Ammoides pusilla Aerial Part: GC-MS Profiling and Evaluation of In Vitro Antioxidant and Biological Activities

Abstract

The study of Ammoides pusilla, a Tunisian medicinal plant, explored its chemical composition and biological activities, highlighting its under-exploited therapeutic potential. The essential oil, obtained by steam distillation, reveals twenty major compounds, including perilic aldehyde, β-phellandrene, and o-cymene. Two new natural constituents were identified in the cyclohexane extract and four in the dichloromethane extract. DPPH and ABTS tests showed that methanol extract exhibited the highest antioxidant activity, giving values of 78.9% and 65.5%, respectively, at 50 µg/mL. Its anti-diabetic activity (IC50 = 25.0 µg/mL) exceeds that of acarbose. The anti-SOD activity of methanol extract also showed promise, at 73.3% at 50 µg/mL. Essential oil and ethyl acetate extract showed notable inhibition of xanthine oxidase activity, reaching 69.0%. In addition, the essential oil demonstrated strong anti-AChE (63.23% at 50 µg/mL) and anti-inflammatory (IC50 = 31.0 µg/mL) activity. In terms of cytotoxicity, the methanol extract was effective against the HCT116 cell line (IC50 = 20.9 µg/mL), and all extracts showed activity against MCF7, OVCAR-3, and IGROV-1 cells, with IC50 values ranging from 4.0 to 25.0 µg/mL. This result underlines the potential of Ammoides pusilla extracts as important sources of bioactive compounds for therapeutic applications. Further research is needed to fully exploit these activities in drug development.

1. Introduction

Metabolism and cellular respiration constantly produce free radicals in the human body; otherwise, this physiological production is already controlled by efficient internal “anti-oxidant” defense systems. In some cases, imbalances crop up either because of anti-oxidant deficiency or excessive release of free radicals. This phenomenon is called oxidative stress, which promotes many diseases, including cancer and other diseases [1]. The objective of the current study was to identify bioactive molecules having anti-oxidant effects from medicinal plants. In Algeria, the Ammoides genus (Apiaceae) comprises two distinct species. The first, Ammoides atlantica (Coss. et Dur.) Wolf, is found exclusively in Algeria, while the second, Ammoides pusilla (Brot.) Breistr., is widely distributed throughout the Mediterranean region. In traditional medicine, Ammoides pusilla (A. pusilla) is used as an infusion to treat a variety of ailments, including headaches, fever, flu, and diarrhea [2,3].

In the literature, we found works treating essential oils without any studies related to extracts. Using the disk diffusion method, the in vitro antibacterial activity of Algerian A. pusilla essential oil was evaluated against various bacterial strains. This essential oil was also analyzed using Gas Chromatography with Flame Ionization Detection (GC-FID) and Mass Spectrometry (GC-MS) [4,5]. The principal compounds present were thymol (53.2%), γ-terpinene (19.4%), and p-cymene (10.6%) [2]. Furthermore, the chemical composition of Ptychotis verticillata (P. verticillate) essential oil from Morocco was analyzed by GC-FID and GC-MS. Carvacrol (44.6%) and thymol (3.4%) are the most important compounds [6]. In a third study, at least 10 different compounds were detected in the essential oil extract of P. verticillata. The main compounds were thymol, gamma-terpinene, D-limonene, and m-cymene, accounting for 95.86% of the oil. They also demonstrated that P. verticillata exhibited antimicrobial activity in vitro against five Gram-positive bacteria [7]. A recent study investigated the chemical composition and antioxidant, antimicrobial, and antiproliferative activities of A. pusilla essential oil [8].

In this work, we focused only on the essential oil and organic extracts of A. pusilla. We were interested in: (a) chemical characterization of the essential oil using gas chromatography (GC-MS and GC-FID) and organic extracts (with and without derivatization by GC-MS), total phenolics, flavonoids, tannin, anthocyanins, and total reducing sugars; (b) antioxidant activity (DPPH and ABTS assays) of the essential oil and extracts; (c) evaluating biological activities (for the first time): anti-inflammatory, anti-SOD, anti-XOD, anti-diabetic, and cytotoxic effects with different cell lines HCT116: colorectal carcinoma; IGROV-1: ovarian cancer; MCF7: mammary gland/breast cancer; OVCAR-3: ovarian cancer.

2. Materials and Methods

2.1. Extraction

2.1.1. Organic Extract

The aerial parts of A. pusilla used in this report were collected during the flowering phase in Beja, a town located in the north of Tunisia (May, 2013). The plant has been identified by Professor Mohamed Bousaid from the Department of Biology (National Institute of Science, Application, and Technology) in Tunisia. The specimen reference under MB042013 was registered in the same department. The aerial part (stems, leaves, and flowers) of A. pusilla, dried, was ground to produce a fine powder, which was successively extracted with solvents of increasing polarity (Cyclohexane (Cyclo), Dichlorometane (DCM), Ethyl acetate (EtOAc), and finally Methanol (MeOH)). A total of 200 g of leaf powder was thus soaked in cyclohexane (2000 mL) for 4 h with frequent stirring at room temperature under pressure. Afterwards, all the mixture was filtered via Wattman paper (GF/A, 110 mm). Then, the solvent was evaporated by rotary vacuum evaporation at 35 °C (IKA, RV 10 auto V, Staufen Germany). The powder extracted with cyclohexane was then reused with the next solvent, dichloromethane, at the same conditions. The same process was followed for ethyl acetate and methanol. All extracts were then stored in sealed amber vials at 4 °C for further biological test activities and chemical composition analysis.

2.1.2. Essential Oil

The dry aerial parts of A. pusilla (200 g) were hydrodistilled for 3 h by means of a Clevenger-type apparatus. A yellowish oil was obtained, dried over a sodium sulfate anhydrous, then stored at +4 °C in amber vials for subsequent analysis. The optical densities were measured with a spectrophotometer (Multiskan Go, F1-01620, Thermo Fisher Scientific, Vantaa, Finland), enabling wavelength selection for 96-well plates and various types of cuvettes.

