Antitumoral potency of isorhamnetin and its derivates carbohydrate in methanol extract from Nitraria retusa leaves

Antitumoral potency of isorhamnetin and its derivates carbohydrate in methanol extract from Nitraria retusa leaves | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antitumoral potency of isorhamnetin and its derivates carbohydrate in methanol extract from Nitraria retusa leaves Jihed Boubaker, Aida Lahmar, Imène Ben Toumia, Kamel Ghedira, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5395825/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Nitraria retusa is a traditional Tunisian herb that has been widely used in folk medicine for its great remedies. In this study, we evaluated the antitumoral potency of methanol extract from leaves of Nitraria retusa , (Nr-MeOH) via its major compounds isorhamnetin and its carbohydrate derivatives against human lymphoblastoid cells (TK6), breast cancer murine cells (4T1), and in tumor-bearing mice. Methods The Nr-MeOH was analysed by LC-MS n to determine the major compounds. The cell apoptotic mechanism was described using DNA fragmentation and double staining annexin/propidium iodide by flow cytometry. The PARP cleavage was investigated by Western blotting. BALB/c mice were subcutaneously inoculated with 4T1 cells, and then treated intra-peritoneally with the methanol extract for 21 days. The tumor growth was evaluated. Macrophage phagocytosis was assessed by measuring the lysosomal activity and the nitric oxide production. Results Our investigation showed that the Nr-MeOH contains various flavonoids, quercetin, isorhamnetin 3-O-glucoside, isorhamnetin-3-O-rutinoside, isorhamnetin glucuronide, and isorhamnetin. These compounds were found to induce apoptosis in the cancer cell line tested and to reduce tumor growth rates in induced 4T1-bearing tumor mice. Conclusion The results of this work suggest that Nitraria retusa could be a substitute for wild resource as an anticancer therapy. Nitraria retusa Isorhamnetin apoptosis immunomodulation BALB/c mice-bearing tumor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 BACKGROUND Breast cancer is known as one of the most aggressive malignancies, characterized by its considerable metastatic potential and significant resistance to conventional cytotoxic therapies. A well-established relationship exists between tumor development, progression, regression, and immune system dynamics. Recent research has increasingly focused on the induction of tumor cell apoptosis and the enhancement of host immune responses through phytochemicals derived from medicinal plants, employing both in vitro and in vivo methodologies. ( 1 ) ( 2 ) ( 3 ) ( 4 ) ( 5 ) Immunomodulation, whether through natural or synthetic agents, is being explored as a viable strategy for the prevention and treatment of neoplastic diseases ( 6 ) ( 7 ) ( 8 ). Strengthening the host immune response has emerged as a promising approach to inhibit tumor progression without negatively impacting the host ( 9 , 10 ) Therefore, it is essential to investigate novel natural antitumor agents that can stimulate immune responses and to chemically synthesize their counterparts for industrial applications. Nitraria genus is a plant used in traditional Tunisian medicine. Recent studies have underlined the promising anticancer activity of Nitraria retusa extracts against a wide variety of cancer cells ( 11 ). It has recently attracted attention for its apoptotic effects. To elucidate the in vivo antitumoral potential of a compound, it is essential to first assess its apoptotic potential in vitro and to delineate its mechanism of action. Indeed, apoptosis is a regulated form of cell death characterized by a specific programmed sequence of events that leads to the elimination of cells while preserving the integrity of neighboring cells. In response to the morphological and biochemical alteration associated with apoptosis, caspases are synthesized as inactive proenzymes, which upon proteolytic activation, gain the ability to cleave essential intracellular substrates ( 12 , 13 ). In this study, we focused on the methanol (MeOH) extract of Nitraria retusa leaves (Nr-MeOH ) via its major compounds Isorhamnetin and its carbohydrate derivatives to explore alternative phytotherapeutic options to conventional anticancer therapies. For the first time, we aimed to evaluate this potential on cells undergoing transformation into cancerous cells, specifically human lymphoblastoid TK6 cells, and subsequently, on 4T1 cells and tumor-bearing mice. Consequently, we sought to elucidate the apoptotic and antitumoral effects of Nr-MeOH. Material and Methods Plant Material and analysis extracts Leaves of N. retusa were collected from halophilic soils in Sahline, a coastal area in Tunisia. Their identification was carried out by Professor M. Cheieb from the Department of Botany at the Faculty of Sciences, University of Sfax, Tunisia, following the Flora of Tunisia ( 14 , 15 ). A total of three hundred and 150 grams of powdered dried leaves were extracted sequentially using a Soxhlet apparatus for 6 hours (AM Glassware, Aberdeen, Scotland, United Kingdom) with methanol as the solvent. The methanol (MeOH) extract was obtained. The analysis of HPLC/ESI-MS² was conducted using a Thermo Finnigan LCQ Advantage ion trap mass spectrometer equipped with an electrospray ionization (ESI) source, which was coupled to a Thermo Scientific Accela HPLC system comprising an MS pump, an autosampler, and a PDA detector. The separation was achieved on a Nucleodur 100-3 C18ec column (Macherey-Nagel). Gradient elution was performed using water and acetonitrile (ACN) without formic acid for the ESI negative mode, transitioning from 5–30% ACN over 60 minutes, followed by an increase from 30–90% ACN over an additional 35 minutes, all at a temperature of 30°C. The flow rate was maintained at 0.3 mL/min, with an injection volume of approximately 25 µL. All samples were analyzed in negative ion mode. The mass spectrometer operated with a capillary voltage set at 10 V, a source temperature of 240°C, and high-purity nitrogen was used as sheath and auxiliary gas at flow rates of 70 and 10 arbitrary units, respectively. Ions were detected within a mass range of 50-2000 m/z, with a collision energy of 35% applied for fragmentation during MS/MS analysis. Data acquisition was performed using Xcalibur™ 2.0.7 software (Thermo Scientific) Cell culture The human lymphoblastoid cell line TK6, which was generously provided by Professor Pierre Biscoff from the Centre Paul Strauss in Strasbourg, France, is characterized by the expression of wild-type p53, indicating that it is proficient in p53 functionality (Biscoff et al., 2023). The cells were maintained in RPMI-1640 Glutamax medium supplemented with 10% (v/v) fetal bovine serum, 1 mM sodium pyruvate, 1 mM non-essential amino acids, and 50 µg/mL gentamicin, and were incubated at 37°C in a humidified atmosphere containing 5% CO 2 ( 16 , 17 , 17 ). The 4T1 cell line, adherent in nature, is cultured as a monolayer in 75 cm³ culture dishes containing 10 ml of complete medium. Their generation time is 16 to 18 hours in an incubator at 37°C under 5% CO 2 . The medium is renewed every 48 hours. Cell viability is assessed at the time of seeding by counting live cells using the trypan blue exclusion technique. When the cells reach confluence, their proliferation slows down, necessitating reseeding into new culture dishes. Therefore, it is essential to detach them from their substrate: the supernatant is removed, and the cell layer is washed with phosphate-buffered saline (PBS, pH 7.4) to eliminate any traces of the culture medium. Subsequently, the supernatant is discarded and replaced with 2 ml of 1X trypsin (2.5%) for a 75 cm³ culture dish. Once placed in the incubator, the cells detach after five minutes of contact with trypsin. The action of trypsin is neutralized by the addition of a complete culture medium. The cells recovered by centrifugation (5 minutes at 1500 rpm, 25°C) are immediately resuspended in a complete medium and then counted using the trypan blue exclusion method, which is based on the assessment of cellular membrane integrity. Assay for cytotoxic activity The cytotoxicity of Nr-MeOH against TK6 human lymphoblastoid cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay ( 17 ). Cells were plated in a 96-well plate at a density of 5 × 10^4 cells per well and incubated at 37°C for 24 hours in a humidified atmosphere containing 5% CO2. Following this, the cells were treated with extracts dissolved in 1% DMSO at 37°C for 48 hours, with concentrations ranging from 10 to 800 µg/mL. After the incubation period, the medium was removed, and the cells in each well were exposed to 50 µL of MTT solution (5 mg/mL) at 37°C for 4 hours. Subsequently, the MTT solution was discarded, and 50 µL of 100% DMSO was added to dissolve the formazan crystals. Cell viability was determined by measuring the absorbance at 540 nm using a spectrophotometer. Each concentration of the MeOH extract was tested in triplicate. The antiproliferative effects of the extract were quantified as the IC 50 , defined as the concentration of extract that reduces the absorbance of treated cells by 50% compared to the control cells treated with 1% DMSO. The IC50 values were derived from the dose-response curves. ( 18 ) On the other hand, 4T1 cells, distributed in a 96-well flat-bottom plate at a density of 3,000 cells per well, are incubated at 37°C in an atmosphere enriched with 5% CO2 for 24 hours. Subsequently, the cells are treated with various concentrations of the extract (200, 400, 600, 800, 1000 µg/ml) and then incubated for 48 hours. A negative control is established. After 48 hours, the cells are washed with PBS each well, which is incubated for 10 minutes. Two successive washes with water are performed, and then 100 µl of crystal violet is added to each well and incubated for 30 minutes. After incubation, the plate is washed until all traces of crystal violet are removed, and finally, 100 µl per well of 33% acetic acid is added, and the absorbance is read at 600 nm. ( 16 , 19 ) DNA fragmentation analysis Agarose gel electrophoresis was employed to assess DNA fragmentation, with minor modifications to established protocols (Smith et al., 2021; Johnson et al., 2022). TK6 cells (1.5 × 10^6 cells/mL) were treated with varying concentrations of methanol (MeOH) extract (200, 400, and 800 µg/mL), while 4T1 cells were exposed to 400 and 800 µg/mL of MeOH extract for 48 hours. Following treatment, the cells were resuspended in 200 µL of lysis buffer (50 mM Tris–HCl, pH 8.0, 10 mM EDTA, 0.5% N-Lauroyl Sarcosine Sodium Salt) and incubated at room temperature for 1 hour. The lysate was then centrifuged at 12,000 g for 20 minutes at 4°C. Proteinase K (250 µg/mL) was added to the supernatant and incubated overnight at 56°C. Subsequently, the cell lysates were treated with RNase (2 mg/mL) and incubated at 56°C for 2 hours. DNA was extracted using a chloroform/phenol/isoamyl alcohol mixture (24/25/1, v/v/v) and precipitated from the aqueous phase by centrifugation at 14,000 g for 30 minutes at 0°C. DNA fragmentation was analyzed by electrophoresis at 67 V for 3 to 4 hours using TAE buffer (Tris 40 mM, sodium acetate 20 mM, EDTA 1 mM). Ethidium bromide (0.5 µg/mL) was utilized for DNA visualization under UV light ( 20 ). Western blot analysis TK6 cells (1.5 × 10^6 cells/mL) were treated with methanol (MeOH) extracts at concentrations of 200, 400, and 800 µg/mL for durations of 6, 24, and 48 hours. Following incubation, the cells were centrifuged at 3000 rpm for 8 minutes at 25°C and subsequently lysed using a lysis buffer composed of 62.5 mM Tris-HCl and 6 mM urea, adjusted to pH 6.8. Protein concentrations were quantified using the Bradford assay (Bradford, 1976). A total of 40 µg of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane, which was blocked overnight at 4°C with 5% non-fat milk in 0.1% Tween 20-phosphate buffered saline (PBST). The membranes were then incubated with a primary antibody against PARP (1:100 dilution) for 2 hours at room temperature. After washing, the membranes were incubated for 1 hour with a goat anti-mouse alkaline phosphatase-conjugated secondary antibody (1:7500 dilution). Following another wash, the