Inhibition of Enzymatic Browning and Antioxidant Activities of Marrubium vulgare L Extracts: A Promising Natural Solution

Inhibition of Enzymatic Browning and Antioxidant Activities of Marrubium vulgare L Extracts: A Promising Natural Solution | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inhibition of Enzymatic Browning and Antioxidant Activities of Marrubium vulgare L Extracts: A Promising Natural Solution Karim Tighilet, Messis Abdelaziz, Adjebli Ahmed This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6111477/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jul, 2025 Read the published version in Pharmacological Research - Modern Chinese Medicine → Version 1 posted You are reading this latest preprint version Abstract In recent decades, medicinal plants have attracted significant interest due to their demonstrated therapeutic properties. This study analyzed different extracts of Marrubium vulgare L. (horehound) to assess their phytochemical composition and biological activities. Quantitative analysis revealed that methanolic leaf extracts were the richest in polyphenols, containing 23.48 to 35.36 mg EAG/g DM. These leaf extracts also had a particularly high flavonoid content, ranging from 12.55 ± 0.233 to 20.56 ± 0.54 mg EQ/g DM. In contrast, aqueous stem extracts were abundant in condensed tannins, with 72.82 ± 0.772 and 65.52 ± 1.216 mg EC/g DM in aqueous and methanolic extracts, respectively. The antioxidant potential of the extracts was evaluated through several in vitro assays. Leaf extracts demonstrated the highest antioxidant activity, with methanolic extracts exhibiting stronger antioxidant capacity than aqueous extracts. Furthermore, leaf extracts were capable of inhibiting peroxidase activity by up to 42.68%, although they showed weak inhibition of polyphenol oxidase. These findings suggest that Marrubium vulgare L is a promising source of natural antioxidants and enzyme inhibitors, with potential applications in the development of functional foods, cosmetics, and pharmaceuticals. The observed differences between extraction solvents highlight the importance of considering phytochemical profiles and bioactivities when selecting and optimizing plant-based ingredients. Botany Applied Biochemistry Food Chemistry antioxidant activity phenolic compounds Enzymatic Browning Marrubium vulgare Figures Figure 1 Introduction For centuries, medicinal plants have been an integral part of traditional health systems in many cultures around the world (Bouzouita et al., 2008 ). In recent decades, there has been growing scientific interest in exploring the therapeutic potential of these natural resources, motivated by their proven efficacy and the desire to develop alternative treatments to synthetic pharmaceuticals (Najmi, 2022). Marrubium vulgare L., commonly known as ballote or Merriwa in Algeria, is one such plant that has attracted attention. M. vulgare L. is a perennial plant from the Lamiaceae family, native to Europe, North Africa, Asia, and the Canary Islands (Argyropoulou et al., 2009 ). Historically, various parts of the plant, particularly the leaves and stems, have been utilized in traditional medicine to treat a wide array of ailments, including dyspepsia and loss of appetite (Aćimović et al., 2020 ), gastrointestinal and respiratory disorders (Bellakhdar, 1997 ; El Alami et al., 2016 ; Aćimović et al., 2020 ), inflammations, and diabetes (Vergara-Galicia et al., 2012 ; Hamza et al., 2019 ). Additionally, hypertension (Ahmed et al., 2010 ), cancer (Kozyra et al., 2020 ), as well as hypocholesterolemic and hypotriglyceridemic conditions (Ibrahim et al., 2016 ) are commonly treated with this plant in traditional remedies. The biological activities of M. vulgare are primarily attributed to its rich content of secondary metabolites, which include bitter furanolabdane-series diterpenes and lactone compounds, particularly marrubiin. Marrubiin is considered the main compound responsible for most of the plant’s biological properties (Paolini et al., 2008 ; Boudjelal et al., 2012 ). These metabolites are believed to contribute to the wide range of therapeutic effects, making M. vulgare a valuable plant in folk medicine. In Algeria, the traditional use of M. vulgare L. extends to the treatment of diarrhea, diabetes, rheumatism, colds, and respiratory pain (Belhattab & Larous, 2006; Alkhatib et al., 2010 ). These longstanding traditional uses have prompted researchers to investigate the phytochemical composition and biological activities of this plant in greater detail. One of the main characteristics observed during the preservation of fruits and vegetables is the phenomenon of browning, which is often associated with the oxidation of polyphenolic compounds (Vamos-Vigyázó & Haard, 1981 ). This browning process can affect the biological properties of vegetables and their organoleptic and nutritional qualities, resulting in significant economic losses for the products (Lee & Whitaker, 1995 ). Therefore, it is of interest to investigate the relationship between the enzymatic browning of vegetables and the antioxidant and anti-enzymatic effects of M. vulgare L. extracts. The aim of this study was to quantify the main phytochemicals, such as polyphenols, flavonoids, flavonols, and tannins, present in M. vulgare L. leaf and stem extracts. We then assessed the antioxidant potential of these extracts using various in vitro tests. Finally, we analyzed their ability to inhibit the enzymes responsible for browning, namely peroxidase and polyphenol oxidase, to explore natural alternatives to synthetic additives. Materials and methods Plant Material and Extraction The leaves and stems of M. vulgare L. were collected from the Bir ghbalou region, Bouira province, Algeria. The plant material was initially cleaned under running water to remove any dust or other impurities. It was then shade-dried at room temperature under continuous aeration for 20 days. The dried plant material was then ground to a particle size of less than 250 µm. The powdered plant material (10 g) was extracted by maceration for 24 hours at room temperature with continuous stirring using two solvents: water and 80% methanol, in a 1/10 weight/volume ratio. The extracts were then filtered through Whatman N°3 filter paper, and the solvents were removed in an oven at 40°C. The dry extracts obtained were stored in airtight containers at 4°C until further analysis (Falleh et al., 2008 ; Perez et al., 2010 ). Determination of total polyphenols Total polyphenol content in aqueous and methanolic extracts was determined using the method described by Tighilet et al. ( 2022 ). This method is based on the use of the Folin-Ciocalteu reagent. 200 µL of each extract was mixed with 1.5 mL of Folin-Ciocalteu reagent (diluted 10-fold with distilled water). Subsequently, 1.5 mL sodium carbonate solution (6% w/v) was added after a five-minute interval. The mixture was incubated at room temperature for two hours, after which the absorbance was measured at 765 nm using a UV-Vis spectrophotometer. A calibration curve was prepared using gallic acid as a standard, and total polyphenol content was expressed in milligrams of gallic acid equivalents per gram of dry matter (mg GAE/g DM). Flavonoid assay The flavonoid content of M. vulgare extracts was determined using the colorimetric method described by Khettal et al. ( 2017 ). This method is based on the formation of yellowish complexes between flavonoids and aluminum trichloride (AlCl 3 ) and is used to quantify flavonoid content. Free hydroxyl groups in the C3 or C5 position, as well as oxygen in the C4 position, are responsible for complexation with AlCl3. Briefly, an aliquot of the extract (1 ml) was mixed with 1 ml of a 2% AlCl 3 solution in methanol. The mixture was incubated at room temperature for 10 minutes, and absorbance was measured at 415 nm using a UV-Vis spectrophotometer. A calibration curve was prepared using quercetin as a standard, and flavonoid content was expressed in milligrams of quercetin equivalents per gram dry matter (mg (QE)/g MD). Flavonol Assay The flavonol content in the M. vulgar e extracts was determined using the method described by Kumaran & Karunakaran ( 2007 ). In this method, 250 µL of the extract was mixed with 250 µL of 2% aluminum trichloride (AlCl 3 ) solution, followed by the addition of 1.5 mL of 5% sodium acetate. The mixture was incubated in the dark at room temperature for 2 hours and 30 minutes. After the incubation period, the absorbance was measured at 440 nm using a UV-Vis spectrophotometer. A control was prepared under the same conditions, but without the addition of the plant extract. A calibration curve was prepared using quercetin as a standard, and flavonoid content was expressed in milligrams of quercetin equivalents per gram dry matter (mg (QE)/g MD). All the experiments were carried out three times independently. Condensed tannin content The condensed tannin content of extracts was determined using the method described by Chew et al. ( 2011 ). 