Received: 11/04/2025 Accepted: 24/05/2025 Published: 08/06/2025 1 of 9 https://doi.org/10.52973/rcfcv-e35674 Revista Científica, FCV-LUZ / Vol. XXXV ABSTRACT Origanum majorana L. (Lamiaceae) has been prescribed in folk medicine for therapeutic purposes such as cancer, asthma, dizziness, rheumatism and inflammatory diseases. To evaluate for the first time the decoction (DecE) and hydro–ethanolic (HEE) extracts of the aerial part of O. majorana. The phytochemical analysis (LC–MS/MS), antioxidant, anti–inflammatory and analgesic potentials were analysed. LC–MS/MS analysis identified 17 components in the extracts. The main compounds detected in the aerial parts of O. majorana include rosmarinic acid (≥ 50%), elagic acid (≥ 20%), indomethacin along with flavonoids, phenolics, benzopyrone, hydroquinones, indolyl acetic acid. Both extracts showed potent antioxidant effects in various antioxidant assay models. Through the eggs protein denaturation assay, both plant extracts showed high in vitro anti–inflammatory effect of the order of 86.91 ± 0.00% for DecE and 85.04 ± 0.00 % for HEE (1.4 mg·mL -1 ). Acute toxicity measurement revealed no mortality or behavioral changes during the testing period, confirming the two extracts not toxic with an LD50 > 2 g·kg -1 . The in vivo anti–inflammatory effects were found to be statistically significant (P<0.05), reducing the edematous response by 78.40 ± 2.52 % and 77.36 ± 3.88 % in DecE and HEE, respectively. Additionally, at a dose of 400.0 mg·kg -1 , both extracts exhibited significant analgesics activity (P<0.05) against acetic acid induced abdominal constriction in mice, with inhibition rates of 78.54 ± 3.30% for DecE and 66.99 ± 1.34% for HEE. The results indicate aerial part extracts of O. majorana have potent anti–inflammatory effects and may prove to have potential health benefits. Key words: Origanum majorana L; phytochemical analysis; toxicity; anti–inflammatory activity; analgesic activity RESUMEN Origanum majorana L. (Lamiaceae) se ha prescrito en la medicina popular con fines terapéuticos para tratar cáncer, el asma, el mareo, el reumatismo y enfermedades inflamatorias. Aquí se evalúa por primera vez los extractos de decocción (DecE) e hidroetanólico (HEE) de la parte aérea de la O. majorana. Se analizó el potencial antioxidante, antiinflamatorio y analgésico mediante análisis fitoquímico (LC–MS/MS). El análisis LC–MS/ MS identificó 17 componentes en los extractos. Los principales compuestos detectados en la parte aérea de O. majorana incluyen ácido rosmarínico (≥ 50 %), ácido elágico (≥ 20 %), indometacina, flavonoides, compuestos fenólicos, benzopirona, hidroquinonas y ácido indolil acético. Ambos extractos mostraron potentes efectos antioxidantes en diversos modelos de ensayo antioxidante. Mediante el ensayo de desnaturalización de proteínas de huevo, ambos extractos vegetales mostraron un alto efecto antiinflamatorio in vitro, del orden del 86.91 ± 0.00 % para DecE y del 85.04 ± 0.00 % para HEE (1.4 mg·mL -1 ). La medición de toxicidad aguda no reveló mortalidad ni cambios en el comportamiento durante el período de prueba, lo que confirmó la no toxicidad de ambos extractos con una DL50 > 2 g·kg -1 . Los efectos antiinflamatorios in vivo resultaron estadísticamente significativos (P<0.05), reduciendo la respuesta edematosa en un 78.40 ± 2.52 % y un 77.36 ± 3.88 % en DecE y HEE, respectivamente. Además, a una dosis de 400,0 mg·kg -1 , ambos extractos mostraron una actividad analgésica significativa (P<0.05) contra la constricción abdominal inducida por ácido acético en ratones, con índices de inhibición del 78.54 ± 3.30 % para DecE y del 66.99 ± 1.34 % para HEE. Los resultados indican que los extractos de la parte aérea de O. majorana tienen potentes efectos antiinflamatorios y podrían tener beneficios potenciales para la salud. Palabras clave: Origanum majorana L; análisis fitoquímico; toxicidad; actividad antiinflamatoria; actividad analgésica Liquid chromatography–mass spectrometry phytochemical analysis, antioxidant, anti–inflammatory and analgesic efficacy of Origanum majorana L. aerial part extracts collected from North–Eastern of Algeria Análisis fitoquímico, cromatografía / espectrometría de masas, eficacia antioxidante, antiinflamatoria y analgésica de extractos de partes aéreas de Origanum majorana L. recolectados en el noreste de Argelia Ahlem Karbab 1 * , Noureddine Charef 1 , Salima Amari 1 , Areej Jaber 2 , Ayat Siedat 3 , Alaa Sanabrah 2 , Solomon Derese 4 1 Setif-1 University Ferhat Abbas, Faculty of Natural and Life Sciences, Laboratory of Applied Biochemistry. Setif-1, Algeria. 2 Al–Ahliyya Amman University, Faculty of Pharmacy, Pharmacological and Diagnostic Research Center. Amman, Jordan. 3 University of Jordan, Department of Chemistry. Amman, Jordan. 4 University of Nairobi, Department of Chemistry. Nairobi, Kenya. *Corresponding author: ahlem.karbab@univ–setif.dz, karbabal2@gmail.com