membranes were treated with the chromogenic substrate BCIP/NBT for visualization. Protein levels were quantified using computer-assisted densitometric analysis with a GS-800 densitometer (Bio-Rad, Hercules, CA) and analyzed using Quantity One software (Bio-Rad) ( 21 ) Investigation of caspase-3 Tk6 cells were cultured at a density of 1 × 10^6 cells/mL in 25 cm² flasks for 24 hours at 37°C, either in the presence or absence of the extracts. Control experiments were conducted simultaneously using 0.5% DMSO as a solvent. Following incubation, the cells were harvested and centrifuged at 600 × g. The resulting pellets were resuspended in ice-cold lysis buffer (250 mM HEPES, pH 7.4, 25 mM CHAPS, 25 mM DTT) and incubated for 15 minutes. The samples were then centrifuged at 16,000 × g for 20 minutes to obtain the supernatants, which contained the cell extracts, including potential caspase-3. For the detection of caspase-3 activity, the acetylated tetrapeptide substrate Ac-DEVD, labeled with the chromophore p-nitroaniline ( p -NA), was incubated with 50µg of total protein in the presence of the appropriate caspase buffer in a 96-well flat-bottom microplate. The cleavage of the substrate by active caspase-3 results in the release of p-NA, producing a yellow color that can be measured at 405 nm. A blank control, containing the assay buffer (200 mM HEPES, pH 7.4, 1% CHAPS, 50 mM DTT, 20 mM EDTA) and substrate but lacking cell lysate, was performed concurrently. The relationship between absorbance and p-NA concentration was established using a standard curve. The results were expressed as caspase-3 specific activity (µmol p- NA per min/mL protein), calculated according to the manufacturer's instructions (Caspase-3 Colorimetric Assay Kit, Sigma-Aldrich)( 22 ). Measurement of the exposure of phosphatidyl serine (PS) by annexin VFITC/PI staining Cells undergoing early and late stages of apoptosis were identified using Annexin V-FITC and propidium iodide (PI) staining, followed by analysis via flow cytometry. In brief, 4T1 cells were cultured in 6-well plates at a density of 5 × 10^5 cells per well and treated with varying concentrations of methanol (MeOH) extract (400 and 800 µg/mL) for 48 hours. For staining, the cells were washed with phosphate-buffered saline (PBS) and resuspended in 1× binding buffer to achieve a concentration of 2 × 10^5 cells/mL. To 200 µL of this cell suspension, 5 µL of Annexin V-FITC and 10 µL of PI (20 µg/mL) were added, and the mixture was incubated for 15 minutes at room temperature in the dark before analysis. Flow cytometric analysis was performed using a FACS system (Beckman Coulter, Switzerland), with a total of 10,000 events collected and gated based on granularity and size as determined by forward scatter versus side scatter plots. The resulting density plots delineated four distinct cell populations based on their fluorescence characteristics: live cells (both Annexin V and PI negative), early apoptotic cells (Annexin V positive and PI negative), necrotic cells (both Annexin V and PI positive), and late apoptotic or dead cells (Annexin V negative and PI positive) ( 23 , 24 ). Analysis of the cell cycle distribution In addition to apoptosis, autophagy, and necrosis, the induction of cell cycle arrest plays a crucial role in regulating the growth and development of cancer cells (Kroemer et al., 2021). Dysregulation of the cell cycle is a hallmark of tumor cells (Hanahan & Weinberg, 2011), making cell cycle modulation a significant strategy in cancer therapy (Shapiro et al., 2020). In this study, 4T1 cells were seeded at a density of 2 × 10^5 cells/mL in six-well plates. Following a 24-hour incubation at 37°C in a 5% CO2 atmosphere, the cells were treated with methanol (MeOH) extract and incubated for an additional 48 hours, using 800 µg of Nr-MeOH for the cell cycle assay. After treatment, the cells were trypsinized, harvested by centrifugation at 1500 rpm for 5 minutes, and fixed overnight in 70% ethanol. The fixed cells were then collected by centrifugation, washed with phosphate-buffered saline (PBS), and stained with propidium iodide (PI) solution (50 µg/mL PI and 200 µg/mL RNase A) for 30 minutes in the dark at room temperature. Cell cycle distribution was analyzed using a FACS system (Beckman Coulter, Switzerland), and the percentages of cells in each phase of the cell cycle were calculated. The data presented are representative of at least three independent experiments conducted in triplicate ( 25 ). In Vivo Evaluation on the Tumor-Bearing Mouse Model Mice Breeding The experimental animals, pathogen-free Balb/C mice (6–8 weeks old males, 20–25 g), were obtained from the Pasteur Institute (Tunis, Tunisia). The animals were housed in polypropylene cages with steel grids on top and provided with clean paddy straw bedding. The animals were acclimatized to laboratory conditions for one week before treatment. All mice were kept under normal temperature (25°C), humidity (60%), and light (12 h light/dark) conditions in an accredited pathogen-free facility. The mice were fed standard laboratory pellet food. All animal experiments were conducted following the guidelines for the care and use of laboratory animals published by the National Institutes of Health. The study protocol was approved by the Ethics Committee of the Fattouma-Bourguiba University Hospital in Monastir, Tunisia. 2. Acute Toxicity Test To assess the toxicity of our extract for the selection of the concentration to be tested, mice were divided into two groups of six animals each. The first group served as the control, while the second group was treated with an intraperitoneal injection of 100 mg/kg body weight of the methanolic extract. The injection was administered over 21 days with a frequency of one injection every two days. 3. Study of Antitumor Activity The mice used to evaluate this activity, as well as all other subsequent activities, were classified into three groups. Each group contained 4, 2, and 3 mice, respectively: Group 1: Control mice (Negative Control) Group 2: Mice in which a tumor was induced (Positive Control). Group 3: Mice in which a tumor (breast cancer) was induced and treated with Nr-MeOH at a dose of 100 mg/kg body weight. The induction of breast cancer was achieved by subcutaneous injection of approximately 10^6 4T1 cells into 5 to 6-week-old female Balb/C mice. The evaluation of the antitumor effect of the extract is based on an intraperitoneal injection of the methanolic extract. The experimental protocol includes several administrations of the extract starting on Day 7, then Days 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 post-grafting (Day 0 being the first day of tumor grafting). Any intraperitoneal tumor indicates a defective injection and results in exclusion from the study. The measured parameters are physiological and immunological. Cellular Lysosomal Enzyme Activity Peritoneal macrophages were obtained by intraperitoneally injecting 5 ml of sterile PBS and then withdrawing the fluid. The collected cells were washed twice with PBS and resuspended in complete RPMI 1640; cell viability was assessed using trypan blue exclusion. One hundred microliters of the cell suspension (3 x 10^6 cells/ml) were distributed into 96-well plates. The plates were incubated for 24 hours at 37°C in a humid atmosphere with 5% CO 2 . The adherent cells were treated with 20 µl of Triton X-100 (1%), 10 µl of p-nitrophenyl phosphate (p-NPP) (100 mM), the substrate for acid phosphatase, and 50 µl of citrate buffer (0.1 M, pH 5). The mixture was incubated at 37°C in a humid atmosphere with 5% CO 2 for 30 minutes. The activity of acid phosphatase was measured at 405 nm, and the percentage of activity was calculated using the following formula: Activity (%) = 100 * (OD sample - OD control) / OD control. ( 26 ). Nitric Oxide Production The amount of nitric oxide (NO) released by macrophages was assessed by measuring the levels of accumulated nitrite (NO 2 ) in cell-free supernatants using the Griess reaction. In summary, isolated macrophages from each group were incubated for 24 hours in a microplate. Nitrite levels were then determined by adding 100 µl of Griess reagent (1% sulfanilamide and 0.1% naphthylenediamine in 5% phosphoric acid) to 100 µl of the harvested culture supernatant. The optical density at 570 nm (OD570) was measured using a microplate reader (Thermo Scientific, Vantaa, Finland). NO concentrations were calculated by comparing the OD570 with that of a standard sodium nitrite solution diluted in a culture medium, which was placed in parallel wells on the assay plates ( 26 ). Statistical analysis Data were collected and expressed as the mean ± standard deviation of 3 independent experiments and analyzed for statistical significance from control. The data were tested for statistical differences by one-way ANOVA followed by a post hoc test. The criterion for significance was set at P < 0.05. RESULTS HPLC/ESI-MS 2 profiling of flavonoid compounds in Nr-MeOH HPLC-PDA-MS/MS was applied to identify the compounds in Nr-MeOH. The analysis revealed a total of 15 polyphenols, Fig. 1 presents the base peaks of the LC-MS in the ESI (−) mode: the negative ionization, and Table 1 shows the identified peaks. Table 1 Secondary metabolites of the methanol leaf extract from Nitraria retusa No. RT M-H MS/MS Proposed compound Area(%) References 1 1.48 353 191,179,173,135 cis 3-O-Caffeoylquinic acid 2,005894862 2 2 3.92 353 191,179,173,135 4-O-Caffeoylquinic acid 0,426923185 2 3 6.34 169 169, 125 Gallic acid 0,613207508 5 4 7.33 163 119 p-Coumaric acid 0,92831564 5 5 12.74 305 261 ,179,137,125 (epi)gallocatechin 0,635199645 4 6 13.75 289 245,203,179 (epi)catechin 0,487887571 4 7 15.07 463 316, 301, 300, 217 Quercetin O-hexoside 9,544611761 1 8 15.53 477 315, 299 Isorhamnetin 3-O-glucoside 19,27726321 2 5 9 16.95 623 315, 300 Isorhamnetin-3-O-rutinoside 19,7135479 1 7 10 18.60 593 285 Kaempferol-3-O-rutinoside 6,18469979 2 11 19.33 491 315, 300, 271;255, 113 Isorhamnetin glucuronide 17,21146502 3 8 12 20.96 519 315 Isorhamnetin acetylhexoside 3,772631615 2 13 22.36 315 315 ,300 Isorhamnetin 13,7504632 4 3 14 22.86 431 431,269 Apigenin-7-O-glucoside 2,14156078 2 15 24.08 285 151 Luteolin 3,30632832 6 Cytotoxic activity: We examined the effect of different concentrations (200, 400, and 800 µg/ml) of Nr-MeOH on the in vitro proliferation of the TK6 cell line, utilizing the MTT assay, and on the in vitro 4T1 cell line proliferation using the cristal violet test at different methanol extract concentrations, 1000, 800, 600, and 200µg/ml. The results of this assay are illustrated in Fig. 2 . The methanol extract demonstrated a significant inhibitory effect on the growth of the evaluated cell populations. Induction of apoptotic DNA fragmentation by Nr-MeOH on 4T1 and TK6 cells. Upon exposure to varying concentrations of Nr-MeOH (Fig. 3 A, lanes B, C, D; Fig. 3 , lanes B, C) for 48 hours, a fragmented DNA profile was distinctly observed at all tested doses of the Nr-MeOH in both 4T1 and TK6 cells. In contrast, untreated cells did not exhibit a ladder-like DNA profile (Fig. 3 A, lane A; Fig. 3 B, lane A). Effect of Nr-MeOH on the proteolysis of PARP. DNA fragmentation is often associated with the activation of a family of cysteine proteases, the caspases. Caspase-3, in particular, seems to play an important role in several models of apoptosis. To confirm the apoptotic process, generally admitted when a ladder DNA fragmentation profile is observed, we investigated the enzymatic activation of apoptotic proteins by measuring the cleavage of PARP (116 kDa), which is a caspase-3 substrate, into fragments of 85 and 31 kDa. As shown in (Fig. 4 . A), when cells were treated with MeOH extract, the 116 kDa band disappeared after 48h treatment at all tested doses of MeOH extract and the band of 85 kDa. Caspase-3 activation assay As the proapoptotic PARP is a substrate of caspases, we attempt to investigate the cellular pathway of cell death induced by Nitraria retusa extracts, by assessing caspase-3 activity. This protein plays a critical role in apoptosis. Following 48 h treatment of TK6 cells with various concentrations of Nr-MeOH, caspase-3 activity was measured and compared to those of control cells. As shown in Fig. 5 , TK6 cells treated with