2.2. Chemical Composition of Essential Oil

GC-FID and GC-MS were used for quantitative and qualitative analysis of the essential oil [9]. For gas chromatographic analysis, we used a Varian Star (3400) chromatograph (Les Ulis, France) equipped with a DB-5MS fused silica capillary column (5% phenylmethylpolysiloxane, 30 m × 0.25 mm, film thickness 0.25 μm). Initial chromatographic conditions included two gradients: the first increased the temperature from 60 °C to 260 °C at a rate of 5 °C/min, followed by a 15 min isothermal step at 260 °C. A second gradient, applied at 340 °C at a rate of 40 °C/min, completed the chromatographic program in 57 min.

A. pusilla essential oil was diluted in petroleum ether for analysis. A volume of 1 µL of the sample was injected using a 1:10 split mode. The carrier gas used was selected as helium (purity 99.999%) at a flow rate of 1 mL/min. It was kept at 200 °C. Using a mass spectrometer (Varian Saturn GC/MS/MS 4D), with an emission current of 10 μA and an electron multiplier voltage between 1400 and 1500 V. The trap temperature was 220 °C, and the transfer line temperature was 250 °C. The mass scan covered a range from 40 to 650 amu. In order to identify the compounds, their retention times (RTs) were compared with those of C5-C24n alkanes obtained on a non-polar DB-5MS column, and their mass spectra were compared with those in the NIST 08 database. Each compound was identified by comparing its retention time (RT) with that of C5-C24n alkanes obtained on a non-polar DB-5MS column and by comparing its mass spectra with those of the NIST 08 database. The percentage composition of the essential oil was determined by the GC-FID peak area normalization method, applying the same mass response factor to all compounds. Three injections of essential oil were used to establish the mean values for the analyses, which were performed in triplicate.

2.3. Organic Extract Profile by HPLC Analysis

The following methodology was used for obtaining the chromatogram of each standard compound as well as for all organic extracts of A. pusilla. A collection of phenolic standards was purchased from Sigma Aldrich (Saint-Quentin-Fallavier, France). A total of 1 mg of each standard compound (dilution 1/10) or 20 mg of each organic extract were dissolved in 1.5 mL of water (pH 2.6)/acetonitrile (90:10), sonicated, and passed through a nylon membrane filter (0.2 µm) before injection. Analysis of all solutions was performed using an Ultimate 3000 Dionex pump, autosampler thermosepration products, and UV-150 detector thermosepration products (Thermo Fisher Scientific, Waltham, MA, USA). A C18 column (5 µm, 4.6 mm × 250 mm) was used, and 50 µL was injected with a flow rate of 1.2 mL/min. A double solvent gradient elution was used: solvent A (acetic acid in water, pH 2.6) and solvent B (acetic acid in acetonitrile: water, (90:10), pH 2.6. The program of gradient was started with 10% B and increased to 30% B during 35 min. This was followed by a stage of 30% B for the following 5 min. A second gradient was applied between 30% B and 50% B for the following 5 min. Finally, the concentration of B increased up to 99.5% for the following 5 min. UV detection was stopped at 280 nm. The data analysis was performed using Chromeleon 6.8 software.

2.4. Examination of Organic Extracts’ Volatile Components Both with and without Silylation

About 5 to 10 mg of each extract was added to 150 µL of BSTFA, adding 1% of TMCS, vial cap was closed, shaken for 30 s, then heated for 15 min at 40 °C. An additional 1 μL of the silylated mixture was directly analyzed by GC-MS. To avoid oxidation, measurements were taken in a sealed atmosphere and under nitrogen. The leaving group of the silylated compound must be weakly basic and capable of stabilizing a negative charge in the transition state. Samples without silylation were solubilized in their solvent of extraction at 3 mg/mL.

For the chromatographic conditions, we applied two gradients: the first was a temperature rise from 60 °C to 270 °C with a gradient of 15 °C/min and a 6 min isotherm at 270 °C. At 50 °C per minute, the second gradient was applied to 300 °C. The entire chromatographic program lasted 30 min. All other chromatographic conditions of GC-MS are identical to those of the essential oil. Analyses were established in triplicate, and results were determined based on the mean values of three injections of each sample. Identification of compounds was obtained by comparing spectral data obtained from the NIST 05 libraries with a percentage higher than 85% similarity.

2.5. Chemical Families

The quantification of total phenolics, flavonoids, total condensed tannins, and total anthocyanin contents was established according to the method cited in [10].

2.6. Reducing Sugar Determination with the 3,5-Dinitrosalicylic Acid (DNS) Assay

The DNS method is a colorimetric assay, using the reducing properties of glucose [11]. In hot and alkaline conditions, there is a reduction in dinitrosalycilic acid, which serves as an oxidant, and glucose, which is the reducing agent. The resulting compound is 3-amino-5-nitrosalicylic acid, a red compound. The amount of simple reducing sugars contained in each organic extract of A. pusilla is determined with a glucose standard solution (0–80 µg/mL). A total of 150 µL of each glucose solution or all organic extracts of A. pusilla (at 400 µg/mL) was mixed with 150 µL of DNS color reagent solution at 96 mM (5.31 M of 3,5-dinitrosalicylic acid, mixed with sodium potassium tartrate in an aqueous solution of NaOH 2 M). The reaction was conducted in Eppendorf tubes brought to ebullition in a water bath at 100 °C for 5 min. The cooling of the reaction mixture was affected by the addition of 750 µL of distilled water. The absorbance was determined at 540 nm.