0.5 mL of the crude extract was mixed with 3 mL of a 4% vanillin solution. Subsequently, 1.5 mL of hydrochloric acid (HCl) was added. The mixture was kept in the dark at room temperature for 15 minutes. The absorbance of the mixture was measured at 500 nm against a blank using a UV-Vis spectrophotometer. Each extract was analyzed in triplicate and the results expressed as milligrams of catechin equivalents per gram of dry matter (mg CE/g DM). Evaluation of antioxidant capacity DPPH assay In order to determine the free radical scavenging capacity of our extracts by the DPPH assay, we used the method described by (Masuda et al., 1999 ). A volume of 1 ml of DPPH methanolic solution (0.1 Mm) was added to 1 ml of extract at concentrations ranging from 20 to 220 µg/ml. In parallel, a negative control was prepared by mixing 1ml of methanol or distilled water with 1ml of DPPH solution. After 30 min incubation in the dark at room temperature, absorbance was measured at 517 nm. The positive control was a solution of two standard antioxidants, gallic acid and quercetin, whose absorbance was measured under the same conditions. The test was repeated independently three times. The antioxidant power of plant extracts assessed by the DPPH test is expressed as a percentage reduction in the DPPH radical according to the following formula: (%) = [A 0 – (A 1 – A 2 ) / A 0 ] × 100 Where: % = percentage of DPPH radical trapped; A 0 = Absorbance of the control reaction (methanol + DPPH); A 1 = Absorbance of the extract + DPPH; A 2 = Absorbance of the extract with methanol (extract + methanol). The scavenging efficiency of the DPPH radical is determined by calculating the EC 50 , i.e. the concentration of plant extract required to reduce the DPPH- radical by 50%. ABTS •+ cation radical trapping activity. The free radical scavenging activity of the extracts was assessed using the ABTS ˙+ radical scavenging method (Re et al., 1999 ). The ABTS ˙+ radical cation was generated by mixing a 7 mM solution of ABTS with a 2.45 mM solution of potassium persulphate (K 2 S 2 O 8 ). This mixture was kept protected from light and at room temperature for 16 hours. The ABTS ˙+ solution was then diluted with distilled water to give an absorbance of 0.700 ± 0.02 at 734 nm. For the assay, 1.9 ml of ABTS ˙+ solution was mixed with 100 µl of extract solution at different concentrations. After incubation for 7 minutes in the dark at room temperature, absorbance was measured at 734 nm. A positive control was performed using a solution of the standard antioxidant, Trolox, under the same conditions. The test was performed in triplicate for each concentration. The percentage ABTS ˙+ radical scavenging activity of the extracts was calculated as follows: % I = [(A 0 – A 1 ) / A 0 ] × 100 Where: % inhibition: Percentage inhibition of the ABTS ˙+ radical; A 0 : Absorbance of the control (containing only ABTS ˙+ ); A 1 : Absorbance of the extract in the presence of ABTS ˙+ . Results are expressed as µM of Trolox equivalent per g of dry matter (µM TE/ g DM). β-Carotene Bleaching Test The antioxidant test using ß-carotene bleaching was determined according to the protocol previously described by Koleva et al. ( 2002 ). Briefly, 0.5mg of ß-carotene was dissolved in 1ml of chloroform and mixed with 25µl of linoleic acid and 200µl of tween 20. The chloroform was evaporated under vacuum at 40°C, then 100ml of distilled water was added. 2.5ml of the emulsion obtained was transferred to different tubes containing 500µl of the extract. The tubes were immediately incubated in a water bath at 50°C for 120 min and the absorbance was measured at 470 nm before and after the heat treatment. A blank control was run in parallel. BHT was used as a positive control and all tests were performed in triplicate. Evaluation of anti-enzymatic activity Anti-enzymatic activities were assessed using enzymes involved in enzymatic browning, namely polyphenoloxidase and peroxidase, extracted from red cabbage. The enzymes were extracted according to the protocol of Ponce et al ( 2004 ). Red cabbage was first cleaned, then ground and homogenised using a high-speed electric mixer for 3 minutes with 30 ml of distilled water at 4°C. The preparation was then filtered through two layers of gauze and centrifuged at 1000g for 15 minutes at 4°C. The supernatant obtained was used as an enzyme extract. Evaluation of anti-peroxidase activity Peroxidase inhibition by marrubium leaves and stems extracts was measured according to the protocol described by Ponce et al. ( 2004 ). For this, 2.87 ml of substrate solution, consisting of 10 ml 1% guaiacol, 10 ml 0.3% hydrogen peroxide and 100 ml phosphate buffer (0.05 M, pH 6.5), were mixed with 0.03 ml extract and 0.1 ml enzyme extract. Enzyme activity was monitored by measuring the change in absorbance at 470 nm for 5 minutes at room temperature. Evaluation of anti-polyphenoloxidase activity Polyphenoloxidase activity was measured following the protocol of Lee et al. ( 2007 ). The substrate used is pyrocatechol, whose oxidation leads to a red-brown coloration. Briefly, 1 ml marrube extract was mixed with 0.1 ml enzyme extract and 0.9 ml phosphate buffer (50 mM, pH 6.8). The resulting solution was incubated for 5 minutes at 25°C, then 1 ml of 0.2 M pyrocatechol was added. The change in optical density (OD) was measured at 420 nm for 1 minute. The enzymatic inhibition rate, expressed as a percentage, is calculated as follows: Inhibition rate (%) = [(Initial activity* - Residual activity**)/ (Initial activity*)]x 100 where: Initial activity*: Enzyme activity (OD/min) measured in the absence of marrube extract. Residual activity**: Enzymatic activity (ΔDO/min) measured in the presence of marrube extract. Statistical analysis Three replicates were carried out for each sample. Results are presented as mean ± standard deviation, calculated using Microsoft Office Excel 2007. Statistical analysis was performed with the ANOVA/MANOVA test, using STATISTICA 5.5 software. Significant differences were considered at a threshold of p < 0.05. Results and Discussion Quantification of phenolics compounds Colorimetric analyses of marrube extracts reveal that methanolic extracts contain significantly higher concentrations of polyphenols, flavonoids, flavonols and tannins than aqueous extracts, as shown in Table 1 . Polyphenols ranged from 23.48 to 35.36 mg GAE/g DM in the methanolic extracts and from 13.78 to 15.41 mg GAE/g DM in the aqueous extracts, with a higher concentration in the leaves than in the stems. Flavonoids followed the same trend, with leaves showing higher levels (20.56 ± 0.54 and 13.99 ± 0.29 EQ/g DM for methanolic and aqueous extracts respectively) than stems. Flavonoids dominated among the polyphenols, with proportions above 0.5 in the extracts. Flavonol concentrations were also higher in the methanolic extracts than in the aqueous extracts for both parts of the plant. On the other hand, tannin levels were higher in the stems than in the leaves, with significant differences (p < 0.05). These results corroborate previous studies and show that methanol is particularly effective at extracting phenolic compounds from plants. In addition, experimental conditions (choice of solvent, concentration, extraction time and temperature, extraction method) and environmental factors (geographical region, altitude, exposure, harvesting season) have a considerable influence on the concentrations of these compounds. However, it should be noted that the method for determining phenolic compounds can be affected by interference from other substances present in the extracts (Bouterfas et al., 2014 ; Zahedifar et Najafian 2023). Table 1 Phenolic compound content of M. vulgare leaves and stems extracts. Part of plant used Solvents Extraction yield (%) TPC (mg GAE/g DM) Flavonoids (mg QE/g DM) Flavonl (mg QE/g DM) Tannins (mg CE/g DM) Leaves Methanol 20.45 35.36 ± 0.92 a 20.56 ± 0.54 a 23.8 ± 0.81 a 64.46 ± 1.81 b Aqueous 12.96 15.41 ± 0.27 c 13.99 ± 0.29 b 16.88 ± 0.83 b 56.99 ± 0.99 c Stems Methanol 27.4 23.48 ± 0.66 b 12.55 ± 0.23 c 14.7 ± 0.09 c 72.82 ± 0.77 a Aqueous 17.4 13.78 ± 0.51 d 9.34 ± 0.39 d 14.59 ± 0.25 c 65.52 ± 1.21 b TPC: Total phenolic compounds; DM: Dry matter; GAE: Gallic acid equivalent; QE: Quercetin equivalent; CE: Catechine equivalent. Values are mean ± standard deviation (n = 3). Means followed by the same letter are not different according to ANOVA analysis of variance. Antioxidant activity The antioxidant activity of M. vulgare extracts was assessed by three in vitro assays: ABTS, DPPH, and β-carotene bleaching test. These tests provide a comprehensive assessment of the antioxidant efficacy of marrube extracts by quantifying their ability to neutralize various free radicals and protect oxidation-sensitive compounds. DPPH radical scavenging activity The results of the evaluation of the antiradical activity of M. vulgare extracts against the DPPH radical show a dose-dependent response with significant differences between extracts (p < 0.05) (Table 2 ). Methanolic extracts showed high percentages of inhibition, ranging from 79.94% for stems to 93.97% for leaves. In comparison, aqueous extracts had lower inhibition percentages, ranging from 61.27% for stems to 78.38% for leaves. The methanolic leaf extract has the highest antioxidant activity with an IC50 of 20.83 ± 0.62 µg/ml, while the aqueous stem extract displays the lowest activity with an IC50 of 87.5 ± 1.34 µg/ml. This high antioxidant activity can be attributed to the phenylpropanoid glycosides present in the plant, such as forsythoside B, ballottetroside, arenarioside and actenoside, known for their powerful antioxidant properties (Sahpaz et al., 2002 ; Martin-Nizard et al., 2003 ). These results concur with earlier studies, carried out on M. vulgare from Poland, which also showed significant antioxidant activity of methanolic extracts against the DPPH radical. On the other hand, the plant's strong anti-free radical activity is generally linked to the synergistic