LC-MS/MS phytochemical analysis of Origanum majorana L./ Karbab et al.____________________________________________________________ 2 of 9 INTRODUCTION Oxidative stress arises from an imbalance between antioxidant defense systems and pro–oxidants factors [1], whether due to a deficit in defense mechanisms such as antioxidant compounds and antioxidant enzymes [2] or excessive production of free radicals [3]. An antioxidant and anti–inflammatory drugs can prevent or inhibit the generation of toxic oxidants, scavenge those that are already produced, thereby blocking the spreading chain reaction produced by these oxidants [4, 5]. Plants have served as a primary source of medicine, with approximately 80% of the global population relying on herbal remedies for various health issues. The traditional knowledge of plant use varies across different regions, underscoring the importance of documenting this information to preserve it for future generations [6]. In recent years, many pharmaceutical companies have renewed their focus on natural product research, leveraging ethnopharmacological studies to uncover their potential as promising sources of new compounds for therapeutic development [7, 8]. Many of these phytochemicals isolated from plants have potential for drug development, and may act additively, individually, or synergistically to improve health. In contrast, synthetic antioxidants have recently faced approval issues in several developed countries, leading to increased interest in natural anti–inflammatory and antioxidants. This trend has spurred efforts to discover new and beneficial antioxidant and anti–inflammatory agents from medicinal plants [7, 8]. Locally, O. majorana is known as Merdeqouch, Arzema or M’loul. This plant contains a high concentration of beneficial substances such as flavonoids, phenolic compounds, cinnamic acid derivatives and flavanones. The components of the plant are being employed in many impoverished nations as a remedy for treatment of a variety of chronic conditions. In folk medicine, is used to treat indigestion, asthma, headache, cramps, depression, rheumatism, and is also considered for its diuretic activity [9]. Traditionally, marjoram leaves are used to manage diabetes [10]. Various parts of plants have been harnessed for therapeutic purposes [5, 11]. Food manufacturers and researchers are increasingly focusing on whole plants and plant parts that contain bioactive phytochemicals in order to develop pharmaceuticals and functional foods. However, there is a paucity of information regarding the phytochemical analysis, antioxidant, anti–inflammatory and analgesic potentials in aerial parts of Merdeqouch plants. This knowledge gap prompted the current study. Furthermore, the phytochemical composition, bioactivity and toxicity of aerial part of O. majorana remain poorly documented and warrant further study. To date, only a few scientific reports that they are aware have investigated the phytochemical composition, antioxidant, toxicity, anti–inflammatory and analgesic activities and the safe dose of decoction and hydro–ethanolic extracts of the aerial parts of O. majorana. Additionally, there is need to determine whether its use in traditional medicine for inflammation is justified. MATERIALS AND METHODS Plant collection and identification Fresh aerial parts of O. majorana were obtained from Bouandas– Setif located in North–Eastern Algeria (5°06′07″ east longitude and 36°29′41″ N) in March–May 2022 during the flowering stage. The aerial parts were air–dried in the shade, then crushed and pulverized with an electric grinder (Germany). The medicinal herb utilized in this investigation was O. majorana, which belongs to the Lamiaceae family. Prof. Laouer H., a renowned taxonomist at Setif-1 University, Algeria, authenticated and identified the plant under a voucher specimen (060/DBEV/UFA/22). Animals Adult female albino mice (Mus musculus) weighing (Aniphy balance) 25.0–30.0 g were used. The animals were procured from Algiers’ ‘Institut Pasteur d’Algérie’. Mice were housed in cages with a 12:12 light/dark cycle at 25.0 ± 1.0°C for 7 days (d) before to the study. They had unlimited access to water and a regular diet and were kept in compliance with Animals By–Laws No. 425 [12]. Extracts preparation Preparation of aqueous extract The plant extracts were obtained using established decoction method [13]. To prepare the decoction extract, 100.0 g of dried aerial parts of the plant were boiled in 1 L of distilled water for 20 min. The resulting extract was then filtered before centrifugation at 3000 G (Sigma 3-30K, Germany) for 20 min. This dried decoction extract (DecE) was then tested for pharmacological characteristics. Preparation of hydroethanolic extract The hydroethanolic extract (HEE) of the plant was prepared from 100 g of ground aerial part. The powder was soaked in 1 L of ethanol/ water (70/30, v/v) and continuously shaken for 48 h. Then the solution was filtered and the solvent was recovered by evaporation in a rotavapor (Buchi rotavap R-205, Switzeland), at a temperature of 45°C. The dried hydro–ethanolic extract was preserved in 4°C. Determination of total polyphenol, flavonoids and hydrolysable tannins The total phenolic content of the extract was assessed using the Folin–Ciocalteu’s technique [14]. The polyphenol concentration was expressed as microgram (µg) of gallic acid equivalent (GE) per gram (g) of dry extract, with quantification based on a gallic acid standard curve (ranging from 0.00 to 160 µg·mL -1 ). The total flavonoid content was determined using the aluminum chloride method [7]. The total flavonoids were reported as µg of quercetin equivalent (QE) per mg dried extract (DE). The total flavonoids in the extracts were evaluated using a quercetine standard curve (0.00 to 40 µg·mL -1 ). To determine hemoglobin precipitation by tannins, they utilized the method indicated below [15]. The supernatant absorbance was measured at 576 nm, and the precipitation efficiency of the investigated solutions was reported as µg tannic acid equivalent (TE)·mg -1 dried extract. To generate the calibration curve, equal parts hemolyzed blood and tannic acid (50-600 µg·mL -1 ) were combined.

