Iodine and selenium are essential for thyroid hormone synthesis. Iodine and selenium interact. Pregnancy increases the maternal iodine requirement. We previously reported inadequate iodine status in pregnant Dutch women. Since little is known about their selenium intake, we investigated the iodine status and selenium intake in relation to iodine and selenium supplement use during pregnancy. Iodine status was established in 201 apparently healthy pregnant women as 24 h iodine excretion , iodine concentration  and iodine/creatinine ratio . Selenium intake was calculated from 24 h selenium excretion. Iodine status in pregnancy proved insufficient . Only women taking 150 µg iodine/day were sufficient (median 24H-UIE 244 µg). Selenium intake was below the Estimated Average Requirement  in 53.8%, below the Recommended Dietary Allowance  in 77.4% and below the Adequate Intake  in 88.7%. Combined inadequate iodine status and selenium intake <RDA was found in 61%. Women who want to become pregnant should, consistently with WHO and ETA recommendations, be advised to use a 150 µg iodine-containing supplement. Concomitant selenium supplementation should be added to this advice, at least in The Netherlands. Iodine and selenium are essential for thyroid hormone synthesis. Iodine deficiency may cause clinical or subclinical hypothyroidism. Pregnancy augments both maternal thyroid hormone synthesis and iodine needs with approximately 50%. The developing fetal brain needs iodine in the first stage of development. Mild or subclinical hypothyroidism in early pregnancy may adversely impact neurological development ,2. At laThe sodium–iodine symporter (NIS) is present not only in the thyroid, mammary glands and the placenta but also in salivary glands, stomach, rectum, kidneys ,9,  taking place between February 2018 and February 2019. Invitations occurred via verbal contact and by spreading flyers in a.o. midwife practices and during childbirth classes.Apparently healthy pregnant women were eligible to participate. Exclusion criteria were multiple pregnancy, (pregnancy-induced) hypertension or diabetes mellitus, renal dysfunction, thyroid disorders and long-term medication use, notably iodine-containing amiodarone and thyroid hormone (thyrax) and also age below 18 years.All participants received verbal and written information and provided written consent. The Medical Ethical Review Committee of the University Medical Centre Groningen (UMCG) considered this study non-WMO compliant. The study was in agreement with the Declaration of Helsinki of 1975, as revised in 2013.The participants provided information about their education level, gestational age, number of previous pregnancies and liveborn infants, length, pre-pregnancy weight, use of nutritional supplements, alcohol, drugs, medication and tobacco, and also medical conditions. Women who graduated high school, intermediate vocational education or less were categorized as lowly educated. Women who graduated college, university or higher were highly educated. Using the brand name of the supplement, we could retrieve the daily iodine and selenium doses, if applicable. The available supplements contained no, 75 or 150 µg of iodine and 8.2–60 µg of selenium per daily dose.Upon instruction, the participants collected the first morning urine portion and subsequently a 24 h urine sample. The samples were stored in a cool place and transported to the UMCG within 48 h. The participants noted the starting and final time of collection. The 24 h urine collections that were incomplete (as noted by the participant), had a volume <300 mL  or were Based on our pilot study , we estiThe World Health Organization (WHO) advises evaluation of the iodine status of a population by using the iodine concentration of a random spot urine specimen . HoweverSelenium intake was estimated as (100/55) × 24 h urine selenium excretion. This formula is based on 50–60% excretion of the ingested selenium via urine . The calWe used IBM-PASW Statistics 23 software for statistical analysis. The relation between gestational age and the investigated iodine status parameters was investigated with Spearman correlation analysis. Between-group differences were analyzed using the Kruskall–Wallis H test with post hoc paired comparisons. The associations between daily iodine dose from supplements and the iodine status parameters were analyzed using the Jonckheere’s Trend test.A total of 292 apparently healthy pregnant women were willing to participate in the JOZO study. We excluded participants who did not provide written permission, (n = 70), used iodine-containing medication (n = 2) or provided incorrectly collected 24 h urine (n = 19).Two of the 201 included participants showed unexpectedly high 24 h UIC (987 and 1072 µg/L) and high morning UIC . One participant showed a high morning UIC (824 µg/L). Since the exclusion criteria were not applicable, those three were not excluded. Iodine was analyzed in all 201 urine samples, but selenium was merely analyzed in the 24 h urines of 186 participants who provided permission for the measurement of additional analytes.p < 0.011). The Jonckheere’s Trend test showed a positive association of iodine dose with 24 h iodine excretion (p = 0.000), UIC (p = 0.002) and UICR (p = 0.000), and with morning UIC (p = 0.003) and UICR (p = 0.000).p = 0.04) and counterparts using 150 µg iodine supplements . Accordingly, the 24 h UIC of participants who did not use iodine-containing supplements .The 24 h iodine excretion of the participants who did not use iodine-containing supplements  and those who did not use iodine-containing supplements . The 24H-UICR of participants who took 75 µg iodine was significantly higher than the 24H-UICR of those who did not take iodine-containing supplements (p = 0.031).Women who used a 150 µg iodine supplement showed significantly higher 24H-UICR  than did counterparts using 75 µg iodine supplements .The morning UIC  and morning UICR  of women who took a 150 µg iodine supplement were higher than those of counterparts who did not use those supplements ; 24 h UIC, 91 µg/L ; 24 h UICR, 130 µg/g ; morning UIC, 115 µg/L ; and morning UICR, 101 µg/g . Based on all five parameters, the iodine status of those participants proved insufficient.The iodine status of participants who took a 75 µg iodine supplement proved insufficient as well .For participants who took 150 µg iodine supplements, we found the following: median 24 h iodine excretion, 244 µg; 24 h UIC, 114 µg/L; 24 h UICR, 171 µg/g; morning UIC, 156 µg/L; and morning UICR, 143 µg/g. Their iodine status proved sufficient when based on 24 h iodine excretion, 24 h UICR and morning UIC but not when based on 24 h UIC and morning UICR.The median estimated selenium intake amounted to 48 µg/day (range: 12-180). Of the 186 participants, 53.8% showed a estimated selenium intake below the 49 µg/day IOM-EAR, 77.4% a estimated selenium intake below the 60 µg/day RDA  and 88.7% an intake below the 70 µg/day AI . The 78 participants who did not take