2.7. Antioxidant Activity

2.7.1. Free Radical Scavenging Activity by 1-1-Diphenyl 2-Picryl Hydrazyl (DPPH●)

The hydrogen atom-or-electron release ability of the extract was measured by the variation from purple to white in the methanol solution of DPPH●. This method is based on the spectrophotometric assay using the stable radical, 2.2′-diphenylpicrylhydrazyl (DPPH●), as a reagent [12]. A total of 20 µL of each extract of A. pusilla (µg/mL) was mixed with 180 µL of 0.1 mM DPPH● in methanol. In plates, after an incubation of 30 min at room temperature in the dark, absorbance was measured at 517 nm using 200–1000 nm Multiskan Go microplate reader (Thermo Fisher Scientific, Vantaa, Finland). Methanol was used as a blank, and ascorbic acid (0.5–10 µg/mL) was used as the reference compound. The absorbance of solvent and DPPH● radical without extract was measured as a control. The radical-scavenging activities of extracts, expressed as percentage inhibition of DPPH●, were calculated according to the formula:

Inhibition (%) = [(A_517control − A_517sample)/A_517control] × 100

2.7.2. ABTS (2,2′-Azinobis-3-ethylbenzothiazoline-6-sulfonate) Assay

The free radical scavenging capacity of antioxidants for the radical cation ABTS was evaluated using the following method [12]: a 7 mM ABTS solution was prepared at pH 7.4 (composed of 5 mM NaH₂PO₄, 5 mM Na₂HPO_4, and 154 mM NaCl), then potassium persulfate was added at a concentration of 2.5 mM. The mixture was kept at room temperature in the dark for 16 h before use, being prepared the day before the assay. After dilution in persulfate buffer, the absorbance of the mixture was adjusted to 0.70 ± 0.05 units at 734 nm. Next, 20 µL of each test sample was added to 180 µL of fresh ABTS solution, and absorbance was measured 6 min after initial mixing. As a reference standard, ascorbic acid was used. Free radical scavenging capacity was expressed as a percentage inhibition. Percentage inhibition was calculated using the same formula as for the DPPH assay.

2.8. Biological Activities

2.8.1. In Vitro α-Amylase Inhibitory Activity

The absorbance at 540 nm was used to evaluate α-amylase activity. The α-amylase inhibitory activity (%) was defined as the percent decrease in the maltose production rate over the control. Acarbose was used as a positive control. The α-amylase inhibition was expressed as a percentage of inhibition and calculated from the equation below, where A is the absorbance [12].

Inhibition (%) = [(A_540control − A_540sample)/A_540control] × 100

2.8.2. In Vitro Xanthine Oxidase Inhibitory Activity

Xanthine oxidase (XOD or XO) is a form of xanthine oxidoreductase, an enzyme that generates reactive oxygen species. It catalyzes the oxidation of hypoxanthine to xanthine and can also catalyze the oxidation of xanthine to uric acid. The anti-Xanthine oxidase activity was assayed spectrophotometrically under aerobic conditions. The assay mixture consisted of essential oils tested at different concentrations (10, 50, and 100 mg/L). Whereas extracts were tested only at one concentration (50 mg/L), phosphate buffer (pH 7.5), and xanthine oxidase enzyme solution (0.01 units/mL in phosphate buffer, pH 7.5). After 15 min of pre-incubation at 25 °C, the reaction was started by adding the substrate solution (xanthine in the same buffer). At 25 °C, the mixture was incubated for 30 min. The absorbance was measured at 290 nm. The test was performed in triplicate. IC50 values were calculated from the mean values of the data, and for extracts, inhibition was determined at one concentration (50 mg/L) [12].

2.8.3. In Vitro Anti-SOD (Superoxide Dismutase) Activity

This activity was evaluated using the method of Belaiba, M et al. [12]. The auto-oxidation of pyrogallol by atmospheric oxygen could be inhibited by superoxide dismutase (SOD), an enzyme naturally present in the body. Moreover, variations in the rate of pyrogallol auto-oxidation in the presence of SOD have yet to be evaluated. The reaction mixture (200 μL) contains Tris buffer 50 mM. pH 7.5; diethylenetriaminepenta acetic acid 1 mM (DTPA); and superoxide dismutase (SOD). Extracts were tested at a concentration of 50 mg/L, followed by incubation for 37 min at 6 °C. After adding 30 mM of pyrogallocallol to initiate the reaction, the absorbance is measured for 4 min at 325 nm. The auto-oxidation of pyrogallol was tested in the absence of an enzyme. To perform this, pyrogallol was added to a Tris tampon (pH 8.5), and the same processes as an incubation of a positive control (without extracts) were repeated in the presence of 5% DMSO to measure the percentage of SOD inhibitory activity on pyrogallol auto-oxidation. The calculation of percent inhibition is as follows:

%Inhibition= Average (A_extract − A_{controlextract})/(A_Control − A_{controlextract})) × 100.

2.8.4. In Vitro Anti-Cholinesterase Activity

Alzheimer’s disease is a chronic brain disease that alters the intellectual faculties irreversibly. This is due to a decreased level of the neurotransmitter acetylcholine (ACh). Acetylcholinesterase is an enzyme very important in the central nervous system. It catalyzes the cleavage of acetylcholine in the synaptic cleft after depolarization. Inhibitors of AChE, such as galanthamine, are frequently used in pharmacotherapy. Indeed, cells (neurons) in the nervous system release an enzyme called acetylcholinesterase (AChE). This enzyme decomposes, once released, acetylcholine (ACh) and choline acetate, resulting in the gradual reduction in the neurotransmitter acetylcholine (ACh). A simple method for evaluating the activity of AChE is the Ellman method [12]. The enzymatic activity was assessed by a modified colorimetric Ellman’s method.

In this method, 50 µL of Tris-HCl buffer (pH 8), 25 µL of an extract buffer solution at different concentrations and 25 µL of an enzyme solution containing 2.8 U/mL AChE were used to assess the enzymatic activity of the extract. The reaction was then initiated via the addition of 125 µL of 3mM 5-5′-thiobis-2-nitrobenzoic acid (DTNB). After incubation of 15 min at 25 °C, 25 µL of a solution of 15mM ATCI (synthetic substrate for AChE) was added in a microplate of 96wells, and the final volume of each well was 225 µL. The absorbance of the mixture was measured at 412nm after 10min. A control mixture was prepared, using 75 µL of a solution similar to the sample mixture but with the respective solvent instead of extract. Each experiment was performed at least three times. Inhibition (%) was calculated in the following way:

(%) Inhibition = 100 − (A_sample/A_control) × 100.

where A_sample is the absorbance of the extract containing the reaction and A_control is the absorbance of the reaction control. All tests were performed in triplicate. Extract concentrations providing 50% inhibition (IC₅₀) were obtained by plotting the inhibition percentage against extract solution concentrations.