effect between the phenolic compounds and flavonoids present in the extracts (Ghedadba, 2014). Table 2 Antioxidant activity of M. vulgare extracts. Assay DPPH ABTS •+ β-Carotene Sample IC 50 (µg mL − 1 ) Aqueous leaves extract 25.00 ± 0.8 b 76.19 ± 0.68 b 140.62 ± 5.05 b Methanolic leaves extract 20.83 ± 0.62 a 67.14 ± 0.32 a 118.75 ± 3.84 a Aqueous stem extract 87.50 ± 1.34 d 95.71 ± 1.54 d 187.50 ± 7.34 d Methanolic stem extract 45.83 ± 1.01 c 87.14 ± 1.88 c 165.67 ± 6.41 c Gallic acid 1.56 ± 0.07 1.54 ± 0.09 - Quercitin 3.87 ± 0.2 2.73 ± 0.1 - BHT - - 12.22 ± 0.07 ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); BHT: Butylhydroxytoluene; DPPH: 2,2-diphenyl-1-picrylhydrazyl; IC 50 : 50% inhibitory concentration. Values are mean ± standard deviation (n = 3). Means followed by the same letter are not different according to ANOVA analysis of variance. ABTS radical trapping activity The results in Table 2 show that, at a concentration of 1 mg/ml, the methanolic extracts of marrubus showed a percentage inhibition of the ABTS radical of 65.10% for stems and 68.24% for leaves. In contrast, aqueous extracts showed inhibition percentages of 52.26% for stems and 58.11% for leaves. The IC 50 concentrations of the ABTS radical for the various marrube extracts ranged from 67.14 to 95.714 µg/ml. The methanolic and aqueous leaf extracts show high ABTS anti-radical activity, with IC 50 of 67.14 ± 0.32 µg/ml µg/ml and 76.19 ± 0.68 µg/ml, respectively. Whereas, methanolic and aqueous extracts of stems show lower activity, with IC 50 of 87.14 ± 1.88 µg/ml and 95.71 ± 1.54 µg/ml, respectively. Several studies have demonstrated that total polyphenol and flavonoid content is strongly correlated with ABTS radical scavenging activity, with correlation coefficients around 0.8 (Khettal et al., 2017 ). These results suggest that phenolic compounds, and flavonoids in particular, play a major role in the antioxidant activity observed (Floegel et al., 2011 ). However, other research has also highlighted that antioxidant activity can be influenced by non-phenolic compounds, such as lipids, sugars and chlorophyll (Heim et al., 2002 ). β-carotene bleaching test The principle of the β-carotene discoloration test is based on the disappearance of its yellow color, which results from its interaction with free radicals generated by the oxidation of linoleic acid in an emulsion (Ghedadba., et al, 2014). The results of the β-carotene bleaching test in the presence of the extracts and the standard antioxidant (BHA) reveal dose-dependent kinetics (Table 2 ). The most significant inhibition was observed with the methanolic leaf extract (79.74%), followed by the aqueous extract of the same part of the plant (67.65%). Furthermore, the results indicate that leaf extract significantly inhibits β-carotene oxidation compared to stem extract, with an inhibition rate of 56.21% for the methanolic extract and 52.29% for the aqueous extract. The 50% inhibitory concentration values (see Table 2 ) reveal that M. vulgare leaf extract in methanol has the highest activity, with an IC 50 value of 118.75 µg/ml. This is followed by the aqueous leaf extract, with an IC 50 of 140.62 µg/ml. In contrast, the stem extracts show less activity, with IC 50 values of 165.25 µg/ml for the methanolic extract and 187.5 µg/ml for the aqueous extract. Our results concur with those of Ghedadba et al. ( 2014 ), who showed that the methanolic extract of M. vulgare exhibits the highest coefficient of antioxidant activity in the β-carotene bleaching system. This observation is also in line with the work of several other research groups, which have demonstrated a positive correlation between phenolic compound content and antioxidant activity. This activity is influenced by a variety of factors (Ghedadba et al., 2014 ). Relationship between antioxidant activity and phenolic compound content The phenolic compound content of an extract or food is generally linked to enhanced antioxidant activity, thanks to their ability to scavenge or reduce free radicals, thus protecting cells from oxidative damage. Correlation analysis revealed that M. vulgare extracts contain 89% flavonoids and 84% flavonols (Table 3 ). This study also showed a strong correlation between the presence of flavonoids and flavonols and antioxidant activity measured by DPPH, ABTS, and β-carotene assays, with correlation coefficients ranging from 0.67 to 0.95. This indicates that flavonoids are the main contributors to the antioxidant activity of phenolic compounds, confirming their efficacy in protecting the body against oxidative damage... On the other hand, a high correlation was observed between the β-carotene test and the DPPH and ABTS tests, with coefficients of 0.93 and 0.97 respectively. This high correlation is attributed to the presence of bioactive molecules with powerful reducing and free radical scavenging properties. Previous studies, including those by Khettal et al. ( 2017 ), have shown a good correlation between total polyphenols and the antioxidant activity of plant extracts. Table 3 Correlation between M. vulgare phenolic compounds and antioxidant activities. Variables TPC Flavonoides Flavonl Tannins DPPH ABTS •+ β-Carotene TPC 1 Flavonoides 0.89 1 Flavonl 0.84 0.96 1 Tannins 0.28 -0.19 -0.26 1 DPPH -0.6 -0.83 -0.67 0.34 1 ABTS •+ -0.72 -0.95 -0.89 0.42 0.93 1 β-Carotene -0.73 -0.95 -0.89 0.42 0.93 0.97 1 TPC: Total phenolic compounds; ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH: 2,2-diphenyl-1-picrylhydrazyl. Anti-browning enzyme activity Anti-peroxidase activity Enzymatic browning can be controlled by three main methods: inhibition of the enzymes responsible, trapping of quinones, or oxygen limitation (Moon et al., 2020 ). We evaluated the efficacy of methanolic and aqueous extracts of M. vulgare in inhibiting the activity of peroxidase extracted from red cabbage, a key enzyme in the browning of fruit and vegetables. At a concentration of 500 µg/mL (Fig. 1 ), the methanolic extracts showed maximum inhibition of 42.68% for leaves and 28.52% for stems, while the aqueous extracts showed inhibitions of 22.73% and 11.40%, respectively. In comparison, ascorbic acid, used as a control, showed 89.85% inhibition at 100 µg/mL. Previous studies have shown that various plant extracts can inhibit peroxidase activity in several vegetables (Ponce et al., 2004 ). Variations in the efficacy of M. vulgare extracts may be due to the diversity of bioactive substances present, as well as differences between isoenzymes and experimental conditions. The observed inhibitory efficacy could also result from the presence in marrube extracts of compounds analogous to the peroxidase substrate, which could enhance their ability to inhibit this enzyme. Anti-polyphenoloxidase activity The Anti-polyphenoloxidase (anti-PPO) activity of aqueous and methanolic extracts of M. vulgare leaves and stems was evaluated using PPO extracted from red cabbage. The results indicate that these extracts lead to a reduction in PPO activity, this reduction varying according to the part of the plant and the extraction solvent (Table 4 ). Methanolic extracts of leaves and stems inhibited red cabbage PPO activity by 51.40% and 47.68% respectively. In contrast, aqueous extracts showed inhibition rates of 38.31% for leaves and 33.64% for stems. Previous research has shown that natural antioxidants can act as PPO inhibitors in fruit and vegetables. These results are corroborated by the work of Lee et al. ( 2007 ), who observed an inhibition of PPO in taro ranging from 54.8–68.5% by onion extracts. Other studies have also reported similar effects with natural extracts on PPO activity in plants (Vhangani et Van Wyk, 2021; Arnold et Gramza-Michałowska, 2022; Sui et al., 2023 ). The variations in inhibition rates observed between different extracts and enzyme sources may be attributed to the presence of several isoenzymes in the extract, each with distinct structural and physicochemical specificities and properties, or to the nature of the active molecules present in the inhibitory plant extracts (Khettal et al., 2017 ). Table 4 Percentage inhibition of peroxidase and polyphenoloxidase by M. vulgare extracts. Extracts Anti-polyphenoloxidase activity (%) Anti-peroxidase activity (%) Leaves methanolic extract 51.4 42.68 Leaves aqueous extract 38.38 28.52 Stems methanolic extract 47.68 22.73 Stems aqueous extract 33.64 11.40 Ascorbic acid 93.08 89. 85 Conclusion The results of the study showed that methanolic extracts of M. vulgare leaves are particularly rich in phenolic compounds, especially flavonoids, compared with stem extracts. Evaluation of antioxidant activities, through DPPH, ABTS and beta-carotene bleaching tests, as well as analysis of the phenolic content of the extracts, revealed a good correlation between phenolic compound concentration and the performance of the various antioxidant tests. In addition, M. vulgare extracts demonstrated a more marked inhibition of polyphenoloxidase than peroxidase extracted from red cabbage. This inhibition was particularly effective with methanolic leaf extracts, underlining their superior potential in preventing enzymatic browning. 