selenium-containing supplements showed a median calculated intake of 42 (16–90) µg/day. Their intakes did not reach the 49 µg/day EAR in 71.8% and did not reach the 60 µg/day RDA in 97.4%; two subjects (2.6%) had selenium intakes above the 70 µg/day AI. The iodine status of pregnant Dutch women proved inadequate: the medians of all applied iodine status parameters were below their respective cut-off values. Only the use of a supplement containing 150 µg iodine was associated with adequate iodine status during pregnancy. A new finding was that the median estimated selenium intake (48 µg/day) was below the 49 µg/day EAR, that 77.4% of the pregnant women consumed less than the 60 µg selenium RDA and that 88.7% consumed less than the 70 µg/day AI. The selenium intakes of the 78 participants who did not take selenium-containing supplements did not reach the 60 µg RDA in 97.4%. The estimated selenium intake proved independent of the selenium supplemental dose, which made it impossible to estimate how much supplemental selenium is needed to reach an adequate intake. Dependent on the applied cut-offs, 46.8–64.5% of all participants had a combined inadequate iodine status and insufficient selenium intake.The 150 µg/L (UIC) and 150 µg/g (ICR) cut-offs for iodine in random urine portions, as advised by the WHO, are commonly used to establish a population’s iodine status. Although the UIC  proved higher in morning urine than in the 24 h collections, , the morning urine appeared more concentrated, as suggested by its higher creatinine concentration. Consequently, the ICR  in the morning urine was lower than the ICR in the 24 h collections . These data show that the interpretation of the ICR and UIC of random urine samples is difficult. A small study with 20 apparently healthy individuals by us showed that the mean intraindividual coefficients of variation of the UIC and ICR, measured in consecutively collected urine portions during 24 h, amounted to 52 and 26%, respectively (unpublished data). The 24 h urine collections are obviously more representative than random portions ,44,45,46The median 24 h urine iodine excretion of the whole group (185 µg) and of women taking a 150 µg supplement (244 µg) in the current JOZO study were lower than those in our pilot study  . The curThe estimated selenium intake (whole group 48 µg/day (12–180); non-supplement users 42 (16–90) µg/day) of the current 31 (21–43)-year-old pregnant Dutch women proved to be in agreement with the selenium intakes from food and dietary supplements reported by the Dutch National Food Consumption Survey 2012–2016 for 4313 19–79-year-old subjects (P50 (P5-P95): men 55 (33–101); women 44 (24–95) ug/day)) . Since pAlthough selenium excretion at the same supplement dose was higher in pregnant women as compared to that of non-pregnant counterparts , a more Intervention studies do not unambiguously indicate that iodine supplementation during pregnancy improves offspring neurological development , which cNegro et al.  showed tThere are a number of limitations in this study. The studied pregnant women were highly educated. This happened notwithstanding our attempts to recruit a representative sample by including more women than statistically needed. Highly educated women are more likely to use nutritional supplements and usually eat a healthier diet. A positive association between education level and iodine intake was previously found in Dutch adults , and eduThe majority of pregnant Dutch women in our study had inadequate status and an insufficient selenium intake. According to well-established associations in the literature, the mothers may be considered at risk of thyroid disfunction, including thyroid autoimmune disease and cancer, while their offspring are at risk of neurological development and possibly suboptimal growth  The poorIt has become clear that iodine and selenium interact and that many diseases linked to either iodine or selenium in the past are in reality caused by an iodine–selenium disbalance . As seafIt may be wise to educate women who plan pregnancy that iodine needs increase already in early pregnancy and to recommend augmentation of their iodine consumption, preferably from seafood and/or seaweed, prior to conception. This can also be achieved by taking a 150 µg iodine/day supplement, which is in line with the advice of the WHO and ETA ,56. The


Cerebrospinal fluid (CSF) glucose was 64.6 mg/dL (blood glucose 120 mg/dL), microprotein 102.6 mg/dL, and lymphocyte count 4 cells/mm3. The patient was started on doxycycline 2 × 100 mg/day, rifampicin 1 × 600 mg/day, streptomycin 1 g/day, and ceftriaxone 2 × 2 g/day. No regression occurred in the patient’s shoulder and neck pain. The cervical magnetic resonance imaging results are shown in A 59-year-old woman diagnosis with brucellosis was admitted to our clinic with complaints of severe headache, neck pain, and pain and weakness in the left arm despite receiving treatment for 2 weeks. The patient participated in animal husbandry and had a history of making and consuming cheese from unpasteurized milk. Physical examination revealed a body temperature of 37 °C, painful neck vertebrae on local palpation, weakness of the left arm, and nuchal rigidity. Her white cell count was 6.07 × 10Brucella spondylodiscitis represents osteoarticular involvement of brucellosis and rarely involves the cervical regionBrucella-related epidural abscesses and neurobrucellosis are rare complications with a poor prognosis,


Castana sativa) shells as potential active ingredient against OM. Therefore, chestnut shells were extracted at different temperatures (110–180 °C) by Subcritical Water Extraction (SWE), aiming to recover antioxidants. The extracts were also evaluated against microorganisms present in the oral cavity as well as on human oral cell lines (TR146 and HSC3). The highest phenolic content was obtained with the extraction temperature of 110 °C, exhibiting the best antioxidant/antiradical activities and scavenging efficiencies against HOCl (IC50 = 4.47 μg/mL) and ROO• (0.73 μmol TE/mg DW). High concentrations of phenolic acids  and flavanoids  characterized the phenolic profile. The antimicrobial activity against several oral microorganisms present in the oral cavity during OM, such as Streptococcus, Staphylococcus, Enterococcus, and Escherichia, was demonstrated. Finally, the effects on HSC3 and TR146 cell lines revealed that the extract prepared at 110 °C had the lowest IC50 . This study highlights the potential effects of chestnut shells on OM.Oral mucositis (OM), a common side effect of oncological treatment, is an oral mucosal disorder characterized by painful ulcerations and increased risk of infection. The use of natural antioxidants to suppress the redox imbalance responsible for the OM condition has emerged as an interesting approach to prevent/treat OM. This study aims to explore the chestnut ( Castanea sativa, Mill.) production has, as its primary purpose, the natural fruit consumption, or the production of frozen chestnut [Oral mucositis (OM) is one of the most common side effects of antineoplastic treatments, namely chemo- and/or radiotherapy, and is defined as an acute inflammatory, ulcerative, and painful disorder that severely degrades the quality of life of patients ,2,3. Ulcchestnut ,12. Portchestnut . The innchestnut . Shells chestnut ,14.C. sativa shells was the main aim of this study, to produce an optimal extract with promising effects against OM. While the in vitro effects on several buccal cell lines and the antibacterial activities against oral pathogens ensure the final OM effects, the phenolic profiles were evaluated to understand the specific compounds responsible for the antioxidant/antiradical activities. To the best of our knowledge, this is the first research that provides insights on how OM can be treated using a food by-product.Extraction is the most vital phase for the recovery of bioactive compounds from natural matrices. A great challenge to start implementing eco-friendly and effective extraction methods, rather than the use of standard solvent extraction, has arisen in industry ,16. GreeExtraction is a critical step in the phytochemical processing pathway to obtain the highest quantity of bioactive compounds from plant sources. To standardize plant products, it is crucial to use an appropriate extraction method ,25. FurtC. sativa shells and reported extraction yields ranging from 6.52% (25% aqueous extract) to 11.13% (100% ethanol) [As it is possible to observe, no significant extraction differences were observed between the extract’s yields, with the extract prepared at 180 °C achieving the lowest value (20.29%), oppositely to the extract obtained at 120 °C (21.00%). Cacciola et al. 2019) evaluated three different methods  using water as a solvent to recover bioactive compounds from chestnut shells. According to the authors, the extraction yields obtained for conventional liquid extraction, UAE, and MAE were, respectively, 5.2%, 2.2%, and 3.8% , at leasethanol) . The sam evaluateThe influence of sample-to-solvent ratio and mass transport enhancement on chestnut shells extraction was also evaluated in Microwave-Assisted Subcritical Water Extraction (MASWE) using a range of temperatures between 100 °C and 220 °C . AccordiThe SWE extraction yields reported in p = 0.008). Thermal degradation is probably responsible for the decrease in polyphenols content at higher temperatures. Nonetheless, this phenomenon alone cannot explain polyphenol behavior, as in conventional extraction methods, and the polyphenols breakdown can also occur at 80 °C. In fact, the increase in TPC observed at high SWE temperatures could also be attributed to the disruption of lignin–phenolic acid linkages or to the breakdown of lignin itself, resulting in large amounts of phenolic acids [ic acids .C. sativa shells extracted by MAE using different solvents (water and ethanol). The research team concluded that the highest TPC was achieved by the aqueous extract (173.89 mg GAE/g DW) [Pinto et al. (2021) assessed the phenolic profile, bioactivity, and cell viability of AE/g DW) . The samAE/g DW) .50 values for the DPPH assay ranged from 426.88 g/mL (110 °C) to 583.47 μg/mL (180 °C). Statistical analysis demonstrated significant differences between the extracts (p = 0.002), which aligns with the TPC results. At lower temperatures the extracts presented higher phenolic content, resulting in higher antioxidant powers.IC50 results varied between 148.68 μg/mL and 256.59 μg/mL for the extracts obtained at 110 °C and 180 °C, respectively. Significant differences were noted between the extracts obtained at 110 °C, 120 °C, and 140 °C (p < 0.016), as well as between the extracts obtained at 120 °C, 160 °C, and 180 °C (p < 0.05).Regarding the ABTS assay, the ICp = 0.031) were observed between the extracts obtained at 110 °C and 160 °C.According to 50 = 0.031 mg/mL for DPPH; IC50 = 0.284 mg/mL for ABTS). The same extract obtained the highest FRAP (56.23 g GAE/100 g extract), in the accordance with TPC results [50 = 44.1 µg/mL), ABTS (IC50 = 65.4 µg/mL) and FRAP (IC50 = 32.0 µg/mL) assays [Fernández-Agulló et al. (2014) also used DPPH and FRAP assays to evaluate the antiradical and antioxidant properties of chestnut shells and found that the aqueous extract (75 °C) obtained by conventional extraction produced the best results (IC results . In the  results . Regardi) assays . Analyzi) assays .C. sativa shells extracts prepared using SWE are detailed in The phenolic profiles of the As shown in As it is possible to observe, the major phenolic acid present in all extracts is gallic acid, particularly at 110 °C, 120 °C and 140 °C . This compound is extremely important for human health, and several studies have reported its biological and pharmacological activities, including anti-inflammatory, antioxidant, anticancer, and antimicrobial activities . MoreoveOxidative stress is the main factor in the basis development of OM, which induces to the activation of the apoptotic process, leading to the clinical manifestation of mucosal damage. Pro-inflammatory cytokines are in a positive feedback mechanism, conducting to ulceration, which compromises the mucosal integrity, allowing several microorganisms to penetrate tissues and increasing the sepsis risk ,43. The Previous studies have reported discrepancies in the phenolic composition of chestnut shells extracts, which can be explained by the use of diverse extraction techniques and also varied extraction settings. For example, Vásquez et al. (2012) analyzed chestnut shells extracted by organic solvents using conventional methods. The authors reported 11 compounds, with catechin/epicatechin and gallocatechin/epigallocatechin being the principal ones, while hydrolysable tannins, such as galloyl-glucoses and ellagic acid, were also present and identified . Gallic 2•−), peroxyl radical (ROO•), and peroxynitrite (ONOO-), when produced in excess in living cells are responsible, directly, or indirectly, for a number of diseases, including OM and, at last stage, oral cancer [A major global issue is the development of disorders related to oxidative stress caused by an imbalance between reactive species and antioxidants commonly present in the human body, being a key factor for the OM pathogenesis. However, its mechanism remains unclear. Nonetheless, it is known that chemo and/or radiotherapy triggers an increase in ROS generation . Reactivl cancer ,47. One l cancer . Table 350 = 31.14 μg/mL) showed the highest activity in terms of O2•− scavenging capacity, whereas the extract prepared at 180 °C showed the lowest activity (IC50 = 73.18 μg/mL). Gallic acid was used as a positive control for this test, and it produced the best results (IC50 = 23.82 μg/mL). No significant differences (p = 0.123) were observed between the extracts. However, HOCl was effectively scavenged by all extracts. While the extract obtained at 160 °C had the lowest free radical scavenging capacity (IC50 = 22.85 °C), the extract prepared at 110 °C had the best effectiveness (IC50 = 4.47 °C). Significant differences were noted between extracts obtained at 110 °C and 140 °C (p = 0.026), and 160 °C and 180 °C (p = 0.027). Regarding the ROO• quenching, the efficacy increased in the following order: gallic acid >110 °C > 140 °C > 120 °C >160 °C >180 °C. No significant difference (p = 0.379) was observed between extracts.The extract prepared at 140 °C  and O2•− (IC50 = 12.92 µg/mL). However, as summarized in •, as Pinto et al. reported an Ssample/STrolox = 0.084 for the optimal extract [Comparing with other studies, the SWE extracts prepared in the present study showed lower ROS scavenging activity. Lameirão et al. (2021) related that the chestnut shells extract obtained by UAE (at 70 °C during 40 min) achieved an ICectively . Likewis extract .