2.8.5. In Vitro Anti-Inflammatory Activity

Lipoxygenase is known for catalyzing the oxidation of unsaturated fatty acids with 1.4-pentadiene groups. The activity was measured spectrophotometrically at 234 nm using the conjugated diene produced by the oxidation of linoleic acid by the 5-Lipoxygenease enzyme. Individually, 20 µL of essential oil concentrations and extracts were evaluated in sodium phosphate buffer (pH 7.4) with 5-LOX (500 U) and 60 µL of linoleic acid (3.5 mM). After a 10-min incubation at 25 °C, the absorbance at 234 nm was measured. The percentage of enzyme activity was plotted against the concentration of the essential oil. The IC₅₀ value is the concentration of essential oil that causes 50% enzyme inhibition. While we determined only the percentage of inhibition for all extracts, Nordihydroguaiaretic acid (NDGA) (Sigma-Aldrich, Steinheim, Germany) was used as a positive control. The percentage inhibition of enzyme activity was calculated as shown below. All tests were carried out in triplicate [12].

% Inhibition = [(A_Control − A_{extract or essential oil})/A_control] × 100.

2.8.6. Cytotoxic Activity with 3-[4,5-Dimethylthiazol-2yl]-2,5-diphenyl Tetrazolium Bromide (MTT) Assay

The cytotoxic activities of extracts and essential oils against cancer cell lines were evaluated by the MTT assay. The reagent used is the tetrazolium salt MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium). The tetrazolium ring is reduced by mitochondrial succinate dehydrogenase in active living cells to formazan. This forms a purple precipitate in the mitochondria. The amount of precipitate formed is proportional to the number of living cells (but also to the metabolic activity of each cell). A simple determination of optical density at 550 nm spectroscopy allows determining the relative amount of live cells.

The MTT colorimetric assay was carried out using 96-well plates. Cells were planted in a 96-well plate at a density of 10 × 10³ cells/well (HCT116). Adherent cells (MCF7, IGROV-1 and OVAR) were delivered at a density of 12 × 10³ cells/well and incubated overnight at 37 °C in a 5% CO₂ environment. Cells in the exponential growth phase were incubated at 37 °C for 72 h with each tested substance at 50 mg/L. After that, the medium was removed, and cells were treated with 50 µL of MTT solution (3 mg/mL in PBS) at 37 °C for 20 to 40 min.

To dissolve the mitochondria and therefore precipitate violet formazan, we added 80 µL of 100% DMSO. At the 540 nm wavelength, optical density was measured. All tests were established in triplicate. The anti-cancer effect of extracts was estimated in terms of growth inhibition percentage. To compare our results, we used an anti-cancer drug solution reference, tamoxifen (50 mM), at 3 different concentrations to determine the IC50 value. Tamoxifen is a selective estrogen receptor modulator used orally in breast cancer. It is currently the best treatment sold for this cancer [12].

2.9. Statistical Analysis of Data

SPSS (version 20.0) was used to compute significance, and Tukey’s test was utilized to compare statistical differences among the solvents employed in the study. All measurements were carried out in quadruplicate using a one-way analysis of variance (ANOVA). To ascertain the relationship between the biological activities or antioxidants and the (TPC), the linear correlation coefficient (R2) was evaluated. Finally, a principal component analysis (PCA) was performed using XLSTAT (version 5.03) in order to verify how all the variables differed. The threshold for dependability was set at p ≤ 0.05.

3. Results and Discussion

3.1. Extraction Yields

Extraction yields for extracts of the aerial part and essential oil of A. pusilla are shown in

Table 1. Chemical composition and extraction yields of A. Pusilla extracts.

Extracts	Yields (%)	Phenolics (GAE) a	Flavonoids (QE) a	Tannins (CE) a	Anthocyanins (C3GE) b	Reducing Sugar (GE) a
Cyclohexane	5.4	89.3 ± 4.00	3.8 ± 0.10	6.5 ± 0.20	1.90 ± 0.02	2.74 ± 0.00
Dichloromethane	0.7	26.0 ± 2.90	2.2 ± 0.10	6.4 ± 0.40	0.90 ± 0.01	1.84 ± 0.00
Ethyl acetate	0.1	113.7 ± 2.40	47.6 ± 1.00	5.5 ± 0.50	0.10 ± 0.03	4.73 ± 0.00
Methanol	10.6	116.6 ± 2.70	91.4 ± 2.20	1.6 ± 0.20	0.50 ± 0.00	15.07 ± 0.00
Essential oil	4.3

Note: GAE: gallic acid equivalents; QE: quercetin equivalents; CE: catechin equivalents; C3GE: cyanidin-3-glucoside equivalent; and GE: glucose equivalent. The letters ^a,b indicate ^a: g/kg dry mass; ^b: mg/kg dry mass.

. For organic extracts, methanol had the highest yield (10.6%), followed by cyclohexane (5.4%), dichloromethane (0.7%), and ethyl acetate (0.1%). It is important to note that A. pusilla is an endemic plant, and there are no similar publications concerning its extracts. Nevertheless, this methodology is frequently employed by other research groups. For example, a recent study used organic solvents such as hexane, ethyl acetate, and methanol to extract bioactive compounds from eight plants in the same family (Apiaceae) [13]. The yield of essential oil was 4.3%, whereas in other studies, the essential oil yields were 0.81% and 2.3% [7]. These differences may be due to non-constant soil, climate change conditions, and maturity.

3.2. Chemical Composition of Essential Oil

A total of 19 compounds were identified, representing 99.9% of the essential oil (

Table 2. Main natural constituents of A. pusilla essential oil.