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Asian Pac J Trop Med 9(5):453–459 Khettal B, Kadri N, Tighilet K, Adjebli A, Dahmoune F, Maiza-Benabdeslam F (2017) Phenolic compounds from Citrus leaves: Antioxidant activity and enzymatic browning inhibition. J Complement Integr Med 14(1):20160030 Koleva II, Van Beek TA, Linssen JP, Groot AD, Evstatieva LN (2002) Screening of plant extracts for antioxidant activity: A comparative study on three testing methods. Phytochemical Analysis: Int J Plant Chem Biochem Techniques 13(1):8–17 Kozyra M, Korga A, Ostrowska M, Humeniuk E, Adamczuk G, Gieroba R, Makuch-Kocka A, Dudka J (2020) Cytotoxic activity of methanolic fractions of different Marrubium spp. against melanoma cells is independent of antioxidant activity and total phenolic content. FEBS Open Bio 10(1):86–95 Kumaran A, Karunakaran RJ (2007) In vitro antioxidant activities of methanol extracts of five Phyllanthus species from India. LWT-Food Sci Technol 40(2):344–352 Lee CY, Whitaker JR (eds) (1995) Enzymatic browning and its prevention. American Chemical Society Lee MY, Lee MK, Park I (2007) Inhibitory effect of onion extract on polyphenol oxidase and enzymatic browning of taro (Colocasia antiquorum var. esculenta). Food Chem 105(2):528–532 Martin-Nizard F, Sahpaz S, Furman C, Fruchart JC, Duriez P, Bailleul F (2003) Natural phenylpropanoids protect endothelial cells against oxidized LDL-induced cytotoxicity. Planta Med 69(03):207–211 Masuda T, Yonemori S, Oyama Y, Takeda Y, Tanaka T, Andoh T, Shinohara A, Nakata M (1999) Evaluation of the antioxidant activity of environmental plants: Activity of the leaf extracts from seashore plants. J Agric Food Chem 47(4):1749–1754 Moon KM, Kwon EB, Lee B, Kim CY (2020) Recent trends in controlling the enzymatic browning of fruit and vegetable products. Molecules 25(12):2754 Najmi A, Javed SA, Al Bratty M, Alhazmi HA (2022) Modern approaches in the discovery and development of plant-based natural products and their analogues as potential therapeutic agents. Molecules 27(2):349 Paolini J, Tomi P, Bernardini AF, Bradesi P, Casanova J, Kaloustian J (2008) Detailed analysis of the essential oil from Cistus albidus L. by combination of GC/RI, GC/MS and 13C-NMR spectroscopy. Nat Prod Res 22(14):1270–1278 Perez YY, Jimenez-Ferrer E, Alonso D, Botello-Amaro CA, Zamilpa A (2010) Citrus limetta leaves extract antagonizes the hypertensive effect of angiotensin II. J Ethnopharmacol 128(3):611–614 Ponce AG, Del Valle CE, Roura SI (2004) Natural essential oils as reducing agents of peroxidase activity in leafy vegetables. LWT-Food Sci Technol 37(2):199–204 Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26(9–10):1231–1237 Sahpaz S, Hennebelle T, Bailleul F (2002) Marruboside, a new phenylethanoid glycoside from Marrubium vulgare L. Nat Prod Lett 16(3):195–199 Sui X, Meng Z, Dong T, Fan X, Wang Q (2023) Enzymatic browning and polyphenol oxidase control strategies. Curr Opin Biotechnol 81:102921 Tighilet K, Palacios F, Khettal B, Santos J (2022) GC-MS and LC-QTOF-MS phytochemical analysis, antiproliferative and antioxidant activities of stems extracts of Retama monosperma grown in Algeria. Res J Biotechnol 17:1–14 Vamos-Vigyázó L, Haard NF (1981) Polyphenol oxidases and peroxidases in fruits and vegetables. Crit Reviews Food Sci Nutr 15(1):49–127 Vergara-Galicia J, Aguirre-Crespo F, Tun-Suarez A, Aguirre-Crespo A, Estrada-Carrillo M, Jaimes-Huerta I, Flores-Flores A, Estrada-Soto S, Ortiz-Andrade R (2012) Acute hypoglycemic effect of ethanolic extracts from Marrubium vulgare . Phytopharmacology 3(1):54–60 Vhangani LN, Van Wyk J (2021) Heated plant extracts as natural inhibitors of enzymatic browning: A case of the Maillard reaction. J Food Biochem 45(2):13611 Zahedifar M, Najafian S (2023) Variation of antioxidant activity and phenolic compositions of Marrubium vulgare L. as influenced by organic acids. J Med Plants 22(87):77–88 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Published Journal Publication published 04 Jul, 2025 Read the published version in Pharmacological Research - Modern Chinese Medicine → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6111477","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":421154960,"identity":"dec37fec-98a4-458d-924b-d70542906d00","order_by":0,"name":"Karim Tighilet","email":"","orcid":"","institution":"Université de Bouira","correspondingAuthor":false,"prefix":"","firstName":"Karim","middleName":"","lastName":"Tighilet","suffix":""},{"id":421154961,"identity":"b569fd75-be7d-4e32-b0c2-7575fd5a41b4","order_by":1,"name":"Messis Abdelaziz","email":"","orcid":"","institution":"Bordj Bou Arreridj University","correspondingAuthor":false,"prefix":"","firstName":"Messis","middleName":"","lastName":"Abdelaziz","suffix":""},{"id":421154962,"identity":"3aa5428f-e3ae-416b-8d88-30fe45cb0d5f","order_by":2,"name":"Adjebli Ahmed","email":"data:image/png;base64,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","orcid":"https://orcid.org/0009-0003-9721-5656","institution":"Bejaia University","correspondingAuthor":true,"prefix":"","firstName":"Adjebli","middleName":"","lastName":"Ahmed","suffix":""}],"badges":[],"createdAt":"2025-02-26 08:55:22","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6111477/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6111477/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.prmcm.2025.100658","type":"published","date":"2025-07-05T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77319565,"identity":"f31d72f3-4a96-4534-9dcb-d2181a8a2c76","added_by":"auto","created_at":"2025-02-27 11:11:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37683,"visible":true,"origin":"","legend":"\u003cp\u003eVariation in guaiacoquinone absorbance in the presence and absence of methanolic (a) and aqueous (b) extracts of \u003cem\u003eM. vulgare\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6111477/v1/3350b5b71537ac3b269cb970.png"},{"id":86173812,"identity":"e97cab4a-6076-4b1a-aec8-ffd804ef9961","added_by":"auto","created_at":"2025-07-07 14:58:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":921938,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6111477/v1/6faa6ce4-0b7a-42cb-9893-f0f8c9acc40c.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eInhibition of Enzymatic Browning and Antioxidant Activities of \u003cem\u003eMarrubium vulgare\u003c/em\u003e L Extracts: A Promising Natural Solution\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFor centuries, medicinal plants have been an integral part of traditional health systems in many cultures around the world (Bouzouita et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In recent decades, there has been growing scientific interest in exploring the therapeutic potential of these natural resources, motivated by their proven efficacy and the desire to develop alternative treatments to synthetic pharmaceuticals (Najmi, 2022). \u003cem\u003eMarrubium vulgare\u003c/em\u003e L., commonly known as ballote or Merriwa in Algeria, is one such plant that has attracted attention.\u003c/p\u003e \u003cp\u003e \u003cem\u003eM. vulgare\u003c/em\u003e L. is a perennial plant from the Lamiaceae family, native to Europe, North Africa, Asia, and the Canary Islands (Argyropoulou et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Historically, various parts of the plant, particularly the leaves and stems, have been utilized in traditional medicine to treat a wide array of ailments, including dyspepsia and loss of appetite (Aćimović et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), gastrointestinal and respiratory disorders (Bellakhdar, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; El Alami et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Aćimović et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), inflammations, and diabetes (Vergara-Galicia et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hamza et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, hypertension (Ahmed et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), cancer (Kozyra et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), as well as hypocholesterolemic and hypotriglyceridemic conditions (Ibrahim et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) are commonly treated with this plant in traditional remedies.\u003c/p\u003e \u003cp\u003eThe biological activities of \u003cem\u003eM. vulgare\u003c/em\u003e are primarily attributed to its rich content of secondary metabolites, which include bitter furanolabdane-series diterpenes and lactone compounds, particularly marrubiin. Marrubiin is considered the main compound responsible for most of the plant\u0026rsquo;s biological properties (Paolini et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Boudjelal et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These metabolites are believed to contribute to the wide range of therapeutic effects, making M. vulgare a valuable plant in folk medicine.\u003c/p\u003e \u003cp\u003eIn Algeria, the traditional use of \u003cem\u003eM. vulgare\u003c/em\u003e L. extends to the treatment of diarrhea, diabetes, rheumatism, colds, and respiratory pain (Belhattab \u0026amp; Larous, 2006; Alkhatib et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). These longstanding traditional uses have prompted researchers to investigate the phytochemical composition and biological activities of this plant in greater detail.\u003c/p\u003e \u003cp\u003eOne of the main characteristics observed during the preservation of fruits and vegetables is the phenomenon of browning, which is often associated with the oxidation of polyphenolic compounds (Vamos-Vigy\u0026aacute;z\u0026oacute; \u0026amp; Haard, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). This browning process can affect the biological properties of vegetables and their organoleptic and nutritional qualities, resulting in significant economic losses for the products (Lee \u0026amp; Whitaker, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Therefore, it is of interest to investigate the relationship between the enzymatic browning of vegetables and the antioxidant and anti-enzymatic effects of \u003cem\u003eM. vulgare\u003c/em\u003e L. extracts.