_________________________________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol.XXXV 3 of 9 Liquid chromatography–mass spectrometry (LC–MS/MS) phytochemical analysis The analysis of some selected flavonoids and phenolic acids was performed on AB Sciex QTRAP 4500 LC–MS/MS (Japan), utilizing Hypersil GOLD Dia (100 mm × 4 mm, particle size 3 µm, column oven 40°C). The ethanolic and aqueous extracts were freshly prepared (as 1000 µg·mL -1 of each sample separately) by dissolving them in ethanol (99% absolute) and deionized water, respectively. The samples were filtrated by nylon syringe filter (0.45 µm), and 5 µL of each was injected into the QTRAP 4500 system using the developed method. The flow rate was 0.3500 mL·min –1 and the mobile phase consisted of 0.1% formic acid in deionized water and, delivered in gradient elution mode. Sample solutions were produced and promptly analysed for phenolic acids and flavonoids using LC–MS/MS. A stock solution consisting of the screened flavonoids and phenolic acids (17 Std: 3,5–dihydroxy–benzoic acid, salicylic acid, caffeic acid, coumarin, ferulic acid, vanillin, 4–hydroxy benzoic acid, chlorogenic acid, rutin, elagic acid, arbutin, trans–2–hydroxy cinnamic acid, P–commaric acid, 3,4–dihydroxy benzoic acid, naringenin, indomethacin, rosmarinic acid) were prepared in HPLC–grade ethanol. Quantitative data for the phenolic compounds were obtained by comparing the samples to the injected mixed phenolic standards. In vitro anti–oxidant activity DPPH radical scavenging assays The free radical scavenging capacity of the extracts were evaluated using the DPPH assay (2,2’–diphenyl–1–picrylhydrazyl) [13]. Absorbance was measured at 517 nm and the scavenging capacity was calculated with the following equation: Where Ab the absorbance of the blank and At the absorbance of the tested sample. Reducing power assay The reducing power of O. majorana extracts was estimated based on their ability to reduce Fe +3 to Fe +2 ions [16]. After ten minutes of incubation, the colour intensity was assessed at 700 nm. In this assay, a higher value of absorbance of the solution indicates a greater reducing power of the extract. In vivo assays Acute toxicity The acute oral toxicity of DecE and HEE was tested in mice in accordance with OECD guidelines [12]. The mice were divided into 3 groups, each consisting of five animals. After 12 h of fasting, the mice were orally administered with a single dose of the extracts (2.0 g·kg -1 ) body weight. The control group received just distilled water. The mice were closely monitored for any indication of toxicity during the first hour of post treatment and regularly over the next 24 h. Subsequently, daily observations were conducted for 14 d to detect any delayed toxic effects. Xylene–induced ear edema assay The oral anti–edema effect was also investigated employing xylene–induced ear edema assay [15]. The mice were divided into groups of six. One hour after oral administration of varying doses of DecE and HEE (50 and 200 mg·kg -1 ), indomethacin (50 mg·kg -1 ) and distilled water (negative control), edematous was locally provoked in mice ears by applying 30 µL of xylene. A digital caliper (caliper to DIN 862, Germany) was used to measure ear swelling both before and 2 h after edema induction. Analgesic activity The analgesic efficacy of the extracts was estimated by induction of abdomen contraction in mice using acetic acid [15]. The mice were divided into groups of five. The negative control group received distilled water, whereas the positive control was administered aspirin (100 mg·kg -1 ). The test groups were administered with extracts at dosages of 200 and 300 mg·kg -1 . Abdominal writhing was caused intraperitoneally by injecting 0.1% acetic acid and 60 minutes post–treatment for all groups. The number of writhes was recorded for each group, starting 5 min after acetic acid injection and continuing for 30 min. The percentage inhibition of the writhing response was determined by applying the following equation: where Cn and Ct are the mean of constriction’ count in mice in the negative control and the treated groups with different concentrations of extracts or aspirin. Statistical analysis Results from in vitro and in vivo were analyzed using one– way ANOVA to determine significance. Data were generated in triplicate and are presented as the mean ± standard deviation. (SD). GraphPad Prism-5 was used to analyze the data collected during this experiment. Significant differences were defined as P<0.05. RESULTS AND DISCUSSION Extract yield Origanum majorana L. was extracted using decoction and maceration methods. The extraction yields were calculated, with the HEE showing the extraction yielding 18.04 ± 1.24%, while the DecE had a yield of 17.42 ± 1.87%. The yields of extracts reported here for the aerial part extracts were analogous to leaves extract obtained by Qnais et al. [17]. The biological evaluation of O. majorana extracts is essential for determining their health– promoting qualities, underscoring the importance of optimizing extraction conditions [18]. The extracts analyzed in this study contained concentrated active principles, likely due to prior extraction with non–polar solvents or at high temperatures [19]. Quantitative phytochemical analysis Total polyphenols, flavonoids and tannins contents Quantitative tests were performed to determine the concentration of the phytochemicals in the extracts. The levels of polyphenols,

LC-MS/MS phytochemical analysis of Origanum majorana L./ Karbab et al.