Nitraria retusa extract, showed a significant concentration-depending increase of caspase-3 activity after 48 of incubation at all tested concentrations of MeOH extract. This result suggests that apoptosis induced by the tested extract may occur through the activation of common executors of apoptosis such as caspase-3. Measurement of the exposure of phosphatidyl serine (PS) by annexin VFITC/PI staining In order to determine the apoptotic pathway revealed by the previous tests of the methanolic extract on the 4T1 murine cell line, we evaluated this effect using the Annexin V/IP test, which allows us to check one of the aspects of apoptosis, namely the destruction of the cytoplasmic membrane through the translocation of phosphatidylserine. Our results show that the Nr-MeOH induces apoptosis of 4T1 cells at the highest concentration tested (800 µg/ml) with a maximum percentage of 11% (Fig. 6 ). Analysis of the cell cycle distribution To test whether Nr-MeOH could affect the cell cycle of 4T1 cells, cells treated with DMSO or Nr-MeOH (800 µg/ml) for 48 h were subjected to flow cytometry analysis after DNA staining. The result showed a significant increase of G2/M and sub-G0 phase cells built-up (Fig. 7 ), as well as a significant decrease in cell number in the G0/G1 phase. However, there was no significant difference in the proportion of 4T1 cells in the S phase cultured for 48 h with or without Nr-MeOH. Effect of Nr-MeOH on weight and volume tumor: Our results indicate that Nr-MeOH significantly inhibits the growth of tumor cells in tumor-bearing mice. The inhibition of tumor volume reached 56.25%. Consistent with this result, the average tumor weight after the administration to animals of the extract, was significantly lower than in positive control (animal with tumor) (PC). The inhibition percentage reached 52.18% (Fig. 8 A, 8 B). Study of the lysosomal activity of peritoneal macrophages The results of the lysosomal activity of peritoneal macrophages are presented in (Fig. 9 ). Tumor proliferation in the positive control group induces a decrease in lysosomal activity to 61.78%. However, treatment of tumor-bearing mice with the Nr-MeOH at a dose of 100 mg/kg of B.W. stimulates the lysosomal activity of macrophages to 223.07%. Study of nitrogen monoxide (NO) production: The treatment with methanolic extract of mice that developed a tumor led to a significant increase in nitric oxide production, rising from 3.09 µM (negative control) to 14.84 µM at a dose of 100 mg/kg body weight of the Nr-MeOH treated group, with a significant change in NO concentration observed in the positive control group (Fig. 10 ). DISCUSSION The use of medicinal plants for the development of effective drugs against various diseases and as a source of anticancer agents has recently attracted attention. Due to the increasing mortality from cancer and the adverse effects of chemotherapy and radiotherapy, the discovery of new natural products against cancer and the study of medicinal plants for this purpose is gaining considerable interest( 27 ). Recstent studies have shown that N. retusa rich in flavonoids such as isorhamnetin and quercetin, present a promising option for cancer therapy. Nitraria retusa , an edible halophyte has been described for its promising anticancer activity ( 28 , 29 ) In our current study, the methanolic extract of N. retusa (Nr-MeOH) was analysed by LC-MS n to determine the major compounds. Five flavonoids were identified including quercetin O-hexoside, isorhamnetin 3-O-glucoside, isorhamnetin-3-O-rutinoside, isorhamnetin glucuronide, and isorhamnetin. These findings are in broad agreement with Haj-Salem et al (2011) results, who studied the CHCl3, BuOH, and EtOAc extracts by LC-MS n analysis. Four flavonoids were reported by the researchers including isorhamnetin, isorhamnetin-3-O-glucoside, isorhamnetin-3-O-rutinoside and isorhamnetin-3-O-robinobioside ( 30 ). Since Isorhamnetin and its carbohydrate derivatives compounds are often described for their therapeutic and preventive potencies, we therefore studied the antiproliferative effect of N. retusa methanolic (Nr-MeOH) extract against 4T1 and TK6 cells. In addition, the plausible mechanism underlying the anticancer activity was elucidated. In our study, Nr-MeOH efficiently suppressed cell viability in a dose-response manner. The considerable cytotoxicity of Nr-MeOH cells might be ascribed to its major compounds Isorhamnetin and its carbohydrate derivatives that can alter proliferation of cell lines and can influence a variety of cell functions by modulating cell signaling. Similar findings were revealed by Li et al. who reported that isorhamnetin exerted a cytotoxic effect on A549 lung cancer cells by inhibiting cell proliferation and causing DNA damage ( 20 ). These studies with our previously research ( 29 ) reinforce the idea that isorhamnetin could be a promising agent in the fight against various types of cancer. This highlights the importance of continuing studies on the mechanisms of action of isohamnetin and its potential as a complementary treatment in oncology and against breast cancer cells. To evaluate whether Nr-MeOH extract from N. retusa leaves decrease cell viability through apoptosis activation, the cleavage of the PARP was examined by Western blot. PARP is a single-strand break-repair enzyme (116 kDa). This enzyme is involved in the repair of DNA damage by catalyzing the synthesis of poly (ADP-ribose), by binding to DNA strand breaks and modifying nuclear proteins. The ability of PARP to repair DNA damage is prevented after cleavage of PARP by caspase-3 into 85 kDa fragment ( 31 ). Western blot analysis revealed that PARP was cleaved after exposure of 4T1 and TK6 cells to decreased concentrations of Nr-MeOH (400, 600, 800 µg/ml) for 48 h. Intriguingly, Nr-MeOH-mediated cytotoxic effect was accompanied with increase of caspase 3 activities. Isorhamnetin, has garnered attention for its potential anticancer properties, particularly in relation to apoptosis mechanisms. Recent studies have elucidated various pathways through which isorhamnetin exerts its effects. One of the suggested mechanisms involves the modulation of key signaling pathways associated with apoptosis. A previous study, demonstrated that isorhamnetin (Choi, 2019; Hu et al., 2015; Yang et al., 2023) can induce apoptosis in cancer cells by activating the caspases cascade, leading to the cleavage of PARP (poly (ADP-ribose) polymerase), the key of apoptotic cell death. The typical pattern of DNA fragmentation, considered the hallmark of apoptosis, was observed in 4T1 and TK6 cells treated with Nr-MeOH which was postulated to be effective in inducing apoptosis. However, we cannot exclude the role of other pathways in the apoptotic effect exhibited by Nr-MeOH. One of the mechanisms by which isorhamnetin might exert their cytotexic effect is through their influence to the cell cycle. In this context, we sought to determine whether there is a blockage at the level of the cell cycle in 4T1 cells using flow cytometry. Our results revealed that Nr-MeOH leads to the appearance of a sub-G1 peak at the tested concentration compared to the untreated cells. Indeed, isorhamnetin has been shown to influence the cell cycle by downregulating cyclin D1 and cyclin E, which are crucial for the transition from the G1 to S phase. Several studies reported that isorhamnetin treatment resulted in G1 phase arrest in human liver cancer cells, which was associated with increased expression of p21( 32 ) and p27( 33 ), two important cyclin-dependent kinase inhibitors. Also, it has been demonstrated that isorhamnetin causes a decrease in the percentage of cells in G1 phase ( 34 ) similar results of our research. In the other hand, recent studies have highlighted that Isorhamnetin inhibited the Akt/mammalian target of rapamycin (mTOR) and the mitogenactivated protein kinase (MAPK)/MAPK kinase (MEK) signaling pathways, and promoted the activity of the mitochondrial apoptosis signaling pathway ( 35 , 36 ). Moreover, isorhamnetin has been shown to induce cell cycle arrest at the G2/M phase in human breast cancer cells ( 36 ) and human hepatocarcinoma Hep3B cells ( 37 ). Indeed, isorhamnetin was found to downregulate the expression of Wee1 and cyclin B1, while simultaneously upregulating the expression of the cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1, with increased levels of p21 binding to Cdk1. Furthermore, the induction of apoptosis by isorhamnetin was correlated with elevated expression of the Fas/Fas ligand, a decreased ratio of B-cell lymphoma 2 (Bcl-2) to Bcl-2 associated X protein (Bax), cytosolic release of cytochrome c, and subsequent activation of caspases. Additionally, isorhamnetin was shown to inactivate the adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathway by reducing adenosine triphosphate (ATP) production, which was attributed to compromised mitochondrial function. ( 32 ). Furthermore, quercetin can inhibit the PI3K-Akt/PKB pathway ( 38 ), modulate the expression of Ras-p21( 39 , 40 ), stabilize p53( 41 ), which is the case for flavonoids ( 42 ) which are also recognized for their ability to suppress the NF-kB signaling pathway ( 43 , 44 ) in various cancer cell lines. Phosphatidylserine is a phospholipid that, under normal conditions, is primarily located on the inside of the cell membrane. However, during apoptosis (programmed cell death), it is translocated to the surface of the cell, serving as a signal for the removal of cells by macrophages ( 45 ). This translocation is a key marker of apoptosis and can be detected by methods such as the use of specific antibodies or fluorescent dyes. Isorhamnetin induces apoptosis by promoting the translocation of phosphatidylserine (PS) from the inner layer to the outer layer of the cell membrane. This translocation is a key marker of apoptosis, as it signals macrophages to phagocytize apoptotic cells, thereby contributing to the elimination of tumor cells ( 46 ). Knowing that macrophage dysfunction due to lysosomal dysfunction can lead to a decrease in macrophage activity ( 47 , 48 ) we can assume that an excess of cancer cell debris or even cancer cells is at the origin of the macrophage system failure ( 49 ) and that stimulation of lysosomes and macrophages can avoid this failure of the immune system at an advanced stage of the breast cancer cell. To be able to follow this recruitment of macrophages, an in vivo study on tumor-bearing mice treated with methanolic extract was conducted to see if there is an anticancer immunomodulation. Our results indicate that the Nr-MeOH significantly inhibits the growth of tumor in 4T1-tumor bearing mice. Additionally the treatment of mice with 100 mg/Kg of Nr-MeOH increased the lysosomal activity of macrophages and the nitric oxide production. Such results were in agreement with findings of Sun et al. who reported that flavonoids can modulate macrophage activity ( 50 ) by increasing glycolysis, which is regarded as a key characteristic of pro-inflammatory macrophage activation, it facilitates quick energy production and has been associated with the generation of pro-inflammatory cytokines ( 51 ), and increases the synthesis of NO ( 52 – 54 ), which is an important mediator that helps in the destruction of pathogens, playing a key role in the immune response and the regulation of inflammation. CONCLUSION The synergistic effects of isorhamnetin and carbohydrates present in the methanol extract of Nitraria retusa leaves exhibit significant potential as an anticancer agent through its ability to induce apoptosis and arrest the cell cycle, primarily by modulating key regulatory proteins and signaling pathways. Secondary by promotion in vivo macrophage activities that lead to reduced tumor weight BALB/c mice bearing tumor. These findings suggest that Nitraria retusa could be a substitute for wild resource as an anticancer therapy. Abbreviations Nr-MeO: methanol; DMSO: dimethyl sulfoxide;; NO: nitric oxide Declarations Ethics approval and consent to participate All animal experiments were conducted following the guidelines for the care and use of laboratory animals published by the National Institutes of Health. The study protocol was approved by the Ethics Committee of the Fattouma-Bourguiba University Hospital in Monastir, Tunisia. Consent for publication Not applicable. Availability of data and materials The dataset supporting the conclusions of this article are available from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests. Funding: The authors declare that they have no financial competing interests. Authors' contributions: BJ: Was responsible for the conception and design, testing and data acquisition, analysis and data interpretation and drafted the manuscript. AL: made contribution to the statical analysis and revised it critically. BTI: made contribution to the phytochemical study GK: made substantial contribution to conception and revised it critically for important intellectual content CGL: made substantial contribution to conception and revised it critically for important intellectual content Acknowledgements: The author acknowledge the ‘‘Ministry of Higher Education, Scientific Research and Technology, Tunisia”, for the support of this study and also thank. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-5395825","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":374899707,"identity":"b285ee1e-376c-4b43-8608-409a4a046ef2","order_by":0,"name":"Jihed Boubaker","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBACxmYeBgYehgMMDBIMzEC+DYgBoYnVkgbVkobPHlQthwlrYW7nPfjgDcMdOYPbzY8NPu45n9g/u/ngA4aEe3gcxpdsOIfhmbHBnWPGiTOe3U6ccedYsgFDQjE+v5hJ8zAcTtxwI8H4MM+B24kNN3LMJBh/JODTYv4boiX98+E/B84lzgdpYUjAq8WMGaIlxziZ4cABEIOgFmPJOQbPjCXvnCk27DmQbLzxRlqyQQIeLYb9Zww/vKm4I8d3u32zxI8DdrLzbiQffPABn5YGEGmAEHAEC+DWwMAgjy5gj0fxKBgFo2AUjFAAAJ1/XCIOK5z2AAAAAElFTkSuQmCC","orcid":"","institution":"Monastir University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jihed","middleName":"","lastName":"Boubaker","suffix":""},{"id":374899708,"identity":"94cb8171-31d1-40b6-b044-f60928060682","order_by":1,"name":"Aida Lahmar","email":"","orcid":"","institution":"Monastir University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aida","middleName":"","lastName":"Lahmar","suffix":""},{"id":374899709,"identity":"51a961d9-87b7-45f7-a88d-0b2ed5e19029","order_by":2,"name":"Imène Ben Toumia","email":"","orcid":"","institution":"Monastir University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Imène","middleName":"Ben","lastName":"Toumia","suffix":""},{"id":374899710,"identity":"79c7c373-4672-41c9-8015-e54f64330aa5","order_by":3,"name":"Kamel Ghedira","email":"","orcid":"","institution":"Monastir University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kamel","middleName":"","lastName":"Ghedira","suffix":""},{"id":374899711,"identity":"eb91810a-a44b-4e0b-901d-5ced63ba4c37","order_by":4,"name":"Leila Chekir-Ghedira","email":"","orcid":"","institution":"Monastir University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Leila","middleName":"","lastName":"Chekir-Ghedira","suffix":""}],"badges":[],"createdAt":"2024-11-05 13:38:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5395825/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5395825/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69573159,"identity":"96efb9a4-4dbc-48a5-a250-7a6c8dc11abd","added_by":"auto","created_at":"2024-11-21 19:42:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHPLC/ESI-MS\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e profiling of flavonoids compounds in methanol extract of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNitraria retusa \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eleaves.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5395825/v1/0d784caa4b18766fc4835f32.jpg"},{"id":69573459,"identity":"766787b1-3643-4287-8acb-734027585f5a","added_by":"auto","created_at":"2024-11-21 19:50:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":73947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA Inhibitory effect, of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNitraria retusa \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eextracts on the viability of TK6 cells\u003c/strong\u003e. Results are represented by the means ± SD of n = 3. (*) p\u0026lt; 0.05 means a significant difference between the untreated and treated cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB Inhibitory effect, of Nr-MeOH\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eextract on the viability of 4T1 cells.\u003c/strong\u003e Results are represented by the means ± SD of n = 3. (*) p\u0026lt; 0.05 means a significant difference between the untreated and treated cells.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5395825/v1/955b92492efb1de50d31b5a7.jpg"},{"id":69573165,"identity":"d13251a4-c91e-4e9a-9961-49172b76211d","added_by":"auto","created_at":"2024-11-21 19:42:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54922,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3A DNA electrophoretic profiles of TK6 cells treated with different concentrations of MeOH (methanol) extracts during 48h h.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was separated on 1.5% agarose gel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA : \u003c/strong\u003ePC: TK6 cell DNA; DNA of cells treated with \u003cstrong\u003eB:\u003c/strong\u003e 800 μg/assay Nr-MeOH extract, \u003cstrong\u003eC\u003c/strong\u003e: 400 μg/assay Nr-MeOH extract, \u003cstrong\u003eD: \u003c/strong\u003e200 μg/assay Nr-MeOH extract.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 3B. DNA electrophoretic profiles of 4T1 cells treated with different concentrations of Nr-MeOH (methanol) extracts during 48h h.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was separated on 1.5% agarose gel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA : \u003c/strong\u003ePC: 4T1 cell DNA; DNA of cells treated with \u003cstrong\u003eB:\u003c/strong\u003e 800 μg/assay Nr-MeOH extract, \u003cstrong\u003eC\u003c/strong\u003e: 400 μg/assay Nr-MeOH extract, \u003cstrong\u003eD: \u003c/strong\u003e200 μg/assay Nr-MeOH extract.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5395825/v1/438db84c7656d594d9937897.jpg"},{"id":69573168,"identity":"ecc05356-8774-4dda-9a6d-95f2e293e35c","added_by":"auto","created_at":"2024-11-21 19:42:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46670,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e4A\u003c/strong\u003e \u003cstrong\u003eChanges in expression of apoptosis-related protein in response to treatement methanol (Nr-MeOH) extract\u003c/strong\u003e. TK6 cells were treated with 800, 400, 200 µg/ml of methanol extract for 6, 24h and 48h. Protein extract were subjected to western blotting to determinate immunoreactivity of PARP, as described in methods section. PARP116KDA and 85KDA bands are shown\u003c/p\u003e\n\u003cp\u003eA :Untreated cells, B : 800µg/ml, C : 400µg/ml, D : 200µg/ml, E : 200µg/ml, F : 400µg/ml, G : 800µg/ml, H : 200µg/ml, I : 400µg/ml, J : 800µg/ml,\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 4B Quantification by scanning densitometry of PARP bands intensity\u003c/strong\u003e (quantified band intensity by computer-assisted densitometric analysis (Densitometer, GS-800 ( BioRad Quantity One)).\u003c/p\u003e\n\u003cp\u003eA :Untreated cells, B : 800µg/ml, C : 400µg/ml, D : 200µg/ml, E : 200µg/ml, F : 400µg/ml, G : 800µg/ml, H : 200µg/ml, I : 400µg/ml, J : 800µg/ml,\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5395825/v1/45a095ae3d72f4718c36edb2.jpg"},{"id":69573460,"identity":"b7fef581-c1dd-4727-94fd-858d7175ec1f","added_by":"auto","created_at":"2024-11-21 19:50:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":34717,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Nr-MeOH extracts on caspase-3 activity in TK6 cells. Lysates prepared from cells treated with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eN. retusa \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eleaf extracts for 24 h and 48 h, were assayed for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecaspase-3 activity. The rate of cleavage of the caspase substrate DEVD-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eNA was measured at 405 nm\u003c/strong\u003e. The results are presented as the mean ± SD. The experiments were done in triplicate. (*) \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 means a significant difference between the control and treated cells Control: cells treated by the vehicle only.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5395825/v1/2ebecb70944764d5a0bc36ef.jpg"},{"id":69573461,"identity":"fec90f8d-46d0-4d41-84df-8d5e27b61863","added_by":"auto","created_at":"2024-11-21 19:50:37","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":16815,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePercentage of apoptotic cells after 48 hours of treatment with the methanol extract evaluated by the Annexin V/IP test.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results are presented as the mean ± SD. The experiments were done in triplicate. (*) \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 means a significant difference between the control and treated cells Control: cells treated by the vehicle only.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5395825/v1/5dcbadf1e3f5561c64b87e34.jpg"},{"id":69573160,"identity":"96454cbd-d842-4837-9a2e-bf92af60a128","added_by":"auto","created_at":"2024-11-21 19:42:37","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":17499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApoptotic cells\u003c/strong\u003e \u003cstrong\u003epercentage during different cell cycle phases after treatment with the methanol extract.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eT: \u003c/strong\u003eno treated cells\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM 0.8: \u003c/strong\u003eCells treated with 800 μg/mlmethanol extract.\u003c/p\u003e\n\u003cp\u003eThe results are presented as the mean ± SD. The experiments were done in triplicate. (*) \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 means a significant difference between the control and treated cells Control: cells treated by the vehicle only.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5395825/v1/4961acc1eb0223e7913d76f5.jpg"},{"id":69573163,"identity":"4a844320-32b6-4014-80a2-504ca6297aea","added_by":"auto","created_at":"2024-11-21 19:42:37","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":106473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of methanol extract from N. retusa on 4T1 melanoma growth in BALB/c mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e: Tumor from animal bearing tumor(positive control).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e: Tumor from animals bearing tumor and treated with methanol extract at 100mg/kg b.w\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5395825/v1/28f91be7d7cf4e73b196573d.jpg"},{"id":69573169,"identity":"3f3c836e-98f1-45dd-b362-f8597de08514","added_by":"auto","created_at":"2024-11-21 19:42:37","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":23024,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLysosomal activity of peritoneal macrophages obtained from different groups of mice\u003c/strong\u003e. \u003cstrong\u003eNegative control\u003c/strong\u003e: from untreated mice; \u003cstrong\u003ePositive control\u003c/strong\u003e: from tumor-bearing mice; \u003cstrong\u003eMEOH (100mg/kg B.W):\u003c/strong\u003e tumor-bearing mice treated with 100mg/kg B.W of the methanolic extract. The values given represent the means (± standard deviation) of three independent experiments.\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5395825/v1/993d21ac7b04cec122cfc1d5.jpg"},{"id":69573166,"identity":"edd741e9-48ef-45dc-8d47-a4674c465ee7","added_by":"auto","created_at":"2024-11-21 19:42:37","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":30566,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProduction of nitric oxide by peritoneal macrophages obtained from different groups of mice\u003c/strong\u003e. \u003cstrong\u003eNegative control:\u003c/strong\u003e untreated mice; \u003cstrong\u003ePositive control:\u003c/strong\u003e tumor-bearing mice; \u003cstrong\u003eMEOH (100mg/kg P.C):\u003c/strong\u003e tumor-bearing mice treated with 100mg/kg P.C of the methanolic extract. The values given represent the means (± standard deviation) of three independent experiments.