\u003c/p\u003e \u003cp\u003eThe aim of this study was to quantify the main phytochemicals, such as polyphenols, flavonoids, flavonols, and tannins, present in \u003cem\u003eM. vulgare\u003c/em\u003e L. leaf and stem extracts. We then assessed the antioxidant potential of these extracts using various \u003cem\u003ein vitro\u003c/em\u003e tests. Finally, we analyzed their ability to inhibit the enzymes responsible for browning, namely peroxidase and polyphenol oxidase, to explore natural alternatives to synthetic additives.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Material and Extraction\u003c/h2\u003e \u003cp\u003eThe leaves and stems of \u003cem\u003eM. vulgare\u003c/em\u003e L. were collected from the Bir ghbalou region, Bouira province, Algeria. The plant material was initially cleaned under running water to remove any dust or other impurities. It was then shade-dried at room temperature under continuous aeration for 20 days. The dried plant material was then ground to a particle size of less than 250 \u0026micro;m.\u003c/p\u003e \u003cp\u003eThe powdered plant material (10 g) was extracted by maceration for 24 hours at room temperature with continuous stirring using two solvents: water and 80% methanol, in a 1/10 weight/volume ratio. The extracts were then filtered through Whatman N\u0026deg;3 filter paper, and the solvents were removed in an oven at 40\u0026deg;C. The dry extracts obtained were stored in airtight containers at 4\u0026deg;C until further analysis (Falleh et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Perez et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of total polyphenols\u003c/h3\u003e\n\u003cp\u003eTotal polyphenol content in aqueous and methanolic extracts was determined using the method described by Tighilet et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This method is based on the use of the Folin-Ciocalteu reagent. 200 \u0026micro;L of each extract was mixed with 1.5 mL of Folin-Ciocalteu reagent (diluted 10-fold with distilled water). Subsequently, 1.5 mL sodium carbonate solution (6% w/v) was added after a five-minute interval. The mixture was incubated at room temperature for two hours, after which the absorbance was measured at 765 nm using a UV-Vis spectrophotometer. A calibration curve was prepared using gallic acid as a standard, and total polyphenol content was expressed in milligrams of gallic acid equivalents per gram of dry matter (mg GAE/g DM).\u003c/p\u003e\n\u003ch3\u003eFlavonoid assay\u003c/h3\u003e\n\u003cp\u003eThe flavonoid content of \u003cem\u003eM. vulgare\u003c/em\u003e extracts was determined using the colorimetric method described by Khettal et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This method is based on the formation of yellowish complexes between flavonoids and aluminum trichloride (AlCl\u003csub\u003e3\u003c/sub\u003e) and is used to quantify flavonoid content. Free hydroxyl groups in the C3 or C5 position, as well as oxygen in the C4 position, are responsible for complexation with AlCl3.\u003c/p\u003e \u003cp\u003eBriefly, an aliquot of the extract (1 ml) was mixed with 1 ml of a 2% AlCl\u003csub\u003e3\u003c/sub\u003e solution in methanol. The mixture was incubated at room temperature for 10 minutes, and absorbance was measured at 415 nm using a UV-Vis spectrophotometer. A calibration curve was prepared using quercetin as a standard, and flavonoid content was expressed in milligrams of quercetin equivalents per gram dry matter (mg (QE)/g MD).\u003c/p\u003e\n\u003ch3\u003eFlavonol Assay\u003c/h3\u003e\n\u003cp\u003eThe flavonol content in the \u003cem\u003eM. vulgar\u003c/em\u003ee extracts was determined using the method described by Kumaran \u0026amp; Karunakaran (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In this method, 250 \u0026micro;L of the extract was mixed with 250 \u0026micro;L of 2% aluminum trichloride (AlCl\u003csub\u003e3\u003c/sub\u003e) solution, followed by the addition of 1.5 mL of 5% sodium acetate. The mixture was incubated in the dark at room temperature for 2 hours and 30 minutes. After the incubation period, the absorbance was measured at 440 nm using a UV-Vis spectrophotometer. A control was prepared under the same conditions, but without the addition of the plant extract. A calibration curve was prepared using quercetin as a standard, and flavonoid content was expressed in milligrams of quercetin equivalents per gram dry matter (mg (QE)/g MD). All the experiments were carried out three times independently.\u003c/p\u003e\n\u003ch3\u003eCondensed tannin content\u003c/h3\u003e\n\u003cp\u003eThe condensed tannin content of extracts was determined using the method described by Chew et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). 0.5 mL of the crude extract was mixed with 3 mL of a 4% vanillin solution. Subsequently, 1.5 mL of hydrochloric acid (HCl) was added. The mixture was kept in the dark at room temperature for 15 minutes. The absorbance of the mixture was measured at 500 nm against a blank using a UV-Vis spectrophotometer. Each extract was analyzed in triplicate and the results expressed as milligrams of catechin equivalents per gram of dry matter (mg CE/g DM).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of antioxidant capacity\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eDPPH assay\u003c/h2\u003e \u003cp\u003eIn order to determine the free radical scavenging capacity of our extracts by the DPPH assay, we used the method described by (Masuda et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA volume of 1 ml of DPPH methanolic solution (0.1 Mm) was added to 1 ml of extract at concentrations ranging from 20 to 220 \u0026micro;g/ml. In parallel, a negative control was prepared by mixing 1ml of methanol or distilled water with 1ml of DPPH solution. After 30 min incubation in the dark at room temperature, absorbance was measured at 517 nm. The positive control was a solution of two standard antioxidants, gallic acid and quercetin, whose absorbance was measured under the same conditions. The test was repeated independently three times.\u003c/p\u003e \u003cp\u003e The antioxidant power of plant extracts assessed by the DPPH test is expressed as a percentage reduction in the DPPH radical according to the following formula:\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003cp\u003e(%) = [A\u003csub\u003e0\u003c/sub\u003e \u0026ndash; (A\u003csub\u003e1\u003c/sub\u003e \u0026ndash; A\u003csub\u003e2\u003c/sub\u003e) / A\u003csub\u003e0\u003c/sub\u003e] \u0026times; 100\u003c/p\u003e\n\u003cp\u003eWhere: % = percentage of DPPH radical trapped;\u003c/p\u003e \u003cp\u003eA\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;Absorbance of the control reaction (methanol\u0026thinsp;+\u0026thinsp;DPPH);\u003c/p\u003e \u003cp\u003eA\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;Absorbance of the extract\u0026thinsp;+\u0026thinsp;DPPH;\u003c/p\u003e \u003cp\u003eA\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;Absorbance of the extract with methanol (extract\u0026thinsp;+\u0026thinsp;methanol).\u003c/p\u003e \u003cp\u003eThe scavenging efficiency of the DPPH radical is determined by calculating the EC\u003csub\u003e50\u003c/sub\u003e, i.e. the concentration of plant extract required to reduce the DPPH- radical by 50%.\u003c/p\u003e \u003cp\u003e \u003cb\u003eABTS\u003c/b\u003e \u003csup\u003e \u003cb\u003e\u0026bull;+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003ecation radical trapping activity.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe free radical scavenging activity of the extracts was assessed using the ABTS\u003csup\u003e\u003cb\u003e˙+\u003c/b\u003e\u003c/sup\u003e radical scavenging method (Re et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The ABTS\u003csup\u003e\u003cb\u003e˙+\u003c/b\u003e\u003c/sup\u003e radical cation was generated by mixing a 7 mM solution of ABTS with a 2.45 mM solution of potassium persulphate (K\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e). This mixture was kept protected from light and at room temperature for 16 hours. The ABTS\u003csup\u003e\u003cb\u003e˙+\u003c/b\u003e\u003c/sup\u003e solution was then diluted with distilled water to give an absorbance of 0.700\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 at 734 nm.