____________________________________________________________ 4 of 9 flavonoids and tannins were quantified using spectrometric methods. Specifically, the total phenolic content (TPC), total flavonoids content (TFC) and total condensed tannins content (CTC) of different Origanum majorana L. hydro–ethanolic and decoction extracts were evaluated employing the Folin–Ciocalteu’s reagent, aluminium chloride and haemoglobin precipitation methods, respectively. The results are presented in TABLE I. Based on the findings, it is evident that the hydroethanolic extract exhibited the highest concentrations of total phenolics, flavonoids and tannins with values of 302.86 ± 3.90 µg·GE -1 ; 10.69 ± 0.60 µg·GE -1 and 103.87 ± 3.06 µg TE·mg DE -1 , respectively. LC–MS/MS analysis Seventeen phenolic compounds, including phenolics and flavonoids, which are widespread in edible plants, were analyzed in phenolic–rich the hydro–ethanolic and decoction extracts (TABLE II). Based on the LC–MS/MS results from the current study, the hydroethanolic and decoction extracts of O. majorana had very rich phenolic content primarily due to their significant concentrations of rosmarinic acid, elagic acid and other flavonoids and phenolic compounds. Rosmarinic acid was identified as the predominant compound in both extracts, with HEE and DecE showing respective relative areas of 52.46 and 50.16%. Ellagic acid was the second most abundant compound, with an area of 21.05 in DecE and 19.09% in HEE. Notably, 4–hydroxy benzoic acid was not detected in the DecE, unlike in the HEE, (TABLE II). The present findings revealed the presence 17 phytochemicals in the two extracts: 3,5–dihydroxy–benzoic acid, salicylic acid, caffeic acid, coumarin, ferulic acid, vanillin, chlorogenic acid, rutin, ellagic acid, arbutin, trans–2–hydroxy cinnamic acid, p–commaric acid, 3,4–dihydroxy benzoic acid, naringenin, indomethacin, rosmarinic acid.Notably, 4–hydroxybenzoic acid which was present only in HEE. In contrast to the present results, Çarıkçı et al. [23] reported the absence of vanillin and hydroxy benzoic acid in the different extracts from the aerial part of O majorana. Conversely, compounds such as caffeic acid, gallic acid, rosmarinic acid, and chlorogenic acid were present in some extracts by other studies. Additionally, prior research has identified several phenolics and flavonoids from the ethyl acetate extract of the aerial parts of O. majorana, including hesperetin, rosmarinic acid, 5, 6, 3′–Trihydroxy–7,8,4′–trimethoxyflavone, hydroquinone, and arbutin [24]. Furthermore, Amaghnouje et al. [25] revealed the presence of 12 phytochemicals in the leaves of O. majorana: rosmarinic acid, arbutin, quercetin–3–O–glucoside, ursolic acid, luteolin–7–O–glucoside, quercetin–7–O–glucuronic acid, kaempferol–3–0–pentose, kaempferol–3–0–glucuronic acid, catechin, caffeic acid, rutin and quercetin. The genus Origanum serves as an excellent source for isolating a wide range of bioactive compounds. Consequently, this genus exhibits significant biological activities and holds potential for addressing a variety of ailments. Phenols, flavonoids, and tannins are among the major phytochemicals responsible for antioxidant activity and a variety of biological activities. It is worth noting that phenolics were found to be the most abundant phytoconstituents measured in this study, followed by flavonoids and tannins in HEE when compared to the DecE extracts. The total phenolic content (TPC) of the aerial parts extracts obtained in this study was higher than values reported in previous studies on two oregano leaves [17, 18, 20], aerial part extracts [21] and seeds extracts reported by Dhull et al. [22]. Similarly, the total tannin content (TC) of both extracts in this study exceeded that of seeds extracts from O. majorana reported by Dhull et al. [22]. TABLE I Total polyphenols, flavonoids and tannins contents of Origanum majorana L. extracts Extracts Total phenolic content (a) Total flavonoids content (b) Tannins content(c) DecE 283.84 ± 3.18 7.23 ± 0.37 74.14 ± 0.93 HEE 302.86 ± 3.90 10.69 ± 0.60 103.87 ± 3.06 (a): gallic acid equivalent (GE µg) per mg dried extract (DE), (b): quercetin equivalent (QE µg) per mg dried extract (DE), (c): tannic acid equivalent (TE µg) per mg dried extract (DE). DecE: Decoction extract, HEE: hydroethanol extract. TABLE II LC–MS/MS data for phenolic and flavonoids compounds detected in the decoction and hydro–ethanolic aerial part extracts of Origanum majorana L. and their percentage N° Compounds Chemical Class Molecular Formula Structure Rt (min) Area % DecE HEE DecE HEE 1 3,5-dihydroxy– benzoic acid Phenolic acid C7H6O4 4.756 4.759 4.31 3.56 2 Salicylic acid Phenolic acid C₇H₆O₃ 3.292 3.309 3.31 2.390 3 Caffeic acid Phenolic acid C9H8O4 10.060 9.986 1.34 1.53