-) scavenging activity was evaluated in the absence and in presence of hydrogen carbonate (HCO3−) to mimic the in vivo environment. Although the results presented in 50 calculated was 1.41 µg/mL (for the extract prepared at 120 °C) and 1.89 µg/mL (for the extract obtained at 160 °C) in the presence and absence of HCO3−, respectively. However, the gallic acid achieved best results . To the best of our knowledge, this is the first peroxynitrite assay performed on chestnut shells extracts. The phenolic profile, and thus the potential for compounds to degrade, as previously mentioned, could play a role in the observed discrepancies. No significant differences between the extracts were observed in the presence (p = 0.285) or absence (p = 0.357) of HCO3−. However, the radical scavenging capacity calculations, particularly for HOCl and ROO•, are generally consistent with the spectrophotometric results , temperature (37 °C), soft tissues , and hard tissues (teeth) . The oraherichia ,52.C. sativa shells extracts was analyzed against several Gram-negative and Gram-positive bacteria, the majority of which are present in the oral cavity in case of OM [The antimicrobial capacity of SWE se of OM ,53. MIC As can be observed, the chestnut shells extracts obtained at different temperatures exhibited diverse effects on the growth of microorganisms, which is probably due to the phenolic content of the extracts.S. aureus (Gram-positive) at the concentrations tested. However, the extract showed a significant antimicrobial activity against two drug-resistant bacteria, S. aureus MRSA and E. coli CTX M2 (MIC of 4 mg/mL and MBC of 32 mg/mL in S. aureus MRSA). This extract also exhibited MIC in E. coli ATCC 8739, E. coli ATCC 25922, and E. faecalis  and MBC of 64 mg/mL in E. coli ATCC 25922. E. coli CTX M2 was resistant to the extract obtained at 180 °C. Compared to the 110 °C extract, the extract prepared at 180 °C presented higher MIC values for the two non-resistant strains of E. coli (8 mg/mL), but a lower MIC for E. faecalis (16 mg/mL). At this temperature, the extract exhibited MBC values for all strains that had growth inhibition. As shown in E. coli CTX M2.The extract prepared at 110 °C did not present MIC and MBC values for S. mutans and E. coli [S. aureus (Gram-positive), possibly through an aggregation mechanism. Nevertheless, phenolic acids, such as gallic acid, also showed antibacterial activity against S. aureus and E. coli. Although these compounds present weaker efficiency in bacterial growth inhibition when compared to flavonoids, they still prove to be more efficient than standard antibiotics, such as gentamicin and streptomycin [Rhus chinensis L., and reported MIC values of 8 mg/mL for S. mutans. The authors also reported in vitro inhibition of S. mutans biofilms, demonstrating the antimicrobial activity of gallic acid [L. ferrea, which exhibited significant antimicrobial activity and inhibition of adhesion of S. mutans biofilms [Phenolic compounds may affect bacterial cells by damaging their cellular membranes, binding to the cell wall, and leading to enzyme inactivation and DNA damage . Flavano E. coli . Flavonoptomycin ,56. Kanglic acid . Likewisbiofilms .Previous research has demonstrated a correlation between phenolic compounds and the antimicrobial activity, as the quantity of phenolic compounds has a proportional inhibitory effect on the microorganism growth . Though,C. sativa extracts in OM treatment, it is particularly important to assess their effects in in vitro models. In this study, SWE C. sativa shells extracts were evaluated on HSC3 and TR146 cell lines by MTT assay. Both cell lines mimic the normal human buccal epithelium and are tumorigenic, making them suitable as oral mucosal models for testing medication absorption. In particular, the TR146 cell line was established from a neck metastasis of a buccal carcinomic tumor, and the HSC3 cell line was derived from tumors of metastatic lymph nodes originating from tongue squamous cell carcinoma [50 values.Considering the potential use of SWE arcinoma ,61,62. Fp = 0.044) and 180 °C . These results might be explained by the fact that the extract prepared at 110 °C contained the highest concentration of phenolic acids, which was in accordance with p = 0.022) and 180 °C . These results proved that TR146 cells were the most sensitive to the SWE chestnut shells extracts  and in HSC3 (468.15 µg/mL). This may be due to the higher content of phenolic compounds, as shown in Regarding the HSC3 results, the extracts obtained at 160 °C and 180 °C presented viabilities of 87.16% and 76.03%, respectively, after exposure with the low concentrations tested (250 µg/mL). The extracts obtained at 110 °C, 120 °C, and 140 °C showed viability reductions at concentrations of 500, 1000, and 2000 µg/mL, respectively. Significant differences were noted for the different concentrations of the extracts obtained at 110 °C , β-nicotinamide adenine dinucleotide (NADH), dihydrorhodamine 123 (DHR), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), sodium hypochlorite solution with 4% available chlorine, phenazine methosulphate (PMS), nitroblue tetrazolium chloride (NBT), fluorescein sodium salt and Trolox were obtained from Sigma-Aldrich . Catechin, gallic acid, Folin–Ciocalteu’s reagent and Triton X-100 were purchased from Sigma Chemical Co. .O-caffeyolquinic acid (≥98%), vanillic acid (≥97%), caffeic acid (≥98%), syringic acid (≥98%), p-coumaric acid (≥98%), trans-ferulic acid (≥99%), sinapic acid (≥99%), 3,5-di-O-caffeyolquinic acid (≥95%), ellagic acid (≥95%), 4,5-di-O-caffeyolquinic acid (≥90%), (+)-catechin (≥98%), (-)-epicatechin (≥90%), naringin (≥95%), quecetin-3-O-galactoside (≥97%), quercetin-3-O-glucopyranoside (≥99%), rutin (≥94%), phloridzin (99%), trans-polydatin (≥98%), resveratrol (≥99%) and caffeine (≥95%), used for the identification and quantification in extracts were purchased from Sigma-Aldrich . Dulbecco’s Modified Eagle Medium (DMEM), penicillin, trypsin-EDTA and streptomycin were provided by Invitrogen Corporation . Dimethyl sulfoxide (DMSO) was purchased by AppliChem .For microbial culture, Mueller Hinton Broth (MHB) medium was purchase to Liofilmchem , Tryptic Soy Broth (TSB), and Tryptic Soy Agar (TSA) medium