N°	Compound	RI	Percentage (%)
1	tricyclene	924	0.4
2	alpha-thujene	933	0.2
3	sabinene	972	0.7
4	beta-pinene	976	0.2
5	myrcene	988	0.6
6	3-carene	1014	0.5
7	o-cymene	1022	14.4
8	beta-phellandrene	1054	27.4
9	kewda ether	1080	0.2
10	trans-sabinene hydrate	1089	0.2
11	cis-rose oxide	1111	0.1
12	lavandulol	1162	0.7
13	terpinen-4-ol	1176	0.2
14	eugenol	1206	5.5
15	perilla aldehyde	1268	47.5
16	trans-carvyl acetate	1346	0.3
17	alpha-copaene	1377	0.2
18	methyl eugenol	1435	0.4
19	p-butylphenol	1793	0.1
	Total		99.9
	Monoterpene hydrocarbons (%)		42.7
	Oxygenated monoterpenes (%)		54.6
	Sesquiterpenes (%)		0.2
	Others (%)		2.4
	Total identified		99.9

). It consisted of 54.6% of oxygenated monoterpenes, 42.7% of monoterpene hydrocarbons, and 0.2% of sesquiterpenes. The main compounds in this broad range were peril aldehyde (47.5%), beta-phellandrene (27.4%), o-cymene (14.4%), and eugenol (5.5%). Ten compounds: peril aldehyde, tricyclene, 3-carene, kewda ether, cis-rose oxide, lavandulol, trans-carvyl acetate, alpha-copaene, and methyl eugenol were founded for the first time A. pusilla essential oil. Peril aldehyde was the predominant constituent in the essential oil of Perilla frutescens (72.07%).

In a previous study [2], a different composition from that of our sample was revealed, characterized by a predominance of thymol (53.2%), gamma-terpinene (19.4%), and p-cymene (10.6%) as the main constituents of the oil. In contrast, essential oil extracted from the aerial parts of A. pusilla in Morocco showed an abundance of phenolic compounds (48.0%), mainly carvacrol (44.6%) and thymol (3.4%) [6].

A recent study established the composition using the same method, and they found beta-phellandrene at a trace percentage (<0.05%), whereas in our study, it was considered the second major compound. Beta-pinene and alpha-thujene were found at the same percentage (0.2%). Sabinene and trans-sabinene hydrate were founded in trace percentages, whereas we founded them at 0.7 and 0.2%, respectively [8]. Such variations in chemical composition may be due to climatic conditions, geographical origin, and the plant’s stage of development, all of which can have an impact on the chemical composition of its essential oil.

3.3. Profile of the Molecular Composition of A. pusilla Extracts before and after Silylation

The chemical composition of A. pusilla organic extracts with and without silylation has been established for the first time and has never been studied before. Analyses of cyclohexane extract (

Table 3. Chemical composition of A. pusilla aerial part extracts before/after derivatization.

Compounds/Structure	Retention Time (min)	Samples
		Essential Oil	Cyclohexane	Dichloromethane	Ethyl Acetate	Methanol
Before derivatization
o-Cymene	7.20	+	+
Undecane	7.72				+
3-Carene	7.94	+	+
Terpinen-4-ol	10.72	+	+
Perilla aldehyde	12.94	+++	++
Thymol	13.50					+
Cyclopropane, 1 methoxy 2,2-dimethyl-3-(3,3–dimethyl-1-propynyl)	16.34		++
Dodecanal	16.84				+
2-Allyl-1,4-dimethoxybenzene	18.73		+
2-Dodecenal (E)	20.77				+
D-Verbenone X30CDRA01	31.75		+
Thiocarbamic acid, N,N-dimethyl, S-1,3-diphenyl-2-butenyl ester	39.23		+
9-Octadecenoic acid, (2-phenyl-1,3-dioxolan-4-yl)methyl ester, cis	39.75		+
After derivatization
Glycerol	11.42			+	+
Carvacrol	11.90				+
2-methyl-alpha-D-Glucofuranoside	15.91			+
2-Deoxy arabinohexonic acid	16.03			+
2-Deoxy-ribopyranose	16.29			++
2-methyl-beta-D-Mannopyranoside	16.71			+

Note: +++: High presence; ++: average presence; +: low presence.

) demonstrated the presence of nine compounds, of which four were already identified in our essential oil (perilla aldehyde, o-cymene, 3-carene, and terpinen-4-ol). Furthermore, we found in this extract: thiocarbamic acid, N,N-dimethyl-S-1,3-diphenyl-2-butenyl ester, identified in the methanol extract of wild mushroom [14], 9-octadecenoic acid, (2-phenyl-1,3-dioxolan-4-yl)methyl ester, cis, which was already identified in Lavandula coronopifolia essential oil [15], and D-verbenone [16].

In addition, two new compounds (Figure 1), never identified in natural substances, were presented in the cyclohexane extract: 2-allyl-1,4-dimethoxybenzene and cyclopropane-1-methoxy-2,2-dimethyl-3-(3,3–dimethyl-1-propynyl). 2-Dodecenal (E), dodecanal, and undecane were identified in the ethyl acetate extract. Dodecanal and 2-Dodecenal (E) were detected in the essential oil of Coriandrum sativum, belonging to the Apiaceae family [17].

Thymol was identified in methanol extract; it was also identified in the essential oil of A. pusilla [3]. It was found that thymol has not been identified in our essential oil. This may be due to both its low amount and its bioavailability; it was probably trapped in the cells of plants, and we could extract it with the fourth organic solvent.

After the silylation, we identified five compounds in the dichloromethane extract: four compounds were determined for the first time in the literature of natural products: 2-methyl-α-D-glucofuranoside, 2-Deoxy-arabinohexonic acid, 2-deoxy-ribopyranose, and 2-methyl-beta-D-mannopyranoside (Figure 2). We also noted the presence of glycerol in this derivate extract. In the ethyl acetate derivatives extract, we noted the presence of two compounds: 5-isopropyl-2-methylphenoxy (carvacrol) and glycerol. Carvacrol was founded as a major compound in the essential oil of A. pusilla from Morocco [5], at 44.6%. With this method of rapid analysis (without purification) by GC-MS, we identified an interesting number of original compounds.