_________________________________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol.XXXV 5 of 9 4 Coumarin Benzopyrone C9H6O2 8.861 8.774 3.08 3.24 5 Ferulic acid Flavonoid C10H10O4 8.304 8.292 1.52 2.67 6 Vanillin Phenolic acid C8H8O3 7.319 7.285 2.57 3.17 7 4-hydroxy benzoic acid Hydroxybenzoic acid derivatives C7H6O3 ND 6.194 ND 1.44 8 Chlorogenic acid Phenolic acid C16H18O9 6.161 6.157 7.46×10 -1 1.55 9 Rutin Flavonoid C27H30O16 8.675 8.670 1.64 4.06 10 Elagic acid Tannins C14H6O8 11.997 11.980 21.05 19.09 11 Arbutin Hydroquinone C12H16O7 11.576 11.583 4.03 3.90×10 -1 12 Trans-2-hydroxy cinnamic acid Phenylpropanoids C9H8O3 9.759 9.751 2.11×10 -1 2.45×10 -1 13 p–Commaric acid Phenolic acid C9H8O3 7.959 7.953 01.30 01.29 14 3,4-dihydroxy benzoic acid Phenolic acid C7H6O4 29.189 29.184 06.69*10 -1 06.61*10 -1 15 Naringenin Flavonoid C15H12O5 11.576 11.581 03.26 03.27*10 -1 16 Indomethacin Indolyl acetic acid C19H16ClNO 12.699 12.653 02.13*10 -2 7.28*10 -5 17 Rosmarinic acid Phenolic acid C18H16O8 8.681 8.680 50.16 52.46 DecE: Decoction extract, HEE: hydroethanol extract, ND: Not detected TABLE II cont... LC–MS/MS data for phenolic and flavonoids compounds detected in the decoction and hydro–ethanolic aerial part extracts of Origanum majorana L. and their percentage

LC-MS/MS phytochemical analysis of Origanum majorana L./ Karbab et al.____________________________________________________________ 6 of 9 Anti–oxidant effects DPPH radical scavenging activity Many plant extracts exhibit significant antioxidant properties due to the presence of phytoconstituents such as flavonoids and phenolic acids. The antioxidant activity of the hydroethanolic (HEE) and decoction extracts (DecE) from the aerial part of O. majorana was evaluated against the DPPH assay, with absorbance measured spectrophotometrically at 517 nm. As shown in TABLE III, both HEE and DecE demonstrated high radical scavenging activity with IC50 values of 24.55 ± 0.00 and 26.06 ± 0.00 µg·mL -1 , respectively compared to the standard. This strong antioxidant activity can be attributed to the high contents of rosmarinic acid (≥50%) and elagic acid (≥20%). Ellagic acid is a potent bioactive compound with numerous industrial and pharmacological uses [26]. exhibited lesser reduction power, in agreement with the current study. By making the conversion or equivalences in the reported units of expression, comparisons can be made, as the majority of the studies has been documented results in terms of absorbance values. Despite this, all extracts demonstrated potential reducing power, likely due to the presence of oxidizable components. In vivo studies Acute toxicity The present results revealed that oral administration of DecE and HEE from O. majorana did not cause any mortality in the treated mice over the 14 d experimental period. Furthermore, no behavioural changes or visible signs of acute toxicity were observed. Consequently, the LD50 of both extracts was determined to exceed 2 g·kg -1 of body weight for mice. Similarly, Qnais et al. [17] reported that a single oral administration of methanol extract had an LD50, higher than 2 g·kg -1 . In addition, it has been reported that methanolic extract of O. majorana leaves is safe and shows no toxicity at the tested doses [25]. However, the administration of high doses of O. majorana extracts should be approached with caution. Xylene–induced ear edema activity The results of this investigation demonstrated that DecE and HEE, at dosages of 50 and 200 mg·kg -1 exhibit strong anti–edema activity in the xylene–induced ear edema assay, as illustrated in FIG. 1. Both extracts exerted significant (P<0.05) and dose–dependent inhibitory effects against the edema reaction produced by xylene at the tested concentrations (50 and 200 mg·kg -1 ). Additionally, the current findings indicated that DecE and HEE from the aerial part of O. majorana, administered at a dose of 200 mg·kg -1 , reduces the edema response by 78.40 ± 2.52 and 77.36 ± 3.88%, respectively. These results were comparable to the standard non–steroidal anti– inflammatory drug indomethacin, which exhibited an inhibition rate of 79.01 ± 6.36% at a dose of 50 mg·kg -1 . The current findings show that DecE and HEE obtained from the aerial part of O. majorana administered at a dose of 200 mg·kg -1 , In agreement with our findings on DPPH and ABTS assays, Erenler et al. [24] reported that hydroquinone, hesperetin, rosmarinic acid and arbutin from O. majorana exhibited a potent antioxidant in of ABTS •+ , DPPH • , and reducing capacities assays. Similarly, Vasudeva et al. [27] reported findings consistent with the present DPPH results, showing that ethanol extracts of root and stem of O. majorana also showed high scavenging activity, with an IC50 values of 21.05 µg·mL -1 and 84.98 µg·mL -1 , respectively. Possibly, the high content of rosmarinic acid is the phenolic acid that contributes to the greater antioxidant activity in the hydromethanolic extract of O. majorana, as evaluated using FRAP and DPPH [28]. In contrast, Vallverdú–Queralt et al. [29] identified protocatechuic acid, syringic acid, and caffeic acid as the primary components in a hydro–ethanolic leaves extract of O. majorana, but did not detect rosmarinic acid. Reducing power assay TABLE III depicts the antioxidant activity curves for extracts with reducing properties. In this experiment, all extracts indicated the capacity to donate electrons, reducing Fe 3+ to Fe 2+ . Among the tested extracts, HEE showed the highest reducing potential with an IC50 = 29.50 ± 0.00 µg·mL -1 followed by DecE, with an IC50 of value of 39.43 ± 0.00 µg·mL -1 , respectively. The reducing power of the extracts serves as a strong indicator of their antioxidant activity. Reductions complete the free radical chain reaction by providing hydrogen atoms to the radical molecules. Vasudeva et al. [27] found that the ethanol extracts of the root and stem of O. majorana TABLE III Antioxidant activity of decoction and hydroethanolic Origanum majorana extracts Antioxidant activity / Inhibition concentration (IC50) Extracts / standards DPPH radical scavenging Reducing power DecE 26.06 ± 0.00 *** 39.43 ± 0.00 *** HEE 24.55 ± 0.00 *** 29.50 ± 0.00 *** BHT 87.26 ± 0.00 ND Vitamin C ND 21. 