was obtained from Biolife . HPLC solvents were delivered by Sigma-Aldrich . Individual’s standards of phenolic compounds used for the identification and quantification in extracts were purchased from Sigma-Aldrich . HPLC solvents were delivered by Sigma-Aldrich . Individual’s standards of phenolic compounds, namely gallic acid (≥99%), protocatechuic acid (99.63%), neochlorogenic acid (≥98%), caftaric acid (≥97%), chlorogenic acid (>95%), 4-Escherichia coli ATCC 25922, E. coli ATCC 8739, and Staphylococcus aureus ATCC 25913. All other strains, including resistant bacteria, were clinical isolates: Enterococcus faecalis and the resistant bacteria, Escherichia coli CTX M2 and Staphylococcus aureus MRSA.TR146 and HSC3 cell lines were obtained from the American Type Culture Collection . The following reference bacterial strains were obtained from the American Culture Collection (ATCC): Castanea sativa shells were generously provided by Sortegel, located in Sortes, Bragança, Portugal. Dehydration of shells occurred at 41 °C for 24 h  and then were pulverized in a miller to 1 mm particles . Prior to extraction, samples were carefully blended and kept at room temperature (20 °C), in the dark.C. sativa shells was performed in a homemade subcritical batch-type extractor of 1.7 L [Extraction of of 1.7 L . For it,R2 > 0.996) was created using gallic acid solution. Results were expressed as mg of Gallic Acid Equivalents (GAE) per g of dry weight (mg GAE/g DW).The Folin–Ciocalteu method was used to measure the total phenolic content (TPC) using spectrophotometry, with minor changes . A calibR2 > 0.993) was established using a standard ferrous sulfate (FeSO4 ·7H2O) solution at a concentration of 1 mM. Results were expressed in µmol of ferrous sulfate equivalents (FSE) per gram of DW (µmol FSE/g DW).FRAP assay was carried out as described by Benzie and Strain with a few minor changes . A calibR2 > 0.989). Results were presented as the radical scavenging activity’s half-maximal inhibitory concentration .DPPH free radical scavenging assay was performed according to the method described by Pinto et al. . Trolox 2 > 0.998) was ascorbic acid and the results were expressed as IC50 (µg/mL).Extracts were tested for ABTS radical scavenging activity according to Re et al. , with slHPLC was used to identify and quantify polyphenols using photodiode array (PDA) detection . Separat2•−) scavenging assay was performed as described by Gomes et al. [50 of the NBT reduction to diformazan.Superoxide radical  scavenging assay was performed accordingly to Gomes et al. . Gallic •) assay was carried out according to the method described by Ou et al. [• induced oxidation of fluorescein, was used to express the results.Peroxyl radical  scavenging activity. ONOO- was synthesized as described by Whiteman et al. [2 values, parallel tests were run in the presence of 187.5 mM NaHCO3, and the results were represented as IC50.The effects of the studied extracts on the of inhibition of ONOOn et al. . To simuMIC values were determined by the broth microdilution method using a 96-well plate with MHB medium with minor modifications . MicroorThe GR parameter was determined using the broth microdilution method in a 96-well plate using TSB. All different strains of bacteria were exposed to extracts concentrations superior, equal, or inferior to the determined MIC, performing 1 h interval readings of the optical density (OD) at 625 nm, 37 °C, for 24 h . Growth MBC assays were evaluated by the broth dilution method, where MBC was established as the lowest extract concentration that inhibited 99.9% of the bacterial inocula after 24 h of incubation at 37 °C. Briefly, 5 µL was taken from the well obtained from the MIC experiment from each extract and its dilutions and added to a petri dish plate with TSA medium. All the petri dish plates were sealed. The plate was divided into six equal areas, and for all extracts, the area of the lower dilution with no visible colonies was determined as MBC .® CO2 Incubator  for 24 h at 37 °C and exposed to various extract concentrations (125–2000 g/mL). This assay was performed according to Pinto et al. [4 cells/mL). MTT reagent was added, and the cells were incubated for 3 h at 37 °C, the number of viable cells was estimated, and the crystals were solubilized using DMSO. The positive and negative control were DMEM and 1% (w/v) Triton X-100. The background absorbance (630 nm) was subtracted at the 590 nm reading. Cell viability results were expressed as percentages.A cell viability assay was performed using two human oral cancer cell lines, namely TR146 and HSC3. Passages 28 and 6 were used for TR146 and HSC3, respectively. The cells were cultured in a CellCultureo et al. . Extractn = 3). IBM SPSS Statistics 27.0 software  was used to conduct statistical analysis. Tukey’s HSD test was used for post hoc comparisons of the means, and one-way ANOVA was used to analyze sample differences. p values lower than 0.05 (p < 0.05) were recognized as significant.Results are expressed as mean ± standard deviation , with a particular focus on gallic acid, protocatechuic acid, catechin, epicatechin, caffeine, and rutin. Furthermore, antimicrobial activity assays demonstrated that, in general, all extracts significantly inhibited bacterial growth against strains commonly found in OM patients. The chestnut shells extracts induced cytotoxic effects in HSC3 and TR146 cells, with a particular focus on TR146 cell line, and the extract obtained at 110 °C exhibited the lowest IC50 values for both cell lines. All the biological properties exhibited by the SWE C. sativa shells extract, particularly the extract obtained at 110 °C, may target the oxidative stress caused by anticancer treatments through the antioxidant activity and radical species scavenging capacity and, consequently, the inflammatory state. In addition, in advanced conditions (ulceration), it can manage bacterial proliferation and exert antiproliferative effects in head and neck squamous cell carcinoma. More research is required to reach a conclusion on the effects on the oral mucosa, particularly on the absorption of phenolic compounds via the buccal route. These may include in vitro oral epithelial permeation assays and oxidative stress experiments in cell lines. Furthermore, the sustainable use of chestnut shells will improve the applicability of food by-products, improve their economic value, and contribute to environmental sustainability.The scientific data presented in this study support the valorization of