\u003c/p\u003e \u003cp\u003eFor the assay, 1.9 ml of ABTS\u003csup\u003e\u003cb\u003e˙+\u003c/b\u003e\u003c/sup\u003e solution was mixed with 100 \u0026micro;l of extract solution at different concentrations. After incubation for 7 minutes in the dark at room temperature, absorbance was measured at 734 nm. A positive control was performed using a solution of the standard antioxidant, Trolox, under the same conditions. The test was performed in triplicate for each concentration. The percentage ABTS\u003csup\u003e\u003cb\u003e˙+\u003c/b\u003e\u003c/sup\u003e radical scavenging activity of the extracts was calculated as follows:\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e% I = [(A\u003csub\u003e0\u003c/sub\u003e \u0026ndash; A\u003csub\u003e1\u003c/sub\u003e) / A\u003csub\u003e0\u003c/sub\u003e] \u0026times; 100\u003c/h2\u003e \u003cp\u003eWhere: % inhibition: Percentage inhibition of the ABTS\u003csup\u003e\u003cb\u003e˙+\u003c/b\u003e\u003c/sup\u003e radical;\u003c/p\u003e \u003cp\u003eA\u003csub\u003e0\u003c/sub\u003e: Absorbance of the control (containing only ABTS\u003csup\u003e\u003cb\u003e˙+\u003c/b\u003e\u003c/sup\u003e);\u003c/p\u003e \u003cp\u003eA\u003csub\u003e1\u003c/sub\u003e: Absorbance of the extract in the presence of ABTS\u003csup\u003e\u003cb\u003e˙+\u003c/b\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eResults are expressed as \u0026micro;M of Trolox equivalent per g of dry matter (\u0026micro;M TE/ g DM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eβ-Carotene Bleaching Test\u003c/h2\u003e \u003cp\u003eThe antioxidant test using \u0026szlig;-carotene bleaching was determined according to the protocol previously described by Koleva et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Briefly, 0.5mg of \u0026szlig;-carotene was dissolved in 1ml of chloroform and mixed with 25\u0026micro;l of linoleic acid and 200\u0026micro;l of tween 20. The chloroform was evaporated under vacuum at 40\u0026deg;C, then 100ml of distilled water was added. 2.5ml of the emulsion obtained was transferred to different tubes containing 500\u0026micro;l of the extract. The tubes were immediately incubated in a water bath at 50\u0026deg;C for 120 min and the absorbance was measured at 470 nm before and after the heat treatment. A blank control was run in parallel. BHT was used as a positive control and all tests were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of anti-enzymatic activity\u003c/h2\u003e \u003cp\u003eAnti-enzymatic activities were assessed using enzymes involved in enzymatic browning, namely polyphenoloxidase and peroxidase, extracted from red cabbage. The enzymes were extracted according to the protocol of Ponce et al (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Red cabbage was first cleaned, then ground and homogenised using a high-speed electric mixer for 3 minutes with 30 ml of distilled water at 4\u0026deg;C. The preparation was then filtered through two layers of gauze and centrifuged at 1000g for 15 minutes at 4\u0026deg;C. The supernatant obtained was used as an enzyme extract.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of anti-peroxidase activity\u003c/h2\u003e \u003cp\u003ePeroxidase inhibition by marrubium leaves and stems extracts was measured according to the protocol described by Ponce et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). For this, 2.87 ml of substrate solution, consisting of 10 ml 1% guaiacol, 10 ml 0.3% hydrogen peroxide and 100 ml phosphate buffer (0.05 M, pH 6.5), were mixed with 0.03 ml extract and 0.1 ml enzyme extract. Enzyme activity was monitored by measuring the change in absorbance at 470 nm for 5 minutes at room temperature.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of anti-polyphenoloxidase activity\u003c/h2\u003e \u003cp\u003ePolyphenoloxidase activity was measured following the protocol of Lee et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The substrate used is pyrocatechol, whose oxidation leads to a red-brown coloration. Briefly, 1 ml marrube extract was mixed with 0.1 ml enzyme extract and 0.9 ml phosphate buffer (50 mM, pH 6.8). The resulting solution was incubated for 5 minutes at 25\u0026deg;C, then 1 ml of 0.2 M pyrocatechol was added. The change in optical density (OD) was measured at 420 nm for 1 minute.\u003c/p\u003e \u003cp\u003eThe enzymatic inhibition rate, expressed as a percentage, is calculated as follows:\u003c/p\u003e \u003cp\u003eInhibition rate (%) = [(Initial activity* - Residual activity**)/ (Initial activity*)]x 100\u003c/p\u003e \u003cp\u003ewhere:\u003c/p\u003e \u003cp\u003eInitial activity*: Enzyme activity (OD/min) measured in the absence of marrube extract.\u003c/p\u003e \u003cp\u003eResidual activity**: Enzymatic activity (ΔDO/min) measured in the presence of marrube extract.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThree replicates were carried out for each sample. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, calculated using Microsoft Office Excel 2007.\u003c/p\u003e \u003cp\u003eStatistical analysis was performed with the ANOVA/MANOVA test, using STATISTICA 5.5 software. Significant differences were considered at a threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of phenolics compounds\u003c/h2\u003e \u003cp\u003eColorimetric analyses of marrube extracts reveal that methanolic extracts contain significantly higher concentrations of polyphenols, flavonoids, flavonols and tannins than aqueous extracts, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Polyphenols ranged from 23.48 to 35.36 mg GAE/g DM in the methanolic extracts and from 13.78 to 15.41 mg GAE/g DM in the aqueous extracts, with a higher concentration in the leaves than in the stems. Flavonoids followed the same trend, with leaves showing higher levels (20.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 and 13.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 EQ/g DM for methanolic and aqueous extracts respectively) than stems. Flavonoids dominated among the polyphenols, with proportions above 0.5 in the extracts. Flavonol concentrations were also higher in the methanolic extracts than in the aqueous extracts for both parts of the plant. On the other hand, tannin levels were higher in the stems than in the leaves, with significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results corroborate previous studies and show that methanol is particularly effective at extracting phenolic compounds from plants. In addition, experimental conditions (choice of solvent, concentration, extraction time and temperature, extraction method) and environmental factors (geographical region, altitude, exposure, harvesting season) have a considerable influence on the concentrations of these compounds. However, it should be noted that the method for determining phenolic compounds can be affected by interference from other substances present in the extracts (Bouterfas et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zahedifar et Najafian 2023).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhenolic compound content of \u003cem\u003eM. vulgare\u003c/em\u003e leaves and stems extracts.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePart of plant used\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSolvents\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExtraction yield (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTPC (mg GAE/g DM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFlavonoids (mg QE/g DM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFlavonl (mg QE/g DM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTannins (mg CE/g DM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLeaves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e64.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.81\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAqueous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e56.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStems\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e72.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAqueous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e65.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTPC: Total phenolic compounds; DM: Dry matter; GAE: Gallic acid equivalent; QE: Quercetin equivalent; CE: Catechine equivalent. Values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3). Means followed by the same letter are not different according to ANOVA analysis of variance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant activity\u003c/h2\u003e \u003cp\u003eThe antioxidant activity of \u003cem\u003eM. vulgare\u003c/em\u003e extracts was assessed by three \u003cem\u003ein vitro\u003c/em\u003e assays: ABTS, DPPH, and β-carotene bleaching test. These tests provide a comprehensive assessment of the antioxidant efficacy of marrube extracts by quantifying their ability to neutralize various free radicals and protect oxidation-sensitive compounds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDPPH radical scavenging activity\u003c/h2\u003e \u003cp\u003eThe results of the evaluation of the antiradical activity of \u003cem\u003eM. vulgare\u003c/em\u003e extracts against the DPPH radical show a dose-dependent response with significant differences between extracts (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Methanolic extracts showed high percentages of inhibition, ranging from 79.94% for stems to 93.97% for leaves. In comparison, aqueous extracts had lower inhibition percentages, ranging from 61.27% for stems to 78.38% for leaves. The methanolic leaf extract has the highest antioxidant activity with an IC50 of 20.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62 \u0026micro;g/ml, while the aqueous stem extract displays the lowest activity with an IC50 of 87.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34 \u0026micro;g/ml. This high antioxidant activity can be attributed to the phenylpropanoid glycosides present in the plant, such as forsythoside B, ballottetroside, arenarioside and actenoside, known for their powerful antioxidant properties (Sahpaz et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Martin-Nizard et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). These results concur with earlier studies, carried out on \u003cem\u003eM. vulgare\u003c/em\u003e from Poland, which also showed significant antioxidant activity of methanolic extracts against the DPPH radical. On the other hand, the plant's strong anti-free radical activity is generally linked to the synergistic effect between the phenolic compounds and flavonoids present in the extracts (Ghedadba, 2014).