91 ± 0.48 DecE: Decoction extract, HEE: hydroethanol extract Indom 50 HEE 50 HEE 200 DecE 50 DecE 200 0 20 40 60 80 100 Inhibition (%) FIGURE 1. Antiinflammatory activity of DecE and HEE by xylene–induced ear edema in mice. Indom 50: Indomethacin: 50 mg·kg -1 , HEE 50 / HEE 200: Hydroethanol extract, 50 mg·kg -1 / 200 mg·kg -1 respectively. DecE 50 / DecE 200: Decoction extract, 50 mg·kg -1 / 200 mg·kg -1 respectively. The results were expressed as means ± SEM, n= 6

_________________________________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol.XXXV 7 of 9 significantly reduced the edema response by more than 75% compared to the nonsteroidal antiinflammatory drug indomethacin (79.01%). In contrast, Ravishankar et al. [30] reported lower maximal inhibition (<40%) for O. majorana fractions using the carrageenan– induced inflammation model. The anti–inflammatory effect of indomethacin can be attributed to its ability to reduce the production of pro–inflammatory prostaglandins. A variety of flavonoids with diverse chemical structures have been associated with different antiinflammatory mechanistic actions [31]. Additionally, natural products have shown potential in reducing inflammation in various systems, including the skin, joint, cardiovascular, lungs, neuro, and gastrointestinal tract. Furthermore, research exploring the structure– activity relationship of flavonoids and their antiinflammatory properties have been published [32]. Xylene induces vasodilation, increased edema and blood vessel permeability, contributing to inflammation. The chemical and biological mechanisms underlying xylene–induced inflammation include sensory neurons that are sensitive to capsaicin, a phenomena referred to as neurogenic inflammation. This suggests that the extracts may reduce substance P release or decrease its activity [33], potentially through the inhibition of phospholipase A2, which plays a key role in the pathophysiology of xylene–induced inflammation [34]. Analgesic activity The present findings indicate that DecE and HEE possess analgesic properties against acetic acid induced abdominal tightness. The results presented in FIG. 2 show that these extracts have a strong antinociceptive efficacy against acetic acid–induced writhing in mice in a dose–dependent manner (P<0.05). At a dose of 400.0 mg·kg -1 , HEE and DecE, significantly reduced abdominal constriction by 78.54 ± 3.30% and 66.99 ± 1.34%, respectively. For comparison, the conventional medication, aspirin (100 mg·kg -1 ), demonstrated a clear inhibitory effect, with a rate of 79.32%. Injection of acetic acid induces peritoneal irritation, leading to characteristic writhing behaviour. Research has shown that acetic acid indirectly promotes the release of endogenous pain mediators such as prostaglandins, kinins, and histamine, which excite nociceptive neurons that are sensitive to nonsteroidal antiinflammatory drugs [8, 15]. Previous studies have demonstrated that methanolic extract from leaves had a lower antinociceptive effect (≤ 40 %) compared to reference drug [19]. In contrast, our findings revealed a significant antinociceptive effect (≥ 60 %) at a dose of 200 mg·kg -1 for both DecE and HEE. These extracts effectively attenuated writhing responses, suggesting that they possess notable antinociceptive properties. Based on the findings of this study, it can be inferred that DecE and HEE obtained from the aerial parts of O. majorana have significant antinociceptive properties. Consequently, it is proposed that the observed antinociceptive effects of DecE and HEE could be attributed to their bioactive constituents. In addition, they are likely attributed to the presence of rosmarinic acid, elagic acid, indomethacin, salicylic acid, vanillin, arbutin and other flavonoid and phenolic acids detected in O. majorana. Phenolic molecules with both antiinflammatory and antioxidant properties are highly desirable when developing pharmaceutical formulations from any natural resource. Considering these findings, it is evident that O. majorana present an antinociceptive properties, which could pave the way for the recovery of natural analgesics. CONCLUSION Finally, it is worthwhile to screen plants commonly used in the local flora for various biological activities, as some may contain novel bioactive substances. The present investigation revealed that natural extracts the aerial parts O. majorana hold promise as antioxidant and anti–inflammatory compounds for treatment human diseases. These results confirm that different phytochemical components exhibit distinct qualitative and quantitative characteristics. The observed biological effects of O. majorana aerial part extracts may be attributed to the high