Verbena officinalis L. is a traditionally important medicinal herb that has a rich source of bioactive phytoconstituents with biological benefits. The objective of this study was to assess the metabolic profile and in vitro biological potential of V. officinalis. The bioactive phytoconstituents were evaluated by preliminary phytochemical studies, estimation of polyphenolic contents, and gas chromatography-mass spectrometry (GC-MS) analysis of all fractions  of V. officinalis. The biological investigation was performed by different assays including antioxidant assays , enzyme inhibition assays (urease and α-glucosidase), and hemolytic activity. The ethyl acetate extract had the maximum concentration of total phenolic and total flavonoid contents . Significant antioxidant potential was observed in all fractions by all four antioxidant methods. Maximum urease inhibitory activity in terms of IC50 value was shown by ethyl acetate fraction (10 ± 1.60 µg mL−1) in comparison to standard hydroxy urea (9.8 ± 1.20 µg·mL−1). The n-hexane extract showed good α-glucosidase inhibitory efficacy (420 ± 20 µg·mL−1) as compared to other extract/fractions. Minimum hemolytic activity was found in crude methanolic fraction (6.5 ± 0.94%) in comparison to positive standard Triton X-100 (93.5 ± 0.48%). The GC-MS analysis of all extract/fractions of V. officinalis including crude methanolic, n-hexane, ethyl acetate, and n-butanol fractions, resulted in the identification of 24, 56, 25, and 9 bioactive compounds, respectively, with 80% quality index. Furthermore, the bioactive compounds identified by GC-MS were analyzed using in silico molecular docking studies to determine the binding affinity between ligands and enzymes (urease and α-glucosidase). In conclusion, V. officinalis possesses multiple therapeutical potentials, and further research is needed to explore its use in the treatment of chronic diseases. The existence of a wide range of secondary metabolites in medicinal plants has led to extensive investigation of these plants in recent years to identify the lead compound that can contribute to the management of chronic diseases and therapeutic effectiveness . There hHelicobacter pylori is ureases. The H. pylori infection can cause gastrointestinal inflammation, which raises the risk of chronic disorders, such as duodenal and gastric ulcers, gastric adenocarcinoma, and gastric lymphoma [Research on enzyme inhibition has expanded significantly over the last two decades . Urease lymphoma ,12,13. Rlymphoma . So, thelymphoma .The enzyme α-Glucosidase, found on the intestinal cell membrane surface, catalyzes the breakage of α-glycosidic linkage present in oligosaccharides to make monosaccharides. Hence, inhibitors of α-glucosidase can postpone the generation of d-glucose from complex carbohydrates thus slowing down glucose absorption, and lowering the level of postprandial plasma glucose . To decrVerbena officinalis L. (Verbenaceae) is known as the herb of grace, pigeons’ grass and vervain. It is primarily found in North Africa, Asia and all over Europe. It is mostly distributed in wastelands and near water in cultivated fields in the northern as well as western regions of Pakistan. V. officinalis is a perennial erect small herb that grows up to 25–100 cm in height with lobed and serrated leaves. Pink or purple is the color of flowers [V. officinalis has been utilized to alleviate several ailments in the folk medicinal system, including rheumatic pain, thyroid problem and wounds [V. officinalis has been studied for its important bioactivities such as antioxidant [V. officinalis, including flavonoids [V. officinalis [ flowers . V. offid wounds , gastricd wounds , cough ad wounds , depressioxidant , diuretiioxidant ,28, antiioxidant , antifunioxidant , antibacioxidant ,31, antiioxidant ,33, antiioxidant , neuroprioxidant  antiprolioxidant  and antiioxidant  effects.avonoids , phenyleavonoids , sterolsavonoids , and ursavonoids  explainsicinalis .V. officinalis (CRVO) was prepared, then fractionated using various solvents in ascending order of their polarity to produce different fractions; n-hexane (NHVO), ethyl acetate (EAVO) and n-butanol (NBVO). The methanolic crude extract along with its various fractions was evaluated for its total phenolic content (TPC) and total flavonoid content (TFC), urease and α-glucosidase inhibition assays, and antioxidant assays by different methods . Metabolic profiles of all fractions of the whole plant of V. officinalis were performed by gas chromatography-mass spectrometry (GC-MS) to identify the tentative secondary metabolites in the respective fractions. In silico molecular docking studies were also conducted for the bioactive compounds identified in all fractions by GC-MS.In account of this, a crude methanolic extract of V. officinalis were performed. This analysis confirmed the presence of many bioactive primary and secondary metabolites, as shown in Preliminary phytochemical testing of CRVO, NHVO, EAVO, and NBVO fractions of the whole plant of Total phenolic content (TPC)−1 DE) and the minimum amount was observed in CRVO (89.07 ± 1.88 mg GAE·g−1 DE)  (The maximum amount of TPC was observed in EAVO (394.30 ± 2.50 mg GAE·gextract) .Total flavonoid content (TFC)−1 DE  and the CRVO sample exhibited the lowest amount of TFC with a value of 66.26 ± 1.42 mg QE·g−1 DE . Tentatively identified compounds in CRVO, NHVO, EAVO and NBVO fractions of V. officinalis were 112, 112, 90, and 46, respectively. Compounds with a quality index of more than 80% were finally selected and represented in V. officinalis using GC-MS were shown in V. officinalis were exhibited in CRVO, NHVO, EAVO, and NBVO fractions of the whole plant of V. officinalis were evaluated for their biological potential using different approaches such as antioxidant assay, enzyme inhibition and hemolytic activities.CRVO, NHVO, EAVO, and NBVO fractions of the whole plant of Radical scavenging potentialV. officinalis was determined using DPPH and ABTS methods. The order of activity of different fractions was as follows; EAVO > NBVO > CRVO > NHVO for DPPH and EAVO > NBVO > CRVO > NHVO for ABTS. The highest scavenging potential estimated by the DPPH method was shown by EAVO (161.21 ± 2.02 mg TE·g−1 DE), and the minimum value was shown by NHVO (34.30 ± 2.02 mg TE·g−1 DE). The maximum free radical scavenging activity calculated by the ABTS method was shown by EAVO (178.57 ± 0.83 mg TE·g−1 DE), while the lowest value was observed in NHVO (51.77 ± 0.36 mg TE·g−1 DE)  .Reducing power antioxidant assayV. officinalis was evaluated by two methods namely CUPRAC and FRAP assays. The results were as follows: EAVO > NHVO > NBVO > CRVO for CUPRAC assay and EAVO > NBVO > NHVO > CRVO for FRAP assay. EAVO showed the maximum reducing potential with a value of 592.88 ± 2.44 mg TE·g−1 DE and CRVO showed the minimum reducing capacity value of 223.11 ± 1.55 mg TE·g−1 DE for CUPRAC assay. The highest reducing activity was calculated for FRAP assay for EAVO with a value of 360.18 ± 2.68 mg TE·g−1 DE, and the lowest was determined for CRVO, with a value of 152.171 ± 2.68 mg TE·g−1 DE -4-hydroxy-, methyl ester showed the maximum binding affinity i.e., −6.8 Kcal·mol−1 against urease and α-glucosidase. ar-Turmerone showed a −5.8 Kcal·mol−1 binding affinity against urease, and it showed a −6.5 Kcal·mol−1 binding affinity against α-glucosidase. Curlone showed a −5.6 Kcal·mol−1 binding affinity against urease while it showed a −5.9 Kcal·mol−1 binding affinity against α-glucosidase. 