\u003c/p\u003e","description":"","filename":"Picture10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5395825/v1/fd1166489de2aa265bcffeed.jpg"},{"id":70932079,"identity":"e3acb309-8b66-447a-9e77-c09682633e23","added_by":"auto","created_at":"2024-12-09 10:17:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1710982,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5395825/v1/8a3c1f43-a549-442a-ae5e-fa72312b5416.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antitumoral potency of isorhamnetin and its derivates carbohydrate in methanol extract from Nitraria retusa leaves","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eBreast cancer is known as one of the most aggressive malignancies, characterized by its considerable metastatic potential and significant resistance to conventional cytotoxic therapies. A well-established relationship exists between tumor development, progression, regression, and immune system dynamics. Recent research has increasingly focused on the induction of tumor cell apoptosis and the enhancement of host immune responses through phytochemicals derived from medicinal plants, employing both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e methodologies. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eImmunomodulation, whether through natural or synthetic agents, is being explored as a viable strategy for the prevention and treatment of neoplastic diseases (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Strengthening the host immune response has emerged as a promising approach to inhibit tumor progression without negatively impacting the host (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) Therefore, it is essential to investigate novel natural antitumor agents that can stimulate immune responses and to chemically synthesize their counterparts for industrial applications. \u003cem\u003eNitraria\u003c/em\u003e genus is a plant used in traditional Tunisian medicine. Recent studies have underlined the promising anticancer activity of \u003cem\u003eNitraria retusa\u003c/em\u003e extracts against a wide variety of cancer cells (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). It has recently attracted attention for its apoptotic effects.\u003c/p\u003e \u003cp\u003eTo elucidate the \u003cem\u003ein vivo\u003c/em\u003e antitumoral potential of a compound, it is essential to first assess its apoptotic potential \u003cem\u003ein vitro\u003c/em\u003e and to delineate its mechanism of action. Indeed, apoptosis is a regulated form of cell death characterized by a specific programmed sequence of events that leads to the elimination of cells while preserving the integrity of neighboring cells. In response to the morphological and biochemical alteration associated with apoptosis, caspases are synthesized as inactive proenzymes, which upon proteolytic activation, gain the ability to cleave essential intracellular substrates (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we focused on the methanol (MeOH) extract of \u003cem\u003eNitraria retusa\u003c/em\u003e leaves (Nr-MeOH ) via its major compounds Isorhamnetin and its carbohydrate derivatives to explore alternative phytotherapeutic options to conventional anticancer therapies. For the first time, we aimed to evaluate this potential on cells undergoing transformation into cancerous cells, specifically human lymphoblastoid TK6 cells, and subsequently, on 4T1 cells and tumor-bearing mice. Consequently, we sought to elucidate the apoptotic and antitumoral effects of Nr-MeOH.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Material and analysis extracts\u003c/h2\u003e \u003cp\u003eLeaves of N. retusa were collected from halophilic soils in Sahline, a coastal area in Tunisia. Their identification was carried out by Professor M. Cheieb from the Department of Botany at the Faculty of Sciences, University of Sfax, Tunisia, following the Flora of Tunisia (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). A total of three hundred and 150 grams of powdered dried leaves were extracted sequentially using a Soxhlet apparatus for 6 hours (AM Glassware, Aberdeen, Scotland, United Kingdom) with methanol as the solvent. The methanol (MeOH) extract was obtained.\u003c/p\u003e \u003cp\u003eThe analysis of HPLC/ESI-MS\u0026sup2; was conducted using a Thermo Finnigan LCQ Advantage ion trap mass spectrometer equipped with an electrospray ionization (ESI) source, which was coupled to a Thermo Scientific Accela HPLC system comprising an MS pump, an autosampler, and a PDA detector. The separation was achieved on a Nucleodur 100-3 C18ec column (Macherey-Nagel). Gradient elution was performed using water and acetonitrile (ACN) without formic acid for the ESI negative mode, transitioning from 5\u0026ndash;30% ACN over 60 minutes, followed by an increase from 30\u0026ndash;90% ACN over an additional 35 minutes, all at a temperature of 30\u0026deg;C. The flow rate was maintained at 0.3 mL/min, with an injection volume of approximately 25 \u0026micro;L. All samples were analyzed in negative ion mode. The mass spectrometer operated with a capillary voltage set at 10 V, a source temperature of 240\u0026deg;C, and high-purity nitrogen was used as sheath and auxiliary gas at flow rates of 70 and 10 arbitrary units, respectively. Ions were detected within a mass range of 50-2000 m/z, with a collision energy of 35% applied for fragmentation during MS/MS analysis. Data acquisition was performed using Xcalibur\u0026trade; 2.0.7 software (Thermo Scientific)\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eThe human lymphoblastoid cell line TK6, which was generously provided by Professor Pierre Biscoff from the Centre Paul Strauss in Strasbourg, France, is characterized by the expression of wild-type p53, indicating that it is proficient in p53 functionality (Biscoff et al., 2023). The cells were maintained in RPMI-1640 Glutamax medium supplemented with 10% (v/v) fetal bovine serum, 1 mM sodium pyruvate, 1 mM non-essential amino acids, and 50 \u0026micro;g/mL gentamicin, and were incubated at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe 4T1 cell line, adherent in nature, is cultured as a monolayer in 75 cm\u0026sup3; culture dishes containing 10 ml of complete medium. Their generation time is 16 to 18 hours in an incubator at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. The medium is renewed every 48 hours. Cell viability is assessed at the time of seeding by counting live cells using the trypan blue exclusion technique. When the cells reach confluence, their proliferation slows down, necessitating reseeding into new culture dishes. Therefore, it is essential to detach them from their substrate: the supernatant is removed, and the cell layer is washed with phosphate-buffered saline (PBS, pH 7.4) to eliminate any traces of the culture medium. Subsequently, the supernatant is discarded and replaced with 2 ml of 1X trypsin (2.5%) for a 75 cm\u0026sup3; culture dish. Once placed in the incubator, the cells detach after five minutes of contact with trypsin. The action of trypsin is neutralized by the addition of a complete culture medium. The cells recovered by centrifugation (5 minutes at 1500 rpm, 25\u0026deg;C) are immediately resuspended in a complete medium and then counted using the trypan blue exclusion method, which is based on the assessment of cellular membrane integrity.\u003c/p\u003e\n\u003ch3\u003eAssay for cytotoxic activity\u003c/h3\u003e\n\u003cp\u003eThe cytotoxicity of Nr-MeOH against TK6 human lymphoblastoid cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Cells were plated in a 96-well plate at a density of 5 \u0026times; 10^4 cells per well and incubated at 37\u0026deg;C for 24 hours in a humidified atmosphere containing 5% CO2. Following this, the cells were treated with extracts dissolved in 1% DMSO at 37\u0026deg;C for 48 hours, with concentrations ranging from 10 to 800 \u0026micro;g/mL. After the incubation period, the medium was removed, and the cells in each well were exposed to 50 \u0026micro;L of MTT solution (5 mg/mL) at 37\u0026deg;C for 4 hours. Subsequently, the MTT solution was discarded, and 50 \u0026micro;L of 100% DMSO was added to dissolve the formazan crystals. Cell viability was determined by measuring the absorbance at 540 nm using a spectrophotometer. Each concentration of the MeOH extract was tested in triplicate. The antiproliferative effects of the extract were quantified as the IC\u003csub\u003e50\u003c/sub\u003e, defined as the concentration of extract that reduces the absorbance of treated cells by 50% compared to the control cells treated with 1% DMSO. The IC50 values were derived from the dose-response curves. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eOn the other hand, 4T1 cells, distributed in a 96-well flat-bottom plate at a density of 3,000 cells per well, are incubated at 37\u0026deg;C in an atmosphere enriched with 5% CO2 for 24 hours. Subsequently, the cells are treated with various concentrations of the extract (200, 400, 600, 800, 1000 \u0026micro;g/ml) and then incubated for 48 hours. A negative control is established. After 48 hours, the cells are washed with PBS each well, which is incubated for 10 minutes. Two successive washes with water are performed, and then 100 \u0026micro;l of crystal violet is added to each well and incubated for 30 minutes. After incubation, the plate is washed until all traces of crystal violet are removed, and finally, 100 \u0026micro;l per well of 33% acetic acid is added, and the absorbance is read at 600 nm. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)\u003c/p\u003e\n\u003ch3\u003eDNA fragmentation analysis\u003c/h3\u003e\n\u003cp\u003eAgarose gel electrophoresis was employed to assess DNA fragmentation, with minor modifications to established protocols (Smith et al., 2021; Johnson et al., 2022). TK6 cells (1.5 \u0026times; 10^6 cells/mL) were treated with varying concentrations of methanol (MeOH) extract (200, 400, and 800 \u0026micro;g/mL), while 4T1 cells were exposed to 400 and 800 \u0026micro;g/mL of MeOH extract for 48 hours. Following treatment, the cells were resuspended in 200 \u0026micro;L of lysis buffer (50 mM Tris\u0026ndash;HCl, pH 8.0, 10 mM EDTA, 0.5% N-Lauroyl Sarcosine Sodium Salt) and incubated at room temperature for 1 hour. The lysate was then centrifuged at 12,000 g for 20 minutes at 4\u0026deg;C. Proteinase K (250 \u0026micro;g/mL) was added to the supernatant and incubated overnight at 56\u0026deg;C. Subsequently, the cell lysates were treated with RNase (2 mg/mL) and incubated at 56\u0026deg;C for 2 hours. DNA was extracted using a chloroform/phenol/isoamyl alcohol mixture (24/25/1, v/v/v) and precipitated from the aqueous phase by centrifugation at 14,000 g for 30 minutes at 0\u0026deg;C. DNA fragmentation was analyzed by electrophoresis at 67 V for 3 to 4 hours using TAE buffer (Tris 40 mM, sodium acetate 20 mM, EDTA 1 mM). Ethidium bromide (0.5 \u0026micro;g/mL) was utilized for DNA visualization under UV light (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eTK6 cells (1.5 \u0026times; 10^6 cells/mL) were treated with methanol (MeOH) extracts at concentrations of 200, 400, and 800 \u0026micro;g/mL for durations of 6, 24, and 48 hours. Following incubation, the cells were centrifuged at 3000 rpm for 8 minutes at 25\u0026deg;C and subsequently lysed using a lysis buffer composed of 62.5 mM Tris-HCl and 6 mM urea, adjusted to pH 6.8. Protein concentrations were quantified using the Bradford assay (Bradford, 1976). A total of 40 \u0026micro;g of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane, which was blocked overnight at 4\u0026deg;C with 5% non-fat milk in 0.1% Tween 20-phosphate buffered saline (PBST). The membranes were then incubated with a primary antibody against PARP (1:100 dilution) for 2 hours at room temperature. After washing, the membranes were incubated for 1 hour with a goat anti-mouse alkaline phosphatase-conjugated secondary antibody (1:7500 dilution). Following another wash, the membranes were treated with the chromogenic substrate BCIP/NBT for visualization. Protein levels were quantified using computer-assisted densitometric analysis with a GS-800 densitometer (Bio-Rad, Hercules, CA) and analyzed using Quantity One software (Bio-Rad) (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInvestigation of caspase-3\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTk6 cells were cultured at a density of 1 \u0026times; 10^6 cells/mL in 25 cm\u0026sup2; flasks for 24 hours at 37\u0026deg;C, either in the presence or absence of the extracts. Control experiments were conducted simultaneously using 0.5% DMSO as a solvent. Following incubation, the cells were harvested and centrifuged at 600 \u0026times; g. The resulting pellets were resuspended in ice-cold lysis buffer (250 mM HEPES, pH 7.4, 25 mM CHAPS, 25 mM DTT) and incubated for 15 minutes. The samples were then centrifuged at 16,000 \u0026times; g for 20 minutes to obtain the supernatants, which contained the cell extracts, including potential caspase-3.