3.4. Phenolics, Flavonoids, Tannins, and Anthocyanins

Table 1 shows the total phenolic content, flavonoids, tannins, and anthocyanins in extracts from the aerial part of A. pusilla. The highest amount of phenolic compounds was found in the methanol extract (116.6 ± 2.7 mg GAE/g dry weight), followed by the ethyl acetate extract (113.7 ± 2.4 mg GAE/g dry weight). For flavonoids, the methanol extract had the highest content (91.4 ± 2.2 mg EQ/g dry weight), while the ethyl acetate extract contained 47.6 ± 1.0 mg EQ/g dry weight. Total tannin content ranged from 1.6 ± 0.2 to 6.5 ± 0.2 mg/g dry weight catechin equivalent per gram dry weight. In terms of tannins, the cyclohexane extract had the highest concentration (6.5 ± 0.2 CE mg/g dry mass), followed by the dichloromethane extract (6.4 ± 0.4 CE g/kg dry mass). Ethyl acetate extract contained 5.5 ± 0.5 CE mg/g dry mass, while methanolic extract had the lowest concentration at 1.6 ± 0.2 CE mg/g dry mass. Anthocyanin was detected in lower quantities than in the other groups. The cyclohexane extract (1.9 ± 0.0 C3GE mg/kg dry mass) contained the highest concentration of anthocyanin. The chemical composition of several extracts revealed that the aerial part of A. pusilla is extremely rich in phenolic compounds. The total phenolics, tannins, flavonoids, and anthocyanin content of A. pusilla were determined for the first time.

3.5. Quantification of Sugar

The DNS assay gives us an idea of the total reducing sugar in the organic extracts. Results (Table 1) demonstrated that methanol extract was the richest extract in reducing sugar with 15.07 GE mg/g dry mass). Cyclohexane, dichloromethane, and ethyl acetate extracts reducing sugar contents to only 2.74, 1.84, and 4.73 GE mg/g dry mass, respectively. This assay has been established for the first time for A. pusilla aerial organics extracts.

3.6. HPLC Profile of Organic Extract and Comparison with Standards

HPLC analysis conducted on organic extracts revealed the presence of aromatic compounds (probably phenolics). These phytochemical compounds were compared to a standard solution injected under the same conditions. The identification of compounds was not established. This result showed that the phenolic structures present in extracts are little known. Chromatogram (280 nm) profile was mentioned in Figure 3. All the extracts were injected at 20 mg/mL.

Considering the total phenolic content in the cyclohexane extract, the two intense compounds (intensity towards 2500) and the non-polar eluted between 46 and 47 min were phenolic derivatives. Their structures must include a significant apolar group, which explains their late elution. The chromatogram of dichloromethane extract had a low intensity (I < 500). This was correlated with the total phenolics since it showed the lowest quantity. With regard to the ethyl acetate extract, the maximum intensity is 1400, and it has fairly polar (20 and 30 min) and non-polar compounds (40 and 50 min). Finally, the methanol extract gave a chromatogram that saturates in I = 2500 for the polar compound at 7.8 min. The other compounds between 20 and 50 min were also observed in the ethyl acetate extract. Thus, chromatogram intensity and total phenolic compound content in both extracts (ethyl acetate and methanol) were analogous.

3.7. Biological Properties

3.7.1. Anti-Oxidant Activity

By DPPH, methanol extract exhibited the best anti-radical power at 50 µg/mL with a percentage of 78.9 ± 0.2% (Figure 4), followed by ethyl acetate with 77.2 ± 0.5%, then cyclohexane with 56.8 ± 1.9%, and finally dichloromethane with 51.0 ± 4.8%. The essential oil tested at 50 µg/mL had 61.5 ± 1.2% inhibition for the DPPH assay.

The anti-oxidant activity of extracts assayed using the ABTS assay is presented in Figure 4. At 50 µg/mL, the most effective extracts were methanol (78.9 ± 0.2%) and ethyl acetate (77.2 ± 0.5%). Dichloromethane and cyclohexane were less important at 50 µg/mL (51.0 ± 4.8 and 56.8 ± 1.9%, respectively). Essential oil tested at 50 µg/mL had 58.3 ± 2.3% inhibition for the ABTS assay.

As shown in Figure 5, significant correlation coefficients (R²) between ABTS assay data and total phenolics were observed (R² = 0.99). In addition, the correlation coefficient R² between DPPH assay data and total phenolic contents was 0.78. Those results approve previous studies on the significant contribution of phenolics to anti-oxidant assays. Numerous studies have demonstrated that plants with a chemical composition similar to that of A. pusilla possess various biological activities, including anti-oxidant properties [18]. DPPH and ABTS test results show that A. pusilla essential oil and extracts (cyclohexane, dichloromethane, ethyl acetate, and methanol) have a significant capacity to neutralize free radicals, suggesting their potential as sources of natural anti-oxidants.

3.7.2. Anti-Diabetic Activity

In the current study, we evaluated the α-amylase inhibition activity of the essential oil and organic extracts (

Table 4. Summary of biological activities of A. pusilla extracts and essential oil at 50 mg/L.

Activity	Anti-Diabetic		Anti-Alzheimer	Anti-Inflammatory	Anti-XOD	Anti-SOD
	(%)	(IC50)	(%)	(%)	(%)	(%)
Extracts	α-Amylase		AchE	5-Lox
Cyclohexane	17.2 ± 0.1	>50	na	30.0 ± 0.1	na	5.0 ± 2.1
Dichloromethane	na	>50	na	34.3 ± 0.1	na	16.4 ± 0.9
Ethyl acetate	15.2 ± 0.2	>50	na	35.4 ± 0.1	69.0 ± 2.3	30.0 ± 0.5
Methanol	94.2 ± 0.0	25.0 ± 0.1	na	45.0 ± 0.0	54.8 ± 1.0	73.3 ± 0.9
Essential oil		na	63.2 ± 0.2	31.0 ± 0.0 *	69.0 ± 1.7	na
Acarbose *		100.0 ± 0.1
Galantamin *			1.0 ± 0.2
NDGA *				2.5 ± 0.4
Allupurinol *					1.3 ± 0.1

Note: * IC50 (µg/mL); na: not active.