3-pyrazolidinone, 4,4-dimethyl-1-phenyl has a binding affinity of −5.7 Kcal·mol−1 against urease and −5.8 Kcal·mol−1 against α-glucosidase while the binding affinity of hydroxy urea and quercetin (standards) for these enzymes (urease and α-glucosidase) was −4.1 and −7.9 Kcal·mol−1, respectively.Molecular docking studies were performed for both urease (PDB DOI: 10.2210/pdb1E9Z/pdb) and α-glucosidase (PDB DOI: 10.2210/pdb5ZCB/pdb). All the compounds from GC-MS profiles of methanolic, The molecular docking study was validated by redocking of urease, α-glucosidase, and selected ligands with Autodock-1.5.6. Additionally, the same results were found in terms of the binding affinity and RMSD values. The docking results of the four ligands with both enzymes are depicted in V. officinalis extracts exhibited plant has a variety of phytochemicals such as phenols, flavonoids, glycosides, alkaloids tannins, saponins, resins, and terpenes. Previous studies showed the presence of these chemicals in the leaves of V. officinalis [V. officinalis may be attributable to the presence of these phytoconstituents.Plants naturally contain substances called phytochemicals, which can have beneficial or harmful effects on human health . The mosicinalis . The resV. officinalis previously [−1 DE and 137.35 ± 0.94 mg QE·g−1 DE, respectively while the crude methanolic fraction showed the minimum values of both phenolic and flavonoid contents having values of 89.07 ± 1.88 mg GAE·g−1 DE and 66.26 ± 1.42 mg QE·g−1 DE, respectively. Previous studies reported the presence of flavonoids  and phenolic constituents  in V. officinalis aerial parts contributing their role in the antioxidant potential of V. officinalis [Polyphenols are bioactive substances that are frequently found in food products made from plants, such as fruits, seeds, and cereals. Polyphenols help to prevent the possibility of chronic diseases including cancer, neurodegenerative and cardiovascular diseases . Severaleviously . Flavonoeviously . The ethicinalis .2/ARE (Nuclear factor erythroid 2-related factor 2/Antioxidant response element) and deactivation of the NF-кB (Nuclear factor kappa B) pathways, directly involved in the inflammatory reaction [Oxidation processes are essential to provide the energy to support biological activity in living organisms. As a result, the uncontrolled production of oxygen reactive species (ROS) is associated with many chronic diseases, including cancer, atherosclerosis, rheumatoid disease, and degenerative processes linked with aging . Synthetreaction . Additioreaction .V. officinalis plant extracts has been established in various scientific studies, which is essential in the prevention of heart disease and cancer [50 was 21.04 ± 1.61 µg·mL−1 and 33.8 ± 0.43 µg·mL−1 for ethanolic and aqueous extract, respectively. Xanthan oxidase is an enzyme that induces the production of oxygen radicals and was likewise inhibited by the solutions. The fraction including verbascoside and small quantities of luteolin 7-glucoside, isoverbascoside, and 1,5- and 4,5-dicaffeoylquinic acid had the highest antioxidant activity [V. officinalis as well as some compounds isolated from this plant [−1 DE and 137.35 ± 0.94 mg QE·g−1 DE), respectively. As a result, EAVO may be a source of free radical scavengers that naturally combat high ROS burdens. Phenolic compounds demonstrate redox characteristics, with the ability to act as antioxidants [n-hexane fraction, ethyl acetate fraction and n-butanol fraction of the whole plant of V. officinalis.The antioxidant activity of d cancer . A studyactivity . Researcis plant . The amois plant , and EAVoxidants . There wHelicobacter pylori is one of the causes of dyspepsia and extra-digestive problems linked with peptic ulcers worldwide [H. pylori is a class one carcinogen for gastric carcinoma and that it was determined that carcinogenic infections, including H. pylori, were responsible for 12% of malignancies detected in 2012. The success of synthetic drugs to cure gastric ulcers is overshadowed due to the toxicity risks associated with such drugs. Additionally, H. pylori resistance to antibiotics was among the list of antibiotic-resistant major priority diseases that encouraged researchers to find new antibiotics to eradicate this pathogen [V. officinalis as there is no comprehensive literature found on the urease inhibitory activity of the whole plant of V. officinalis. EAVO and NBVO fractions exhibited the highest urease inhibitory potential with IC50 of 10 ± 1.60 µg·mL−1 and 30 ± 2.40 µg·mL−1, respectively as compared to hydroxy urea IC50 value of 9.8 ± 1.20 µg·mL−1. The NHVO and CRVO fractions showed moderate results with IC50 values of 324 ± 16.40 µg·mL−1 and 465 ± 20.20 µg·mL−1, respectively , is the dissolution or breakage of the integrity of the RBC membrane . The proV. officinalis was presented in V. officinalis.The hemolytic activity of different fractions of the whole plant of n-hexane, ethyl acetate, and n-butanol fractions were docked against urease and α-glucosidase enzymes, along with hydroxy urea and quercetin (standards) docked against urease and α-glucosidase enzymes. The in silico molecular docking results depict the interaction of urease and α-glucosidase with the ligands benzenepropanoic acid, 3,5-bis-4-hydroxy-, methyl ester, ar-turmerone, curlone, 3-pyrazolidinone, and 4,4-dimethyl-1-phenyl detected in GC-MS analysis, which conclusively supports our observation of the plant extract in terms of urease and alpha-glucosidase inhibitory assays. The favorable in vitro potential of any extract should always be followed by toxicological experiments to determine the safety level and beneficial effects on animal models and it will be included in future studies [Additionally, it is possible to make significant advancements to in vitro research techniques for the quick screening of enzyme inhibitors utilizing molecular modeling. Therefore, to assess the biological activities of the extract and fraction, a combination of bioinformatics simulation and in vitro study will be helpful. Docking is a method of molecular modeling used to foretell how proteins (enzymes) will interact with small molecules (binders or ligands) . Therefo studies .V. officinalis was collected during the flowering season from 31°10′35″ N 72°42′13″ E Chak NO. 363 JB, Tehsil Gojra, District Toba Tek Singh, Punjab, Pakistan from November 2017 to March 2018. The taxonomic status of the plant was verified by Botanist Government College University, Lahore, Pakistan. The plant specimen with a voucher number of 3514 was deposited in the Botany department of the university. The solvents and chemicals of methanol, n-hexane, ethyl acetate, n-butanol, Folin-Ciocalteu reagent, sodium carbonate, gallic acid, aluminum chloride, quercetin, DPPH solution, Trolox, 2,2-azinobis(3-ethylbenothiazoline) 6-sulfonic acid, potassium persulfate, CuCl2, Neocuprion, ferric chloride, 2,4,6-tris(2-pyridyl)-s-triazine TPTZ, urease solution, α-glucosidase, and p-nitro-α-D-glucopyranoside were purchased from Sigma-Aldrich