]. The experimental data revealed that the other mineral concentrations in A. campestris decreased in the following order: Cu > Zn > Li > Mn > Sr > Ti > Ni > Co > Mo with a concentration of 70.22 ± 0.94 mg/Kg. A. campestris had a high Cu content above the WHO allowable limits. As per FAO/WHO, the permitted limit of Cu content for fruit samples is 4.5 mg/kg [56]. It is therefore advisable for consumers of A. campestris to take note of this to avoid exposure to Cu in excessive doses; although, the plant is usually consumed in smaller quantities, thus reducing the chance of excess exposure. Other essential elements such as zinc and manganese were detected at significant levels. Consequently, A. campestris can be considered an excellent source of zinc and manganese. Pb, Cr, and Cd were also detected, and have been shown to be of toxicological concern in other studies. According to the World Health Organization's ADI for lead, the allowed limit for lead concentration in medicinal plants is 10 mg/kg of the plant matter [57]. At 0.68 ± 0.305 mg/Kg, the A. campestris lead content is within the acceptable range [58]. In addition, our results are lower than the maximum permissible limits (MPL) set by the WHO for cadmium, which is 0.3 mg/kg. Interestingly, A. campestris contains large concentrations of macroelements such as Ca, Mg, and Na. This analysis demonstrated the enormous potential of plants in producing a wide range of minerals to benefit the treatment of various diseases.
AcDE, AcEAE contained sesquiterpene lactone glucosides with the molecular ion at m/z 481, which were identified as 15-O-β-D-glucopyranosyl-11β, and 13-dihydro urospemal A [39]. The majority of tentatively identified compounds are already known from the Artemisia genus.
In plants, phenolic chemicals constitute a major category of secondary metabolites with important pharmacological effects on the human body; these compounds provide protection against stressors via various mechanisms [59]. The number of polyphenols in A. campestris extracts was determined by examining the extracts with solvents at different polarities. Variable levels of polyphenols and flavonoids were found in this quantitative study. The AcEAE and AcDE yielded the highest total phenolics and flavonoids. Our findings were much greater than those obtained by Megdiche et al. [60] and Djeridane et al. [61]. According to our findings, polar organic solvents proved to be the most efficient in the process of extracting phenolic and flavonoid components. Several factors, including the solvents (degrees of polarity) utilized, ambient and ecological conditions, extraction and quantification processes, and geographic location, have been documented to influence phytochemical content.
Antioxidants protect against free-radical induced tissue damage by inhibiting radical production, scavenging them or promoting their breakdown [59]. Since a given antioxidant's reaction varies in different testing systems, this led to the assessment of the antioxidant activity of plant extracts using a diversity of approaches, including direct, indirect, competitive procedures, reduction, chelation, and/or inhibition principles [62]. Due to the elevated levels of phenolics and flavonoids in the AcEAE, the above methods show that this extract has a potent antioxidant capacity, which gives AcEAE its distinctive character. Indeed, the strongest antioxidants are phenolic chemicals, which include hydroxyl groups in their structure that allow hydrogen or electrons to be transferred to a reactive molecule.
Synthetic-free radicals termed DPPH and ABTS are commonly used to assess plant extracts and pure compounds for their potential anti-radical ability [63]. The AcEAE had potent antioxidant power against the radical DPPH and the radical ABTS. The change from purple (DPPH − ) to yellow (DPPH − H) was due to the reception of electrons measurable at 515 nm. The ABTS decolorization method is used to quantify the loss of color due to the addition of an antioxidant to the blue-green chromophore 2.2 azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS _+ ). The obtained results appear to be better correlated with the finding of Akrout et al. [64] using infusion and ethanol 50%. On the other hand, the obtained results of DPPH are similar to those reported by Megdich et al. [60]. The antioxidant properties of AcEAE were low and comparable to those of standard antioxidants saved from the bleaching of carotene. However, it was found that AcBE was the only one that moderately prevented the oxidation of β-carotene brought about by the free radicals produced as a by-product of the oxidation of linoleic acid. It is an important property that the ability to operate on the products of lipid peroxidation provides. Comparing our results with those reported previously, AcBE is incredibly effective in β-carotene [65].