). Methanol extract has an interesting anti-diabetic activity at 50 µg/mL; we obtained 94.2 ± 0.0% and an IC50 in order of 25.0 ± 0.1 µg/mL, more interesting than the positive control, acarbose (IC50 = 100 µg/mL). For cyclohexane and ethyl acetate extracts, they were less active, and we obtained only 17.2 ± 0.1 and 15.2 ± 0.2% inhibition at 50 µg/mL. IC50 were more than 50 mg/L for dichloromethane.

This result proved that A. pusilla extracts have an interesting in vitro anti-diabetic activity, encouraging continued study of that specificity and identifying the compound responsible for that activity. Previous studies have demonstrated that. A. pusilla was traditionally used to treat diabetes and hypertension [19,20]. Methanol extract had the best anti-diabetic activity.

3.7.3. Effects of Samples on SOD and XOD Activities

We studied superoxide dismutase (SOD), a crucial anti-oxidant enzyme that scavenges the superoxide radical (${\mathrm{O}}_2^{-}$). Suggested to play a role in tumor suppression, SOD was mainly found in mitochondria, where most of the ${\mathrm{O}}_2^{-}$ was produced during cellular respiration. We revealed that increased SOD expression in ovarian cancer was a response to intrinsic oxidative stress caused by reactive oxygen species (ROS). As superoxide scavengers, SODs reduce oxidative stress and the stimulatory effect of ROS on cancer cell growth [21]. In our study, most of the tested extracts and essential oils of A. pusilla were slightly active on the SOD inhibition activity, except for the, methanol extract, which obtained 73.3 ± 0.9% at 50 µg/mL, as mentioned in Table 4. Among the extracts assayed for anti-XOD activity against 50 µg/mL, only ethyl acetate and methanol extracts showed any efficacy (69.0 ± 2.3 and 54.8 ± 1.0%, respectively). Likewise, the essential oil was very interesting as an XOD inhibitor, with a percentage of 69.0 ± 1.7%. This activity was established for the first time for this plant and has never been studied before. Methanol extract exhibited both anti-SOD and anti-XOD activities. Methanol extract was chosen due to its polarity and because it was anticipated that it would more effectively extract the polyphenols that possessed anti-oxidant activity. In the literature, a total of eighty-four different extracts from 27 medicinal plants and spices traditionally used against gout in Central and Eastern Europe were tested for XOD inhibition in vitro. Of the total, 25 extracts of 13 species showed inhibition in excess of 50%, while 16 extracts of 9 species showed comparable activity at 100 µg/mL. Moreover, both ethanolic extracts and methanolic extracts based on methylene chloride were the best performers [22].

3.7.4. Anti-AChE Activity

Galantamin is considered the most commonly used natural drug in Alzheimer therapy and acts as an enzyme inhibitor. This substance is isolated from the extract of snowdrops [23]. The anti-AChE activity of A. pusilla aerial part extracts and essential oils was evaluated. Only the essential oil exhibited anti-AChE activity with 63.23 ± 0.2% values at 50 µg/mL, and all the extracts were ineffective at this concentration (Table 4). To our knowledge, the anti-AChE activity of A. pusilla essential oil is established here for the first time.

3.7.5. Anti-Inflammatory Activity

5-Lox inhibition activity was evaluated for A. pusilla in Table 4. Essential oil was effective with an IC50 = 31.0 ± 0.0 µg/mL. This value of essential oil (compound mixture) was also comparable with the NDGA reference drug (2.5 ± 0.07 µg/mL). We outlined that most of the solvent extracts allowed us to obtain less than 50% inhibitory activity at a concentration of 50 µg/mL (IC50 > 50 µg/mL). Methanol extract showed the highest anti-inflammatory activity (45%). This suggests that the highest anti-inflammatory activity was achieved by the best anti-oxidant extracts, which contain the highest levels of total phenolics. In the literature, phenolics are considered good Lox inhibitors [24]. Anti-inflammatory in vitro activity was established for the first time in this study.

3.7.6. Cytotoxic Activity of Extracts

Four cancer cell lines, MCF-7, HCT116, IGROV-1, and OVCAR-3, were evaluated using the MTT test, which reliably detects cell proliferation. In this study, the cytotoxic activities of all the extracts were evaluated at 50 µg/mL, and the IC50 was calculated from the mean of the data (

Table 5. In vitro cytotoxic activity of A. pusilla extracts at 50 µg/mL.

Extracts	HCT116		MCF7		OVCAR-3		I-GROV1
	%	IC50	%	IC50	%	IC50	%	IC50
Cyclohexane	na	>50	101.1 ± 1.6	20.0 ± 0.2	92.1 ± 2.4	4.0 ± 0.1	96.3 ± 1.3	15.0 ± 0.2
Dichloromethane	na	>50	99.1 ± 1.5	24.1 ± 0.4	91.3± 4.1	24.0 ± 0.1	91.6 ± 1.7	16.0 ± 0.1
Ethyl acetate	na	>50	94.2 ± 4.0	20.9 ± 0.1	93.3 ± 6.3	23.9 ± 0.1	93.3 ± 2.1	25.0 ± 0.1
Methanol	94.2 ± 2.3	20.9 ± 0.2	95.4 ± 3.6	19.53 ± 0.7	92.3 ± 6.3	24.8 ± 0.1	92.3 ± 2.1	20.8 ± 0.1
Tamoxifen		1.0 ± 0.2		1.0 ± 0.1		1.4 ± 0.3		2.2 ± 0.3

Note: na: not active.

), established in triplicate.