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntioxidant activity of \u003cem\u003eM. vulgare\u003c/em\u003e extracts.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAssay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDPPH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ-Carotene\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSample\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAqueous leaves extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e76.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e140.62\u0026thinsp;\u0026plusmn;\u0026thinsp;5.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethanolic leaves extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e118.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.84\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAqueous stem extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e87.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e187.50\u0026thinsp;\u0026plusmn;\u0026thinsp;7.34\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethanolic stem extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e165.67\u0026thinsp;\u0026plusmn;\u0026thinsp;6.41\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGallic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercitin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBHT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); BHT: Butylhydroxytoluene; DPPH: 2,2-diphenyl-1-picrylhydrazyl; IC\u003csub\u003e50\u003c/sub\u003e: 50% inhibitory concentration. Values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3). Means followed by the same letter are not different according to ANOVA analysis of variance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eABTS radical trapping activity\u003c/h2\u003e \u003cp\u003eThe results in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e show that, at a concentration of 1 mg/ml, the methanolic extracts of marrubus showed a percentage inhibition of the ABTS radical of 65.10% for stems and 68.24% for leaves. In contrast, aqueous extracts showed inhibition percentages of 52.26% for stems and 58.11% for leaves. The IC\u003csub\u003e50\u003c/sub\u003e concentrations of the ABTS radical for the various marrube extracts ranged from 67.14 to 95.714 \u0026micro;g/ml. The methanolic and aqueous leaf extracts show high ABTS anti-radical activity, with IC\u003csub\u003e50\u003c/sub\u003e of 67.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 \u0026micro;g/ml \u0026micro;g/ml and 76.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 \u0026micro;g/ml, respectively. Whereas, methanolic and aqueous extracts of stems show lower activity, with IC\u003csub\u003e50\u003c/sub\u003e of 87.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88 \u0026micro;g/ml and 95.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54 \u0026micro;g/ml, respectively.\u003c/p\u003e \u003cp\u003eSeveral studies have demonstrated that total polyphenol and flavonoid content is strongly correlated with ABTS radical scavenging activity, with correlation coefficients around 0.8 (Khettal et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These results suggest that phenolic compounds, and flavonoids in particular, play a major role in the antioxidant activity observed (Floegel et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, other research has also highlighted that antioxidant activity can be influenced by non-phenolic compounds, such as lipids, sugars and chlorophyll (Heim et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eβ-carotene bleaching test\u003c/h2\u003e \u003cp\u003eThe principle of the β-carotene discoloration test is based on the disappearance of its yellow color, which results from its interaction with free radicals generated by the oxidation of linoleic acid in an emulsion (Ghedadba., et al, 2014).\u003c/p\u003e \u003cp\u003eThe results of the β-carotene bleaching test in the presence of the extracts and the standard antioxidant (BHA) reveal dose-dependent kinetics (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The most significant inhibition was observed with the methanolic leaf extract (79.74%), followed by the aqueous extract of the same part of the plant (67.65%). Furthermore, the results indicate that leaf extract significantly inhibits β-carotene oxidation compared to stem extract, with an inhibition rate of 56.21% for the methanolic extract and 52.29% for the aqueous extract. The 50% inhibitory concentration values (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) reveal that \u003cem\u003eM. vulgare\u003c/em\u003e leaf extract in methanol has the highest activity, with an IC\u003csub\u003e50\u003c/sub\u003e value of 118.75 \u0026micro;g/ml. This is followed by the aqueous leaf extract, with an IC\u003csub\u003e50\u003c/sub\u003e of 140.62 \u0026micro;g/ml. In contrast, the stem extracts show less activity, with IC\u003csub\u003e50\u003c/sub\u003e values of 165.25 \u0026micro;g/ml for the methanolic extract and 187.5 \u0026micro;g/ml for the aqueous extract.\u003c/p\u003e \u003cp\u003eOur results concur with those of Ghedadba et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), who showed that the methanolic extract of \u003cem\u003eM. vulgare\u003c/em\u003e exhibits the highest coefficient of antioxidant activity in the β-carotene bleaching system. This observation is also in line with the work of several other research groups, which have demonstrated a positive correlation between phenolic compound content and antioxidant activity. This activity is influenced by a variety of factors (Ghedadba et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eRelationship between antioxidant activity and phenolic compound content\u003c/h2\u003e \u003cp\u003eThe phenolic compound content of an extract or food is generally linked to enhanced antioxidant activity, thanks to their ability to scavenge or reduce free radicals, thus protecting cells from oxidative damage. Correlation analysis revealed that \u003cem\u003eM. vulgare\u003c/em\u003e extracts contain 89% flavonoids and 84% flavonols (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This study also showed a strong correlation between the presence of flavonoids and flavonols and antioxidant activity measured by DPPH, ABTS, and β-carotene assays, with correlation coefficients ranging from 0.67 to 0.95. This indicates that flavonoids are the main contributors to the antioxidant activity of phenolic compounds, confirming their efficacy in protecting the body against oxidative damage... On the other hand, a high correlation was observed between the β-carotene test and the DPPH and ABTS tests, with coefficients of 0.93 and 0.97 respectively. This high correlation is attributed to the presence of bioactive molecules with powerful reducing and free radical scavenging properties. Previous studies, including those by Khettal et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), have shown a good correlation between total polyphenols and the antioxidant activity of plant extracts.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation between \u003cem\u003eM. vulgare\u003c/em\u003e phenolic compounds and antioxidant activities.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTPC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlavonoides\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFlavonl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTannins\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDPPH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eβ-Carotene\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavonoides\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavonl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTannins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDPPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eABTS\u003csup\u003e\u0026bull;+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-Carotene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTPC: Total phenolic compounds; ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH: 2,2-diphenyl-1-picrylhydrazyl.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAnti-browning enzyme activity\u003c/h2\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eAnti-peroxidase activity\u003c/h2\u003e \u003cp\u003eEnzymatic browning can be controlled by three main methods: inhibition of the enzymes responsible, trapping of quinones, or oxygen limitation (Moon et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). We evaluated the efficacy of methanolic and aqueous extracts of \u003cem\u003eM. vulgare\u003c/em\u003e in inhibiting the activity of peroxidase extracted from red cabbage, a key enzyme in the browning of fruit and vegetables. At a concentration of 500 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), the methanolic extracts showed maximum inhibition of 42.68% for leaves and 28.52% for stems, while the aqueous extracts showed inhibitions of 22.73% and 11.40%, respectively. In comparison, ascorbic acid, used as a control, showed 89.85% inhibition at 100 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that various plant extracts can inhibit peroxidase activity in several vegetables (Ponce et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Variations in the efficacy of \u003cem\u003eM. vulgare\u003c/em\u003e extracts may be due to the diversity of bioactive substances present, as well as differences between isoenzymes and experimental conditions. The observed inhibitory efficacy could also result from the presence in marrube extracts of compounds analogous to the peroxidase substrate, which could enhance their ability to inhibit this enzyme.