The AcEAE of iron and copper ions was reduced by A. campestris with great efficiency, which may be due to their ability to rebuild oxidized molecules and, therefore, break the hydrogen subtract. Our research shows the evaluation of antioxidant activities for the first time using CUPRAC and O-Phenanthroline. Table 5 presents the ferric reducing power A 0 . 5 of A. campestris extracts which represents the speed of reducing Fe 3+ (CN) 6 to Fe 2+ (CN) 6 by direct electron donation. Then, the complexes of ferric ferrous from the reaction mixture experienced high absorption at 700 nm [66]. Comparable to the results reported by Boulanouar et al., A. campestris oil has been previously reported to exhibit a strong reducing power activity [67], with an IC 50 = 0.305 ± 0.006 mg·mL −1 .
Another approach conducted for the first time in the genus of A. campestris is the use of the Galvinoxyl free radical scavenging assay. Similarly, AcEAE demonstrates a strong antioxidant activity which is better than the standard. Consequently, when hydrogen donors interact with the free radical Gox it is reduced to GoxH [29]. This reduction was observed spectrophotometrically as a disappearance of color when they were quenched.
The neurodegenerative disorder known clinically as Alzheimer's disease, is a deadly neurodegenerative disease characterized by increasing brain damage and mental illness [68]. Using cholinesterase inhibitors as a key treatment strategy for AD, acetylcholinesterase and butyrylcholinesterase inhibitors extend the availability of the neurotransmitter acetylcholine to the brain receptors by inhibiting the enzymes that are affected within the hydrolysis of acetylcholine [69]. As demonstrated in Table 6, A. campestris exhibited good inhibitory efficacy for the AcEAE against AChE (IC 50 = 23.16 ± 0.19 µg/mL) afterwards AcPEE (IC 50 = 59.03 ± 0.58 µg/mL). The AcPEE, nonetheless, moderately inhibited BChE activity (IC 50 =93.50 ± 1.60 µg/mL) compared with the Galantamine standard. AcBE and AcAE were reported to be inactive. Compared to the results found for the essential oil of A. campestris reported by Cheraif et al. [70], a significant enzyme inhibitory effect was shown by the AcPEE towards both the AChE and BChE enzymes. For this purpose, we can reach the conclusion that the chemical constituents are capable of behaving as antagonists of AChE and BChE; studies have already demonstrated that particular flavonoids and phenolic acids, such as acacetin and apigenin, possess a significant effect on inhibiting activity towards AChE and BChE, and their existence has already been proven according to the LC-MS/MS results [71]. Natural compounds from multiple sources, such as terpenoids, phenols and alkaloid chemicals have been found to inhibit AchE and BChE in several studies [72]. Secondary metabolites in the AcPEE might well be responsible for acetyl and butyrylcholinesterase activity. Recently, Boukhalkhal et al. [73] identified the following terpenoids in A. campestris L.: Carnosic acid and OH-Rubescensin BI, BII; these compounds may be the source of the anticholinesterase effect investigated in this paper. We have highlighted our findings in light of this, and A. campestris might well have anti-Alzheimer activity that could be beneficial for treating AD.
Chronic hyperglycemia is a characteristic of the metabolic disorder known as diabetes; there are several medicinal strategies for treating type 2 diabetes. One of these processes by which plants exercise their anti-hyperglycaemic action is by inhibiting alpha-amylase in the small intestine, preventing complex carbohydrates from being broken down and preventing their absorption. Starch and other glucose polymers in diabetic individuals hydrolyze (1.4)-D-glycosidic linkages in the presence of this enzyme's inhibitors [74]. A. campestris extracts showed an outstanding anti-diabetic action that was more efficient than acarbose (Table 6), and we found a significant difference in their IC 50 . Molecular docking simulations were used to investigate polyphenols' capacity to inhibit the enzymes alphaglucosidase and alpha-amylase; furthermore, it was hypothesized that substances such as caffeic acid, Naringin, and Rutin might considerably inhibit the enzymes [75], which includes all phenolic chemicals and flavonoids discussed within the profile phytochemicals of A. campestris extracts that are considered crucial in the treatment against diabetes in this study. In contrast, the existence of certain minerals may favorably contribute to the anti-diabetic effects of plants, e.g., by enhancing the effects of insulin [76]. Our research establishes that A. campestris has anti-diabetic potential in vitro by inhibiting alpha-amylase.