presence of phytochemicals such us rosmairinic acid and ellagic acid, and other phenolics and flavonoids compounds. Consequently, the phytopharmaceutical potential of O. majorana aerial parts can be further explored to promote human health. However, further research is necessary to ensure its safe and effective application. Declaration of competing interest The authors declare that they have no conflicts of interest. ACKNOWLEDGEMENTS The authors would like to acknowledge the Algerian Ministry (MESRS) and the Directorate Scientific Research (DGRSDT) for their financial support. Ethical approvals Experimentation Animale’ (http://aasea.asso.dz/articles/) approved experimental assays under statute No. 88-08/1988, which deals with veterinary medical activities and animal health protection (N° JORA: 004/1988). BIBLIOGRAPHIC REFERENCES [1] Meda N T R, Bangou M J, Bakasso S, Millogo–Rasolodimby J, Nacoulma OG. Antioxidant activity of phenolic and flavonoid fractions of Cleome gynandra and Maerua angolensis of Burkina Faso. J. Appl. Pharm. Sci. [Internet]. 2013; 3(2):36- 42. doi: https://doi.org/pqpp Aspirin DecE 200 DecE 400 HEE 200 HEE 400 0 20 40 60 80 100 Inhibition (%) * FIGURE 2. Analgesic effect of DecE and HEE against writhing caused by acetic acid in mice. DecE 200 / DecE 400: Decoction extract, 200 mg·kg -1 / 400 mg·kg -1 respectively. HEE 50 / HEE 200: Hydroethanol extract, 200 mg·kg -1 / 400 mg·kg -1 respectively. The results were expressed as means ± SEM, *: P<0.1, n= 5

LC-MS/MS phytochemical analysis of Origanum majorana L./ Karbab et al.____________________________________________________________ 8 of 9 [2] Albayrak S, Aksoy A, Albayrak S, Sagdic O. In vitro antioxidant and antimicrobial activity of some Lamiaceae species. Iran. J. Sci. [Internet]. 2013; 37(1):1-9. doi: https://doi.org/pqpr [3] Duran–Bedolla J, Rodriguez MH, Saldana–Navor V, Rivas– Arancibia S, Cerbon M, Rodriguez MC. Oxidative stress: production in several processes and organelles during Plasmodium sp development. Oxid. Antioxid. Med. Sci. [Internet]. 2013 [cited 12 Feb 2025]; 2(2): 93-100. Available in: https://goo.su/MY1Y [4] Tang SY, Halliwell B. Medicinal plants and antioxydants: what do we learn from cell culture and Caenorhabditis elegans studies, Biochem. Biophys. Res. Commun. [Internet]. 2010; 394(1):1-5. doi: https://doi.org/dgtgn9 [5] Taha M, Elazab ST, Abdelbagi O, Saati AA, Babateen O, Baokbah TAS, Qusty NF, Mahmoud ME, Ibrahim MM, Badawy AM. Phytochemical analysis of Origanum majorana L. extract and investigation of its antioxidant, anti–inflammatory and immunomodulatory effects against experimentally induced colitis downregulating Th17 cells. J. Ethnopharmacol. [Internet]. 2023; 317:116826. doi: https://doi.org/pqpw [6] Tugume P, Nyakoojo C. Ethno–pharmacological survey of herbal remedies used in the treatment of paediatric diseases in Buhunga parish, Rukungiri District, Uganda. BMC Complement. Altern. Med. [Internet]. 2019; 19:353. doi: https://doi.org/gnffck [7] Amari S, Ahlem K, Arrar L, Noureddine C. Fractionation, phytochemical screening and antioxidant activity of different sub–fractions from leaves and flowers of Erica arborea L. Turkish JAF Sci. Tech. [Internet]. 2023; 11(4):830-837. doi: https://doi.org/pqpz [8] Karbab A, Charef N, Zarga MHA, Qadri MI, Mubarak MS. Ethnomedicinal documentation and anti–inflammatory effects of n–butanol extract and of four compounds isolated from the stems of Pituranthos scoparius: An in vitro and in vivo investigation. J. Ethnopharmacol. [Internet]. 2021; 267:113488. doi: https://doi.org/pqp2 [9] Jun WJ, Han BK, Yu KW, Kimb MS, Chang IS, Kim HY, Cho HY. Antioxidant effects of Origanum majorana L. on superoxide anion radicals. Food Chem. [Internet]. 2001; 75(4):439-444. doi: https://doi.org/crsp2t [10] Vasudeva N, Goel P. Origanum majorana L . Phyto– pharmacological review. Indian. J. Nat. Prod. Resour. [Internet]. 2015 [cited Aug. 5 2014]; 6(4):261-267. Available in: https://goo.su/P1cr8JC [11] Saxena D, Jayant SK, Soni KB, Neekhra K. Origanum Majorana: A potential herbe for functional food. Eur. J. Pharm. Med. Res. [Internet]. 2016 [cited Jan. 20 2025]; 3(2):321-325. Available in: https://goo.su/aFtQw [12] Organisation for Economic Co–operation and Development (OECD). Guidelines for the testing of chemicals. Acute oral toxicity procedure. Up–and–Down–Procedure (UDP). [Internet]. Paris: OECD; 2008 [cited Dec. 12 2024]; 27p. Available in: https://goo.su/s8qor [13] Ahlem K, Noureddin C, Lekhmici A. Phenolic contents, in vitro antioxidant, and in vivo antiinflammatory studies of aqueous extract from Pituranthos scoparius (Coss. & Dur.) growing in Algeria. Iran. J. Pharmacol. Ther. [Internet]. 2019 [cited Dec. 2 2024]; 17(1):1-7. Available in: https://goo.su/IhXjJ [14] Karbab A, Mokhnache K, Arrar L, Baghiani A, Khennouf S, Charef N. Fractionation, phytochemical screening and free radical scavenging capacity of different subfractions from Pituranthos scoparius roots. J. Drug. Delivery. Ther. [Internet]. 2020; 10(3):133-136. doi: https://doi.org/pqqg [15] Amari S, Karbab K, Charef N, Arrar L, Mubarak MS. Anti– urolithiatic, antibacterial, anti–inflammatory and analgesic effects of Erica arborea flowers and leaves hydromethanolic extracts: An ethnopharmacological study. Saudi. J. Biol. Sci. [Internet]. 