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eAnti-polyphenoloxidase activity\u003c/h2\u003e \u003cp\u003eThe Anti-polyphenoloxidase (anti-PPO) activity of aqueous and methanolic extracts of \u003cem\u003eM. vulgare\u003c/em\u003e leaves and stems was evaluated using PPO extracted from red cabbage. The results indicate that these extracts lead to a reduction in PPO activity, this reduction varying according to the part of the plant and the extraction solvent (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Methanolic extracts of leaves and stems inhibited red cabbage PPO activity by 51.40% and 47.68% respectively. In contrast, aqueous extracts showed inhibition rates of 38.31% for leaves and 33.64% for stems.\u003c/p\u003e \u003cp\u003ePrevious research has shown that natural antioxidants can act as PPO inhibitors in fruit and vegetables. These results are corroborated by the work of Lee et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), who observed an inhibition of PPO in taro ranging from 54.8\u0026ndash;68.5% by onion extracts. Other studies have also reported similar effects with natural extracts on PPO activity in plants (Vhangani et Van Wyk, 2021; Arnold et Gramza-Michałowska, 2022; Sui et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe variations in inhibition rates observed between different extracts and enzyme sources may be attributed to the presence of several isoenzymes in the extract, each with distinct structural and physicochemical specificities and properties, or to the nature of the active molecules present in the inhibitory plant extracts (Khettal et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercentage inhibition of peroxidase and polyphenoloxidase by \u003cem\u003eM. vulgare\u003c/em\u003e extracts.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExtracts\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnti-polyphenoloxidase activity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnti-peroxidase activity (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaves methanolic extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e51.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeaves aqueous extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStems methanolic extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e47.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStems aqueous extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e33.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAscorbic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e93.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e89. 85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results of the study showed that methanolic extracts of \u003cem\u003eM. vulgare\u003c/em\u003e leaves are particularly rich in phenolic compounds, especially flavonoids, compared with stem extracts. Evaluation of antioxidant activities, through DPPH, ABTS and beta-carotene bleaching tests, as well as analysis of the phenolic content of the extracts, revealed a good correlation between phenolic compound concentration and the performance of the various antioxidant tests. In addition, \u003cem\u003eM. vulgare\u003c/em\u003e extracts demonstrated a more marked inhibition of polyphenoloxidase than peroxidase extracted from red cabbage. This inhibition was particularly effective with methanolic leaf extracts, underlining their superior potential in preventing enzymatic browning. These results indicate that methanolic extracts of \u003cem\u003eM. vulgare\u003c/em\u003e leaves could be a promising option for antioxidant and anti-browning applications in the food and cosmetics industries.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAćimović M, Jeremić K, Salaj N, Gavarić N, Kiprovski B, Sikora V, Zeremski T (2020) \u003cem\u003eMarrubium vulgare\u003c/em\u003e L.: A phytochemical and pharmacological overview. Molecules 25(12):2898\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmed B, Masoodi MH, Siddique AH, Khan S (2010) A new monoterpene acid from \u003cem\u003eMarrubium vulgare\u003c/em\u003e with potential antihepatotoxic activity. 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Nat Prod Res 22(14):1270\u0026ndash;1278\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez YY, Jimenez-Ferrer E, Alonso D, Botello-Amaro CA, Zamilpa A (2010) Citrus limetta leaves extract antagonizes the hypertensive effect of angiotensin II. J Ethnopharmacol 128(3):611\u0026ndash;614\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePonce AG, Del Valle CE, Roura SI (2004) Natural essential oils as reducing agents of peroxidase activity in leafy vegetables. LWT-Food Sci Technol 37(2):199\u0026ndash;204\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRe R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. 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Crit Reviews Food Sci Nutr 15(1):49\u0026ndash;127\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVergara-Galicia J, Aguirre-Crespo F, Tun-Suarez A, Aguirre-Crespo A, Estrada-Carrillo M, Jaimes-Huerta I, Flores-Flores A, Estrada-Soto S, Ortiz-Andrade R (2012) Acute hypoglycemic effect of ethanolic extracts from \u003cem\u003eMarrubium vulgare\u003c/em\u003e. Phytopharmacology 3(1):54\u0026ndash;60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVhangani LN, Van Wyk J (2021) Heated plant extracts as natural inhibitors of enzymatic browning: A case of the Maillard reaction. J Food Biochem 45(2):13611\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZahedifar M, Najafian S (2023) Variation of antioxidant activity and phenolic compositions of \u003cem\u003eMarrubium vulgare\u003c/em\u003e L. as influenced by organic acids. J Med Plants 22(87):77\u0026ndash;88\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"University of Béjaïa","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"antioxidant activity, phenolic compounds, Enzymatic Browning, Marrubium vulgare","lastPublishedDoi":"10.21203/rs.3.rs-6111477/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6111477/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn recent decades, medicinal plants have attracted significant interest due to their demonstrated therapeutic properties. This study analyzed different extracts of \u003cem\u003eMarrubium vulgare L.\u003c/em\u003e (horehound) to assess their phytochemical composition and biological activities. Quantitative analysis revealed that methanolic leaf extracts were the richest in polyphenols, containing 23.48 to 35.36 mg EAG/g DM. These leaf extracts also had a particularly high flavonoid content, ranging from 12.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.233 to 20.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 mg EQ/g DM. In contrast, aqueous stem extracts were abundant in condensed tannins, with 72.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.772 and 65.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.216 mg EC/g DM in aqueous and methanolic extracts, respectively. The antioxidant potential of the extracts was evaluated through several in vitro assays. Leaf extracts demonstrated the highest antioxidant activity, with methanolic extracts exhibiting stronger antioxidant capacity than aqueous extracts. Furthermore, leaf extracts were capable of inhibiting peroxidase activity by up to 42.68%, although they showed weak inhibition of polyphenol oxidase. These findings suggest that \u003cem\u003eMarrubium vulgare L\u003c/em\u003e is a promising source of natural antioxidants and enzyme inhibitors, with potential applications in the development of functional foods, cosmetics, and pharmaceuticals. The observed differences between extraction solvents highlight the importance of considering phytochemical profiles and bioactivities when selecting and optimizing plant-based ingredients.\u003c/p\u003e","manuscriptTitle":"Inhibition of Enzymatic Browning and Antioxidant Activities of Marrubium vulgare L Extracts: A Promising Natural Solution","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-27 11:11:09","doi":"10.21203/rs.3.rs-6111477/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1061080b-8804-4908-bfd5-2854f69968ce","owner":[],"postedDate":"February 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":44890852,"name":"Botany"},{"id":44890853,"name":"Applied Biochemistry"},{"id":44890854,"name":"Food Chemistry"}],"tags":[],"updatedAt":"2025-07-07T14:58:03+00:00","versionOfRecord":{"articleIdentity":"rs-6111477","link":"https://doi.org/10.1016/j.prmcm.2025.100658","journal":{"identity":"pharmacological-research-modern-chinese-medicine","isVorOnly":true,"title":"Pharmacological Research - Modern Chinese Medicine"},"publishedOn":"2025-07-05 00:00:00","publishedOnDateReadable":"July 5th, 2025"},"versionCreatedAt":"2025-02-27 11:11:09","video":"","vorDoi":"10.1016/j.prmcm.2025.100658","vorDoiUrl":"https://doi.org/10.1016/j.prmcm.2025.100658","workflowStages":[]},"version":"v1","identity":"rs-6111477","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6111477","identity":"rs-6111477","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}