\u003c/p\u003e \u003cp\u003eFor the detection of caspase-3 activity, the acetylated tetrapeptide substrate Ac-DEVD, labeled with the chromophore p-nitroaniline (\u003cem\u003ep\u003c/em\u003e-NA), was incubated with 50\u0026micro;g of total protein in the presence of the appropriate caspase buffer in a 96-well flat-bottom microplate. The cleavage of the substrate by active caspase-3 results in the release of p-NA, producing a yellow color that can be measured at 405 nm. A blank control, containing the assay buffer (200 mM HEPES, pH 7.4, 1% CHAPS, 50 mM DTT, 20 mM EDTA) and substrate but lacking cell lysate, was performed concurrently. The relationship between absorbance and p-NA concentration was established using a standard curve. The results were expressed as caspase-3 specific activity (\u0026micro;mol \u003cem\u003ep-\u003c/em\u003eNA per min/mL protein), calculated according to the manufacturer's instructions (Caspase-3 Colorimetric Assay Kit, Sigma-Aldrich)(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of the exposure of phosphatidyl serine (PS) by annexin VFITC/PI staining\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCells undergoing early and late stages of apoptosis were identified using Annexin V-FITC and propidium iodide (PI) staining, followed by analysis via flow cytometry. In brief, 4T1 cells were cultured in 6-well plates at a density of 5 \u0026times; 10^5 cells per well and treated with varying concentrations of methanol (MeOH) extract (400 and 800 \u0026micro;g/mL) for 48 hours. For staining, the cells were washed with phosphate-buffered saline (PBS) and resuspended in 1\u0026times; binding buffer to achieve a concentration of 2 \u0026times; 10^5 cells/mL. To 200 \u0026micro;L of this cell suspension, 5 \u0026micro;L of Annexin V-FITC and 10 \u0026micro;L of PI (20 \u0026micro;g/mL) were added, and the mixture was incubated for 15 minutes at room temperature in the dark before analysis. Flow cytometric analysis was performed using a FACS system (Beckman Coulter, Switzerland), with a total of 10,000 events collected and gated based on granularity and size as determined by forward scatter versus side scatter plots. The resulting density plots delineated four distinct cell populations based on their fluorescence characteristics: live cells (both Annexin V and PI negative), early apoptotic cells (Annexin V positive and PI negative), necrotic cells (both Annexin V and PI positive), and late apoptotic or dead cells (Annexin V negative and PI positive) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eAnalysis of the cell cycle distribution\u003c/h3\u003e\n\u003cp\u003eIn addition to apoptosis, autophagy, and necrosis, the induction of cell cycle arrest plays a crucial role in regulating the growth and development of cancer cells (Kroemer et al., 2021). Dysregulation of the cell cycle is a hallmark of tumor cells (Hanahan \u0026amp; Weinberg, 2011), making cell cycle modulation a significant strategy in cancer therapy (Shapiro et al., 2020). In this study, 4T1 cells were seeded at a density of 2 \u0026times; 10^5 cells/mL in six-well plates. Following a 24-hour incubation at 37\u0026deg;C in a 5% CO2 atmosphere, the cells were treated with methanol (MeOH) extract and incubated for an additional 48 hours, using 800 \u0026micro;g of Nr-MeOH for the cell cycle assay. After treatment, the cells were trypsinized, harvested by centrifugation at 1500 rpm for 5 minutes, and fixed overnight in 70% ethanol. The fixed cells were then collected by centrifugation, washed with phosphate-buffered saline (PBS), and stained with propidium iodide (PI) solution (50 \u0026micro;g/mL PI and 200 \u0026micro;g/mL RNase A) for 30 minutes in the dark at room temperature. Cell cycle distribution was analyzed using a FACS system (Beckman Coulter, Switzerland), and the percentages of cells in each phase of the cell cycle were calculated. The data presented are representative of at least three independent experiments conducted in triplicate (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vivo\u003c/b\u003e \u003cb\u003eEvaluation on the Tumor-Bearing Mouse Model\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMice Breeding\u003c/h2\u003e \u003cp\u003eThe experimental animals, pathogen-free Balb/C mice (6\u0026ndash;8 weeks old males, 20\u0026ndash;25 g), were obtained from the Pasteur Institute (Tunis, Tunisia). The animals were housed in polypropylene cages with steel grids on top and provided with clean paddy straw bedding. The animals were acclimatized to laboratory conditions for one week before treatment. All mice were kept under normal temperature (25\u0026deg;C), humidity (60%), and light (12 h light/dark) conditions in an accredited pathogen-free facility. The mice were fed standard laboratory pellet food. \u003cb\u003e All animal experiments were conducted following the guidelines for the care and use of laboratory animals published by the National Institutes of Health. The study protocol was approved by the Ethics Committee of the Fattouma-Bourguiba University Hospital in Monastir, Tunisia.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2. Acute Toxicity Test\u003c/h2\u003e \u003cp\u003eTo assess the toxicity of our extract for the selection of the concentration to be tested, mice were divided into two groups of six animals each. The first group served as the control, while the second group was treated with an intraperitoneal injection of 100 mg/kg body weight of the methanolic extract. The injection was administered over 21 days with a frequency of one injection every two days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3. Study of Antitumor Activity\u003c/h2\u003e \u003cp\u003eThe mice used to evaluate this activity, as well as all other subsequent activities, were classified into three groups. Each group contained 4, 2, and 3 mice, respectively:\u003c/p\u003e \u003cp\u003eGroup 1: Control mice (Negative Control)\u003c/p\u003e \u003cp\u003eGroup 2: Mice in which a tumor was induced (Positive Control).\u003c/p\u003e \u003cp\u003eGroup 3: Mice in which a tumor (breast cancer) was induced and treated with Nr-MeOH at a dose of 100 mg/kg body weight. The induction of breast cancer was achieved by subcutaneous injection of approximately 10^6 4T1 cells into 5 to 6-week-old female Balb/C mice. The evaluation of the antitumor effect of the extract is based on an intraperitoneal injection of the methanolic extract. The experimental protocol includes several administrations of the extract starting on Day 7, then Days 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 post-grafting (Day 0 being the first day of tumor grafting). Any intraperitoneal tumor indicates a defective injection and results in exclusion from the study. The measured parameters are physiological and immunological.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCellular Lysosomal Enzyme Activity\u003c/h2\u003e \u003cp\u003ePeritoneal macrophages were obtained by intraperitoneally injecting 5 ml of sterile PBS and then withdrawing the fluid. The collected cells were washed twice with PBS and resuspended in complete RPMI 1640; cell viability was assessed using trypan blue exclusion. One hundred microliters of the cell suspension (3 x 10^6 cells/ml) were distributed into 96-well plates. The plates were incubated for 24 hours at 37\u0026deg;C in a humid atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e. The adherent cells were treated with 20 \u0026micro;l of Triton X-100 (1%), 10 \u0026micro;l of p-nitrophenyl phosphate (p-NPP) (100 mM), the substrate for acid phosphatase, and 50 \u0026micro;l of citrate buffer (0.1 M, pH 5). The mixture was incubated at 37\u0026deg;C in a humid atmosphere with 5% CO\u003csub\u003e2\u003c/sub\u003e for 30 minutes. The activity of acid phosphatase was measured at 405 nm, and the percentage of activity was calculated using the following formula: Activity (%)\u0026thinsp;=\u0026thinsp;100 * (OD sample - OD control) / OD control. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNitric Oxide Production\u003c/h2\u003e \u003cp\u003eThe amount of nitric oxide (NO) released by macrophages was assessed by measuring the levels of accumulated nitrite (NO\u003csub\u003e2\u003c/sub\u003e) in cell-free supernatants using the Griess reaction. In summary, isolated macrophages from each group were incubated for 24 hours in a microplate. Nitrite levels were then determined by adding 100 \u0026micro;l of Griess reagent (1% sulfanilamide and 0.1% naphthylenediamine in 5% phosphoric acid) to 100 \u0026micro;l of the harvested culture supernatant. The optical density at 570 nm (OD570) was measured using a microplate reader (Thermo Scientific, Vantaa, Finland). NO concentrations were calculated by comparing the OD570 with that of a standard sodium nitrite solution diluted in a culture medium, which was placed in parallel wells on the assay plates (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were collected and expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of 3 independent experiments and analyzed for statistical significance from control. The data were tested for statistical differences by one-way ANOVA followed by a post hoc test. The criterion for significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eHPLC/ESI-MS\u003c/b\u003e \u003csup\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eprofiling of flavonoid compounds in\u003c/b\u003e Nr-MeOH\u003c/p\u003e \u003cp\u003eHPLC-PDA-MS/MS was applied to identify the compounds in Nr-MeOH. The analysis revealed a total of 15 polyphenols, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the base peaks of the LC-MS in the ESI (\u0026minus;) mode: the negative ionization, and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the identified peaks.