2023; 30(10):103785. doi: https://doi.org/pqqc [16] Karbab A, Mokhnache K, Arrar L, Charef N. Total phenolic contents and antioxidant capacity of aqueous extract from Pituranthos scoparius (coss. & dur.) growing in Algeria. J. Drug. Delivery Ther. [Internet]. 2020; 10(3):125-127. doi: https://doi.org/pqqf [17] Qnais E, Bseiso Y, Wedyan M, Alkhateeb HC. Comparison of antinociceptive activity of Origanum majorana L. methanol leaf extract in mice in different models. Der Pharma Chemica. [Internet]. 2016 [cited Feb. 20 2025]; 8(13):307-313. Available in: https://goo.su/ch9IhnE [18] Karimi A, Min B, Brownmiller C, Lee SO. Effects of extraction techniques on total phenolic content and antioxidant capacities of two Oregano Leaves. J. Food Res. [Internet]. 2015; 4(1):112-123. doi: https://doi.org/pqqh [19] Hossain MB, Barry–Ryan C, Martin–Diana AB, Brunton NP. Optimisation of accelerated solvent extraction of antioxidant compounds from rosemary (Rosmarinus officinalis L.), marjoram (Origanum majorana L.) and oregano (Origanum vulgare L.) using response surface methodology. Food Chem. [Internet]. 2011; 126(1):339-346. doi: https://doi.org/ct7qwn [20] Roby MHH, Serhan MA, Selim KAH, Khalel KI. Evaluation of antioxidant activity, total phenols and phenolic compounds in thyme (Thymus vulgaris L.), sage (Salvia officinalis L.), and majoram (Origanum majorana L.) extracts. Ind. Crop. Prod. [Internet]. 2013; 43:827-831. doi: https://doi.org/pqqj [21] Çelik SE, Tufan AN, Bekdeser B, Özyürek M, Güçlü K, Apak R. Identification and determination of phenolics in Lamiaceae species by UPLC–DAD–ESI–MS/MS. J. Chromatogr. Sci. [Internet]. 2017; 55(3):291-300. doi: https://doi.org/f9zfp4 [22] Dhull SB, Kaur P, Purewal SS, Phytochemical analysis, phenolic compounds, condensed tannin content and antioxidant potential in Marwa (Origanum majorana) seed extracts. Res. Eff. Technol. [Internet]. 2016; 2(4):168-174. doi: https://doi.org/pqqk [23] Çarıkçı S, Kılıç T, Dirmenci T, Gören A C. Phenolic Compounds from section Majorana (Mill.) Benth of Origanum L. species extracts via validated LC–MS/MS method. J. Chem. Metrol. [Internet]. 2022; 16(2):147-151. doi: https://doi.org/pqqm

_________________________________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol.XXXV 9 of 9 [24] Erenler R, Sen O, Aksit H, Demirtas I, Yaglioglu A S, Elmastas M, Telci I. Isolation and identification of chemical constituents from Origanum majorana and investigation of antiproliferative and antioxidant activities. J. Sci. Food Agric. [Internet]. 2016; 96(3):822-836. doi: https://doi.org/pqqn [25] Amaghnouje A, Mechchate H, Es–safi I, Boukhira S, Aliqahtani S, Noman AM, Nasr OA, Conte F, Calarco R, Bousta AD. Subacute assessment of the toxicity and antidepressant–like effects of Origanum majorana L. polyphenols in swiss albino mice. Molecules [Internet]. 2020; 25(23):5653. doi: https:// doi.org/g7t8fr [26] Sepulveda L, Ascacio A, Rodriguez–Herrera R, Aguilera– Carbo A, Aguilar CN. Ellagic acid: biological properties and biotechnological development for production processes. Afr. J. Biotechnol. [Internet]. 2012; 43(50):4518-4523. doi: https://doi.org/fz9j6r [27] Vasudeva N, Singla P, Das S, Sharma SK. Antigout and antioxidant activity of stem and root of Origanum majorana Linn. Am. J. Drug. Discovery Dev. [Internet]. 2014; 4(2):102- 112. doi: https://doi.org/pqqr [28] Hossain MB, Camphuis G, Aguiló–Aguayo I, Gangopadhyay N, Rai DK. Antioxidant activity guided separation of major polyphenols of marjoram (Origanum majorana L.) using flash chromatography and their identification by liquid chromatography coupled with electrospray ionization tandem mass spectrometry. J. Sep. Sci. [Internet]. 2014; 37(22):3205-3213. doi: https://doi.org/f2v8rf [29] Vallverdú–Queralt A, Regueiro J, Alvarenga JFR, Martinez– Huelamo M, Leal LN, Lamuela–Raventos RM. Characterization of the phenolic and antioxidant profiles of selected culinary herbs and spices: Caraway, turmeric, dill, marjoram and nutmeg. Food Sci. Technol. [Internet]. 2015; 35(1):189- 195. doi: https://doi.org/pqqs [30] Ravishankar K, Nageswara Rao S, Girija Sastry V. Evaluation of antioxidant, anti–inflammatory, analgesic and antipyretic activities of the ethanolic extract along with its organic soluble fractions of Origanum majorana. J. Drug Alcohol Res. 2021 [Accessed 20 Apr 2025]; 10(12):1-18. Available in: https:// goo.su/sCpG5Nk [31] Howes MJR. Chapter 28. Phytochemicals as anti–inflammatory nutraceuticals and phytopharmaceuticals. In: Chatterjee S, Jungraithmayr W, Bagchi D, editors. Immunity and inflammation in health and disease. London (UK): Academic Press; 2018. p. 363-388. [32] Gautam R, Jachak SM. Recent developments in anti– infammatory natural products. Med. Res. Rev. [Internet]. 2009; 29(5):767-820. doi: https://doi.org/fb6sfr [33] Zhou M, Wang H, Suolangjiba S, Kou J, Yu B, Antinociceptive and anti–inflammatory activities of Aquilaria sinensis (Lour.) Gilg. Leaves extract. J. Ethnopharmacol. [Internet]. 2008; 117(2):345-350. doi: https://doi.org/bt7pq3 [34] Rahman SMM, Atikullah MD, Islam MDN, Mohaimenul MD, Ahammad F, Islam MDS, Saha B, Rahman MDH, Clin. Anti–inflammatory, antinociceptive and antidiarrhoeal activities of methanol and ethyl acetate extract of Hemigraphis alternata leaves in mice. Clin. Phytosci. [Internet]. 2019; 5:16. doi: https://doi.org/pqqt