For HCT116, only methanol extract was effective against this line cancer cell (IC50 = 20.9 ± 0.2 µg/mL) and was allowed to inhibit 94.2% of the total cells at 50 µg/mL. For MCF7, all extracts were active, and the IC50 varied from 19.53 to 24.1 µg/mL. Furthermore, all four organic extracts listed allowed inhibition of OVCAR-3, with IC50 values equal to 4.0 ± 0.1, 24.0 ± 0.1, 23.9 ± 0.1, and 24.8 ± 0.1 µg/mL, respectively. Also, for IGROV-1, all tested extracts were active to inhibit their growth, and their IC50 were 15.0 ± 0.2, 16.0 ± 0.1, 25.0 ± 0.1, and 20.8 ± 0.1 mg/L, respectively. We observed that A. pusilla aerial part extracts have different effects on each line of cancer. The study of anti-cancer A. pusilla property has never been cited before in the literature. It is possible that phenolic compounds, specifically flavonoid and tannin, in the aerial part of A. pusilla might be responsible for the cytotoxic activity of organic extracts against HCT116, MCF7, OVCAR-3, and IGROV-1. The better IC50 value observed in cyclohexane extract with 4.0 ± 0.1 µg/mL indicates that the anti-cancer agent has a stronger cytotoxic effect against the ovarian cell line (OVCAR-3).

3.8. Principal Components Analysis (PCA)

Total phenolics, flavonoids, total condensed tannins, total anthocyanin contents, reducing sugar, and biological activity contents of A. pusilla extracts were also analyzed according to PCA (

Table 6. Variable factor contribution to the principal components analysis (%).

	F1	F2	F3
Phenolics	12.249	0.000	0.059
Flavonoids	8.685	3.787	9.496
Tannins	6.790	8.807	2.476
Anthocyanins	4.150	5.458	35.995
Reducing sugar	2.899	15.799	0.983
Cytotoxicity/HCT116	1.026	17.233	8.720
Cytotoxicity/MCF7	12.272	0.142	0.315
Cytotoxicity/OVAR	12.167	0.049	1.699
Cytotoxicity/IGROV	12.353	0.000	0.594
Antidiabetic	1.026	17.233	8.720
Anti-alzheimer	12.263	0.002	0.682
Anti-inflamatory	8.176	6.784	1.218
Anti-XOD	3.231	8.678	29.017
Anti-SOD	2.714	16.027	0.027

). All axes of measurement were excluded from the results obtained.

As indicated in Figure 6, the overall variation was 91.51%. PC1 and PC2 axes were responsible for 57.40% and 34.11% of the variability, respectively.

The loadings in the principal component analysis (PCA) diagram represent both the correlation of the principal components against the original variables and the corresponding correlations between various activities: total phenolics, flavonoids, total condensed tannins, total anthocyanins, and reducing sugar.

Referring to the data in

Table 7. Correlations among variables and factors.

	F1	F2	F3
Phenolics	0.992	−0.002	−0.025
Flavonoids	0.835	0.425	−0.321
Tannins	0.739	−0.649	−0.164
Anthocyanins	0.577	−0.511	0.625
Reducing sugar	0.483	0.869	0.103
Cytotoxicity/HCT116	0.287	0.907	0.307
Cytotoxicity/MCF7	0.993	−0.082	−0.058
Cytotoxicity/OVAR	0.989	−0.048	−0.136
Cytotoxicity/IGROV	0.996	0.001	−0.080
Anti-diabetic	0.287	0.907	0.307
Anti-alzheimer	−0.993	0.010	0.086
Anti-inflamatory	−0.811	0.569	0.115
Anti-XOD	−0.510	0.644	−0.561
Anti-SOD	0.467	0.875	−0.017

, many correlations were identified. The first principal component (PC1) is strongly correlated with total phenolics, total condensed tannins, and total anthocyanins, as well as with cytotoxic activity against IGROV cells (r = 0.996), MCF7 cells (r = 0.993), and OVAR cells (r = 0.989). Furthermore, the second principal component (PC2) correlated well with cytotoxic activity against HCT116 (load = 0.907), anti-diabetic activity (load = 0.907), and anti-SOD activity (load = 0.875). For the third principal component (PC3), a correlation was observed with anti-Alzheimer’s (load = 0.086) and anti-inflammatory (load = 0.115) activities, although these loads were moderate.

In Figure 7, the Biplot shows the position of extracts according to their content of total phenols, flavonoids, total condensed tannins, total anthocyanins, reducing sugars, and biological activities, as known from their specific chemical profiles. The results show that the methanolic extract exhibits strong anti-DPPH activity, attributable to its richness in TPCs [11], underscoring a significant correlation between the availability of phenolic compounds and anti-oxidant activity.

Furthermore, CYHA, EtOAc, and DCM extracts showed cytotoxic activity against IGROV, MCF7, and OVAR cells, suggesting the presence of other constituents such as fatty acids, known for their cytotoxic activity in the literature. A significant correlation was also observed between A. pusilla essential oil (EO) and anti-Alzheimer’s activity.

4. Conclusions

In the present study, we investigated the chemical composition, anti-oxidant, anti-inflammatory, anti-SOD, anti-XOD, anti-diabetic, and cytotoxic effects (HCT116, MCF7, IGROV-1, and OVCAR-3 cell lines) of essential oil and organic extracts of A. pusilla aerial part. The chemical composition of organic extracts demonstrated the presence of thiocarbamic acid, N, N-dimethyl, S-1,3-diphenyl-2-butenyl ester, 9-Octadecenoic acid, (2-phenyl-1,3-dioxolan-4-yl) methyl ester, and cis. D-verbenone, identified in the essential oils of thyme and rosemary and having interesting anti-oxidant and anti-microbial activities, and was also identified here for the first time in A. pusilla and in the Ammoides genre. An interesting anti-diabetic activity was observed in methanol extract at 50 µg/mL; we obtained 94.2 ± 0.0% and an IC50 = 25.0 ± 0.1 µg/mL, four times more active than acarbose (IC50 = 100.0 ± 0.1 µg/mL). The study of the anti-cancer activity of A. pusilla was cited for the first time. The better IC50 value was observed in cyclohexane extract with 4 ± 0.1 µg/mL against the ovarian cell line (OVCAR-3). It will be interesting to discover the molecules responsible for all good activities, with applications in the pharmaceutical industry and further in vivo experiments. For the molecules responsible for these activities, it will be interesting to optimize their extractions using a process/green solvent pair (supercritical CO₂ without or with solvent, Accelerated Solvent Extraction (ASE), water, ethanol, etc.). These subsequent steps will focus on developing environmentally friendly extraction technologies for possible therapeutic uses.