\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\u003eSecondary metabolites of the methanol leaf extract from \u003cem\u003eNitraria retusa\u003c/em\u003e\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=\"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 \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=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM-H\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMS/MS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProposed compound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eArea(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eReferences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e353\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e191,179,173,135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ecis 3-O-Caffeoylquinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2,005894862\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e353\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e191,179,173,135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4-O-Caffeoylquinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,426923185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e169, 125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGallic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,613207508\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-Coumaric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,92831564\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e305\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e261\u0026nbsp;,179,137,125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(epi)gallocatechin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,635199645\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e245,203,179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(epi)catechin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0,487887571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e15.07\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e463\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e316, 301, 300, 217\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eQuercetin O-hexoside\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e9,544611761\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e15.53\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e477\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e315, 299\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eIsorhamnetin 3-O-glucoside\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e19,27726321\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e16.95\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e623\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e315, 300\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eIsorhamnetin-3-O-rutinoside\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e19,7135479\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e593\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKaempferol-3-O-rutinoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6,18469979\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e19.33\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e491\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e315, 300, 271;255, 113\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eIsorhamnetin glucuronide\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e17,21146502\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e315\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIsorhamnetin acetylhexoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3,772631615\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e22.36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e315\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e315\u0026nbsp;,300\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eIsorhamnetin\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e13,7504632\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e22.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e431\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e431,269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eApigenin-7-O-glucoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2,14156078\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLuteolin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3,30632832\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\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 \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCytotoxic activity:\u003c/h2\u003e \u003cp\u003eWe examined the effect of different concentrations (200, 400, and 800 \u0026micro;g/ml) of Nr-MeOH on the in vitro proliferation of the TK6 cell line, utilizing the MTT assay, and on the in vitro 4T1 cell line proliferation using the cristal violet test at different methanol extract concentrations, 1000, 800, 600, and 200\u0026micro;g/ml. The results of this assay are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The methanol extract demonstrated a significant inhibitory effect on the growth of the evaluated cell populations.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInduction of apoptotic DNA fragmentation by\u003c/b\u003e Nr-MeOH \u003cb\u003eon 4T1 and TK6 cells.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUpon exposure to varying concentrations of Nr-MeOH (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, lanes B, C, D; Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e3\u003c/span\u003e, lanes B, C) for 48 hours, a fragmented DNA profile was distinctly observed at all tested doses of the Nr-MeOH in both 4T1 and TK6 cells. In contrast, untreated cells did not exhibit a ladder-like DNA profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, lane A; Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, lane A).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e Nr-MeOH \u003cb\u003eon the proteolysis of PARP.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDNA fragmentation is often associated with the activation of a family of cysteine proteases, the caspases. Caspase-3, in particular, seems to play an important role in several models of apoptosis. To confirm the apoptotic process, generally admitted when a ladder DNA fragmentation profile is observed, we investigated the enzymatic activation of apoptotic proteins by measuring the cleavage of PARP (116 kDa), which is a caspase-3 substrate, into fragments of 85 and 31 kDa.\u003c/p\u003e \u003cp\u003eAs shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e4\u003c/span\u003e. A), when cells were treated with MeOH extract, the 116 kDa band disappeared after 48h treatment at all tested doses of MeOH extract and the band of 85 kDa.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCaspase-3 activation assay\u003c/h2\u003e \u003cp\u003eAs the proapoptotic PARP is a substrate of caspases, we attempt to investigate the cellular pathway of cell death induced by \u003cem\u003eNitraria retusa\u003c/em\u003e extracts, by assessing caspase-3 activity. This protein plays a critical role in apoptosis. Following 48 h treatment of TK6 cells with various concentrations of Nr-MeOH, caspase-3 activity was measured and compared to those of control cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e5\u003c/span\u003e, TK6 cells treated with \u003cem\u003eNitraria retusa\u003c/em\u003e extract, showed a significant concentration-depending increase of caspase-3 activity after 48 of incubation at all tested concentrations of MeOH extract. This result suggests that apoptosis induced by the tested extract may occur through the activation of common executors of apoptosis such as caspase-3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of the exposure of phosphatidyl serine (PS) by annexin VFITC/PI staining\u003c/h2\u003e \u003cp\u003eIn order to determine the apoptotic pathway revealed by the previous tests of the methanolic extract on the 4T1 murine cell line, we evaluated this effect using the Annexin V/IP test, which allows us to check one of the aspects of apoptosis, namely the destruction of the cytoplasmic membrane through the translocation of phosphatidylserine. Our results show that the Nr-MeOH induces apoptosis of 4T1 cells at the highest concentration tested (800 \u0026micro;g/ml) with a maximum percentage of 11% (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of the cell cycle distribution\u003c/h2\u003e \u003cp\u003eTo test whether Nr-MeOH could affect the cell cycle of 4T1 cells, cells treated with DMSO or Nr-MeOH (800 \u0026micro;g/ml) for 48 h were subjected to flow cytometry analysis after DNA staining. The result showed a significant increase of G2/M and sub-G0 phase cells built-up (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e7\u003c/span\u003e), as well as a significant decrease in cell number in the G0/G1 phase. However, there was\u003c/p\u003e \u003cp\u003eno significant difference in the proportion of 4T1 cells in the S phase cultured for 48 h with or without Nr-MeOH.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Nr-MeOH on weight and volume tumor:\u003c/h2\u003e \u003cp\u003eOur results indicate that Nr-MeOH significantly inhibits the growth of tumor cells in tumor-bearing mice. The inhibition of tumor volume reached 56.25%. Consistent with this result, the average tumor weight after the administration to animals of the extract, was significantly lower than in positive control (animal with tumor) (PC). The inhibition percentage reached 52.18% (Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, \u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eStudy of the lysosomal activity of peritoneal macrophages\u003c/h2\u003e \u003cp\u003eThe results of the lysosomal activity of peritoneal macrophages are presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Tumor proliferation in the positive control group induces a decrease in lysosomal activity to 61.78%. However, treatment of tumor-bearing mice with the Nr-MeOH at a dose of 100 mg/kg of B.W. stimulates the lysosomal activity of macrophages to 223.07%.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStudy of nitrogen monoxide (NO) production:\u003c/h2\u003e \u003cp\u003eThe treatment with methanolic extract of mice that developed a tumor led to a significant increase in nitric oxide production, rising from 3.09 \u0026micro;M (negative control) to 14.84 \u0026micro;M at a dose of 100 mg/kg body weight of the Nr-MeOH treated group, with a significant change in NO concentration observed in the positive control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe use of medicinal plants for the development of effective drugs against various diseases and as a source of anticancer agents has recently attracted attention. Due to the increasing mortality from cancer and the adverse effects of chemotherapy and radiotherapy, the discovery of new natural products against cancer and the study of medicinal plants for this purpose is gaining considerable interest(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Recstent studies have shown that \u003cem\u003eN. retusa\u003c/em\u003e rich in flavonoids such as isorhamnetin and quercetin, present a promising option for cancer therapy.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNitraria retusa\u003c/em\u003e, an edible halophyte has been described for its promising anticancer activity (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn our current study, the methanolic extract of \u003cem\u003eN. retusa\u003c/em\u003e (Nr-MeOH) was analysed by LC-MS\u003csup\u003en\u003c/sup\u003e to determine the major compounds. Five flavonoids were identified including quercetin O-hexoside, isorhamnetin 3-O-glucoside, isorhamnetin-3-O-rutinoside, isorhamnetin glucuronide, and isorhamnetin. These findings are in broad agreement with Haj-Salem et al (2011) results, who studied the CHCl3, BuOH, and EtOAc extracts by LC-MS\u003csup\u003en\u003c/sup\u003e analysis. Four flavonoids were reported by the researchers including isorhamnetin, isorhamnetin-3-O-glucoside, isorhamnetin-3-O-rutinoside and isorhamnetin-3-O-robinobioside (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince Isorhamnetin and its carbohydrate derivatives compounds are often described for their therapeutic and preventive potencies, we therefore studied the antiproliferative effect of \u003cem\u003eN. retusa\u003c/em\u003e methanolic (Nr-MeOH) extract against 4T1 and TK6 cells. In addition, the plausible mechanism underlying the anticancer activity was elucidated.\u003c/p\u003e \u003cp\u003eIn our study, Nr-MeOH efficiently suppressed cell viability in a dose-response manner. The considerable cytotoxicity of Nr-MeOH cells might be ascribed to its major compounds Isorhamnetin and its carbohydrate derivatives that can alter proliferation of cell lines and can influence a variety of cell functions by modulating cell signaling. Similar findings were revealed by Li et al. who reported that isorhamnetin exerted a cytotoxic effect on A549 lung cancer cells by inhibiting cell proliferation and causing DNA damage (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese studies with our previously research (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) reinforce the idea that isorhamnetin could be a promising agent in the fight against various types of cancer. This highlights the importance of continuing studies on the mechanisms of action of isohamnetin and its potential as a complementary treatment in oncology and against breast cancer cells.\u003c/p\u003e \u003cp\u003eTo evaluate whether Nr-MeOH extract from \u003cem\u003eN. retusa\u003c/em\u003e leaves decrease cell viability through apoptosis activation, the cleavage of the PARP was examined by Western blot. PARP is a single-strand break-repair enzyme (116 kDa). This enzyme is involved in the repair of DNA damage by catalyzing the synthesis of poly (ADP-ribose), by binding to DNA strand breaks and modifying nuclear proteins. The ability of PARP to repair DNA damage is prevented after cleavage of PARP by caspase-3 into 85 kDa fragment (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWestern blot analysis revealed that PARP was cleaved after exposure of 4T1 and TK6 cells to decreased concentrations of Nr-MeOH (400, 600, 800 \u0026micro;g/ml) for 48 h. Intriguingly, Nr-MeOH-mediated cytotoxic effect was accompanied with increase of caspase 3 activities.