Obesity is a disease characterized by a high ratio of weight to height. One of the leading causes of obesity is an imbalance between the amount of calories a person consumes and the amount of calories their body will burn. Inhibition of pancreatic lipase has become an important approach for the treatment of obesity. The lipolytic products of diacylglycerol, monoglycerides and glycerol, and polyun-saturated fatty acids are produced by the enzyme lipase which catalyses the breakdown of dietary triglycerides [77]. A remarkable effect (IC 50 : 0.061 ± 0.001 µg/mL) has been achieved using orlistat as a result of our investigation; it presents long-term effectiveness as a pancreatic lipase inhibitor, and is one of the most significant and recently proposed treatments for obesity. This inquiry is the first to investigate the anti-lipase activity of the areal parts of the A. campestris extracts. AcPEE was revealed to be the most effective (Table 6), which provides a moderate potential to identify novel anti-obesity agents. Natural compounds present in natural sources are lipase inhibitors as a consequence of the multitude of inhibitors generated from the following classes of compounds: saponins, polyphenolics, and terpenes [77], as was previously discussed in relation to A. campetris.
Prolonged exposure to ultraviolet radiation can be divided into the following three areas: UVA, UVB, and UVC, which can have negative repercussions including the development of skin cancer in certain conditions [78]. Natural substances are frequently applied in traditional medicine and the industries of cosmetics, and several of flavonoids have been shown to provide protection against ultraviolet radiation [79]. The sun protection factor, known as the SPF, is a quantitative assessment of the effectiveness of a sunscreen product. In relation to this concept, it has recently been established that, according to the SPF ratings, the values of SPF [2→12], [12→30], [30→50] and >50 are classified as possessing a minimum, moderate and strong sun protection action, respectively. According to the Commission of European Communities 2006 recommendation [80], the AcME, AcDE, AcEAE and AcBE belong to the high protection class. The excellent photoprotective capabilities of A. campestris extracts might well be mainly attributed to the existence of flavonoids and phenolics. Based on the findings of this study and what was discussed before, A. campestris has a substantial affinity between its photoprotective effect and its total phenolic content. The Saharan climate and geographical area and the plant's daily exposure to sunshine led to the production of more phytochemical compounds to shield the plant from ultraviolet harm, hence improving photoprotective activity. As a consequence, the acquired data demonstrated the considerable antioxidant activity of A. campestris as a prospective source of sunscreen in the cosmetic industry or pharmaceutics formulations.

Conclusions
The phytochemical profile of A. campestris extracts proved the presence of a variety of types of bioactive compounds that have interesting pharmacological effects. This study marked an important comprehensive examination of the biological activity and mineral constituents of A. campetris, an endemic plant species from Algeria. The findings highlight the importance of the biological activities of A. campetris. The prominent antioxidant and anti-enzymatic properties (anti-diabetic; anti-Alzheimer and anti-obesity) of the plant were demonstrated. A. campestris demonstrates high absorption of ultraviolet light. In our study, the ICP-OES technique was used to conduct a mineral analysis of the plant. The findings demonstrate that A. campestris contains a high concentration of minerals that are essential for human health, including calcium, iron, sodium, magnesium, zinc, and manganese, amongst others. Furthermore, the toxic reference values for the potentially toxic elements were much below the World Health Organization's tolerance limits. Our study proved that the plant has distinctive medical, food, nutrition, and pharmaceutical properties. ICP-OES Inductively coupled plasma-optical emission spectrometry TIC + Total ion current in positive mode TIC − Total ion current in negative mode