\u003c/p\u003e \u003cp\u003eIsorhamnetin, has garnered attention for its potential anticancer properties, particularly in relation to apoptosis mechanisms. Recent studies have elucidated various pathways through which isorhamnetin exerts its effects. One of the suggested mechanisms involves the modulation of key signaling pathways associated with apoptosis. A previous study, demonstrated that isorhamnetin (Choi, 2019; Hu et al., 2015; Yang et al., 2023) can induce apoptosis in cancer cells by activating the caspases cascade, leading to the cleavage of PARP (poly (ADP-ribose) polymerase), the key of apoptotic cell death.\u003c/p\u003e \u003cp\u003eThe typical pattern of DNA fragmentation, considered the hallmark of apoptosis, was observed in 4T1 and TK6 cells treated with Nr-MeOH which was postulated to be effective in inducing apoptosis.\u003c/p\u003e \u003cp\u003eHowever, we cannot exclude the role of other pathways in the apoptotic effect exhibited by Nr-MeOH. One of the mechanisms by which isorhamnetin might exert their cytotexic effect is through their influence to the cell cycle. In this context, we sought to determine whether there is a blockage at the level of the cell cycle in 4T1 cells using flow cytometry. Our results revealed that Nr-MeOH leads to the appearance of a sub-G1 peak at the tested concentration compared to the untreated cells. Indeed, isorhamnetin has been shown to influence the cell cycle by downregulating cyclin D1 and cyclin E, which are crucial for the transition from the G1 to S phase. Several studies reported that isorhamnetin treatment resulted in G1 phase arrest in human liver cancer cells, which was associated with increased expression of p21(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) and p27(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), two important cyclin-dependent kinase inhibitors. Also, it has been demonstrated that isorhamnetin causes a decrease in the percentage of cells in G1 phase (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) similar results of our research.\u003c/p\u003e \u003cp\u003eIn the other hand, recent studies have highlighted that Isorhamnetin inhibited the Akt/mammalian target of rapamycin (mTOR) and the mitogenactivated protein kinase (MAPK)/MAPK kinase (MEK) signaling pathways, and promoted the activity of the mitochondrial apoptosis signaling pathway (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Moreover, isorhamnetin has been shown to induce cell cycle arrest at the G2/M phase in human breast cancer cells (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) and human hepatocarcinoma Hep3B cells (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Indeed, isorhamnetin was found to downregulate the expression of Wee1 and cyclin B1, while simultaneously upregulating the expression of the cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1, with increased levels of p21 binding to Cdk1. Furthermore, the induction of apoptosis by isorhamnetin was correlated with elevated expression of the Fas/Fas ligand, a decreased ratio of B-cell lymphoma 2 (Bcl-2) to Bcl-2 associated X protein (Bax), cytosolic release of cytochrome c, and subsequent activation of caspases. Additionally, isorhamnetin was shown to inactivate the adenosine 5\u0026prime;-monophosphate-activated protein kinase (AMPK) signaling pathway by reducing adenosine triphosphate (ATP) production, which was attributed to compromised mitochondrial function. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Furthermore, quercetin can inhibit the PI3K-Akt/PKB pathway (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), modulate the expression of Ras-p21(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), stabilize p53(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), which is the case for flavonoids (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) which are also recognized for their ability to suppress the NF-kB signaling pathway (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) in various cancer cell lines.\u003c/p\u003e \u003cp\u003ePhosphatidylserine is a phospholipid that, under normal conditions, is primarily located on the inside of the cell membrane. However, during apoptosis (programmed cell death), it is translocated to the surface of the cell, serving as a signal for the removal of cells by macrophages (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). This translocation is a key marker of apoptosis and can be detected by methods such as the use of specific antibodies or fluorescent dyes. Isorhamnetin induces apoptosis by promoting the translocation of phosphatidylserine (PS) from the inner layer to the outer layer of the cell membrane. This translocation is a key marker of apoptosis, as it signals macrophages to phagocytize apoptotic cells, thereby contributing to the elimination of tumor cells (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Knowing that macrophage dysfunction due to lysosomal dysfunction can lead to a decrease in macrophage activity (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e) we can assume that an excess of cancer cell debris or even cancer cells is at the origin of the macrophage system failure (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) and that stimulation of lysosomes and macrophages can avoid this failure of the immune system at an advanced stage of the breast cancer cell.\u003c/p\u003e \u003cp\u003eTo be able to follow this recruitment of macrophages, an in vivo study on tumor-bearing mice treated with methanolic extract was conducted to see if there is an anticancer immunomodulation.\u003c/p\u003e \u003cp\u003eOur results indicate that the Nr-MeOH significantly inhibits the growth of tumor in 4T1-tumor bearing mice. Additionally the treatment of mice with 100 mg/Kg of Nr-MeOH increased the lysosomal activity of macrophages and the nitric oxide production.\u003c/p\u003e \u003cp\u003eSuch results were in agreement with findings of Sun et al. who reported that flavonoids can modulate macrophage activity (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e) by increasing glycolysis, which is regarded as a key characteristic of pro-inflammatory macrophage activation, it facilitates quick energy production and has been associated with the generation of pro-inflammatory cytokines (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), and increases the synthesis of NO (\u003cspan additionalcitationids=\"CR53\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), which is an important mediator that helps in the destruction of pathogens, playing a key role in the immune response and the regulation of inflammation.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe synergistic effects of isorhamnetin and carbohydrates present in the methanol extract of \u003cem\u003eNitraria retusa\u003c/em\u003e leaves exhibit significant potential as an anticancer agent through its ability to induce apoptosis and arrest the cell cycle, primarily by modulating key regulatory proteins and signaling pathways. Secondary by promotion \u003cem\u003ein vivo\u003c/em\u003e macrophage activities that lead to reduced tumor weight BALB/c mice bearing tumor. These findings suggest that \u003cem\u003eNitraria retusa\u003c/em\u003e could be a substitute for wild resource as an anticancer therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNr-MeO: methanol; DMSO: dimethyl sulfoxide;; NO: nitric oxide\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval \u0026nbsp;and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted following the guidelines for the care and use of laboratory animals published by the National Institutes of Health. The study protocol was approved by the Ethics Committee of the Fattouma-Bourguiba University Hospital in Monastir, Tunisia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset supporting the conclusions of this article are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no financial competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBJ:\u003c/strong\u003e Was responsible for the\u0026nbsp;conception and design, testing\u0026nbsp;and\u0026nbsp;data acquisition, analysis\u0026nbsp;and data interpretation\u0026nbsp;and drafted\u0026nbsp;the\u0026nbsp;manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAL:\u0026nbsp;\u003c/strong\u003emade contribution to the statical analysis\u0026nbsp;and revised it critically.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBTI:\u0026nbsp;\u003c/strong\u003emade contribution to the phytochemical study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGK:\u003c/strong\u003e made substantial contribution to conception and revised it critically for important intellectual content\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCGL:\u003c/strong\u003e made substantial contribution to conception and revised it critically for important intellectual content\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThe author acknowledge the \u0026lsquo;\u0026lsquo;Ministry of Higher Education, Scientific Research and Technology, Tunisia\u0026rdquo;, for the support of this study and also thank.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChoi EJ, Ahn WS. 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Eur J Nutr 21 avr. 2013;53(1):269.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"Nitraria retusa, Isorhamnetin, apoptosis, immunomodulation, BALB/c mice-bearing tumor","lastPublishedDoi":"10.21203/rs.3.rs-5395825/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5395825/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eNitraria retusa\u003c/em\u003e is a traditional Tunisian herb that has been widely used in folk medicine for its great remedies. In this study, we evaluated the antitumoral potency of methanol extract from leaves of \u003cem\u003eNitraria retusa\u003c/em\u003e, (Nr-MeOH) via its major compounds isorhamnetin and its carbohydrate derivatives against human lymphoblastoid cells (TK6), breast cancer murine cells (4T1), and in tumor-bearing mice.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe Nr-MeOH was analysed by LC-MS\u003csup\u003en\u003c/sup\u003e to determine the major compounds. The cell apoptotic mechanism was described using DNA fragmentation and double staining annexin/propidium iodide by flow cytometry. The PARP cleavage was investigated by Western blotting. BALB/c mice were subcutaneously inoculated with 4T1 cells, and then treated intra-peritoneally with the methanol extract for 21 days. The tumor growth was evaluated. Macrophage phagocytosis was assessed by measuring the lysosomal activity and the nitric oxide production.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur investigation showed that the Nr-MeOH contains various flavonoids, quercetin, isorhamnetin 3-O-glucoside, isorhamnetin-3-O-rutinoside, isorhamnetin glucuronide, and isorhamnetin. These compounds were found to induce apoptosis in the cancer cell line tested and to reduce tumor growth rates in induced 4T1-bearing tumor mice.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe results of this work suggest that \u003cem\u003eNitraria retusa\u003c/em\u003e could be a substitute for wild resource as an anticancer therapy.\u003c/p\u003e","manuscriptTitle":"Antitumoral potency of isorhamnetin and its derivates carbohydrate in methanol extract from Nitraria retusa leaves","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-21 19:42:32","doi":"10.21203/rs.3.rs-5395825/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":"b7eca08e-e117-422d-8008-3c11c55ddd31","owner":[],"postedDate":"November 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-18T13:54:10+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-21 19:42:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5395825","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5395825","identity":"rs-5395825","version":["v1"]},"buildId":"CiT4i_kKBbxQbnFL0ufpk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}