Chemical Co Ltd. . All other reagents used in the study were of analytical and chromatographic grade. Deionized water was used to prepare all solutions.The whole plant of V. officinalis was suspended in 1000 mL of distilled water. n-Hexane (20 g), ethyl acetate (100 g), and n-butanol (95 g) were used as extraction solvents to obtain different solvent fractions. Each fraction was then concentrated by using a rotary evaporator, followed by a 45 °C oven dry extraction. All extracts are kept in the refrigerator in air-tight containers for future assessment [The whole plant was shade dried and then pulverized into a coarse powder. The pulverized powdered material (10 kg) was then macerated in 80% methanol (20 L) for 2 weeks with frequent shaking at room temperature. Filtration of the methanolic extract was completed using Whatman filter paper and dried in a vacuum under reduced pressure at 40 °C by a rotary evaporator to produce a dry crude methanolic extract. The dry crude methanolic extract (460 g) of sessment .V. officinalis to evaluate the primary and secondary groups of metabolites in its methanolic extract along with its different fractions. The identification of primary metabolites including carbohydrates, amino acids, proteins, and starch was completed using Molish’s test, Ninhydrin, Biuret test, and Iodine test, respectively. The screening of secondary metabolites such as saponins (Frothing test), tannins (Ferric-Chloride test), phenols (Lead acetate test), flavonoids (Amyl Alcohol test), alkaloids (Dragendroff’s test), glycosides , resin (Acetic-anhydride test) and Steroids and Terpenes  were also done according to standard methods [Various phytochemical tests were performed for the phytochemical analysis of  methods .Determination of TPC−1 was made in methanol. The volume of 200 µL of sample solution was mixed with 200 µL of Folin Ciocalteu reagent in a 2 mL test tube and was vigorously mixed by the vortex. Then 0.8 mL of sodium carbonate solution (700 µM) was added to the mixture. The mixture was incubated for 2 h at ambient temperature, followed by the transfer of 200 µL of assay sample mixture to a 96-microtiter plate. The absorbance of each sample was recorded at λ 765 nm by using the instrument Biotek-Synergy HT. The same procedure was completed by producing aliquots of gallic acid’s at various concentrations including 10, 20, 40, 60, 80, 100, and 200 µg·mL−1 in methanol and the calibration curve was drawn by recording the absorbance of each aliquot of gallic acid at λ 765 nm. The methanol was used as a negative control. Total phenolic content was expressed in milligrams of gallic acid per gram of dry extract (mg·GAE·g−1 DE).The TPC of the crude methanolic sample and its different fractions were determined using the Folin-Ciocalteu reagent as reported previously with some modifications . The samDetermination of TFC−1, prepared in methanol. A solution mixture was prepared by combining 1 mL of sample extract (1 mg·mL−1), 4 mL of deionized water, 300 µL of sodium nitrite solution (5%), and 300 µL of AlCl3 solution (10%). Two mL of sodium hydroxide solution (1 M) was added, incubated for 6 min, then 2.4 mL of deionized water was added. The absorbance of each sample mixture solution was measured at λ 510 nm using an instrument UV-visible spectrophotometer IRMECO U2020. The same procedure was repeated by preparing the solution of quercetin’s different aliquots including 50, 100, 200, 300, 400, 500, 600, 800, and 1000 µg·mL−1 in methanol. The methanol (solvent) was used as the negative control. The calibration curve of quercetin was acquired by recording the absorbance of each aliquot of quercetin at λ 510 nm. The result of TFC of each sample extract was expressed as milligrams of quercetin per gram of dry extract (mg QE·g−1 DE).The TFC of each sample extract solution, including methanolic and its fractions, was assessed using a modified aluminum chloride method as reported in previous literature . The stoV. officinalis were studied by employing GC-MS. GC-MS was conducted using a gas chromatograph (Agilent 7890B) combined with an Agilent 5977B MSD equipped with mass hunter acquisition software. The system consisted of an HP-5ms ultra inert column with dimensions . The carrier gas was helium at a flow rate of 1.3 mL/min in constant flow mode. The temperature at the front inlet was adjusted to 250 °C. The initial oven temperature was held at 50 °C for 2 min, and then the oven temperature steadily increased from 50 °C to 200 °C at a rate of 15 °C/min. The sample extract was prepared in one microliter solution strength and was injected. MS source and MS Quad temperature were set at 230 °C and 151 °C, respectively. The identification was made using a scanning ranging from 50 to 1000 m/z and metabolites were identified by a comparison of the mass spectrum of each separated metabolite on specific retention time with mass spectrum data stored in the NIST-14 library [CRVO, NHVO, EAVO, and NBVO fractions of the whole plant of  library .V. officinalis was assessed by using DPPH and ABTS assays with minor modifications described in the literature [The radical scavenging potential of different extracts of terature .2,2-diphenyl-1-picrylhydrazyl (DPPH) assay−1). The 50 µL of each extract solution was added to a 96-microtiter plate followed by the addition of 150 µL 200 mM DPPH solution. The mixture was incubated at room temperature in dark for 30 min. The same procedure was repeated for the different concentrations of Trolox between 5–100 µg·mL−1 (positive control) to generate a calibration curve for the calculation of scavenging potential. The same procedure was conducted for the blank (negative control) by adding 50 µL methanol instead of Trolox or sample. The absorbance was measured at λ 517 nm using an instrument Bio Tek Synergy HT reader. The results of antioxidant potential were exhibited in milligram Trolox equivalent per gram of dry extract (mg TE·g−1 DE).Each extract was added into a sufficient amount of methanol to acquire the desired concentration of each sample (0.3127 mg·mL2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay−1) in a glass test tube. The test tube was incubated for 30 min in the dark and absorbance was noted at 734 nm. To generate a calibration curve for Trolox, the same procedure was repeated using 5–80 µg·mL−1 solution of Trolox, with methanol as the negative control. The results were presented in milligram Trolox per gram of dry extract (mg TE·g−1 DE).An equal volume of 2,2-azinobis(3-ethylbenothiazoline) 6-sulfonic acid (2.5 mM) and potassium persulfate (2.45 mM) was mixed, and 2 mL of this mixture was added to a sample solution of 1 mL  and ferric reducing antioxidant power (FRAP) assays in accordance with the modified procedure reported in the literature [The reducing antioxidant potential of different fractions and methanolic extract of terature .CUPRAC assay2 10 mM, neocuprion 7.5 mM, ammonium acetate buffer 1M pH 7, and then 3 mL of this reaction mixture was mixed with a sample solution of the 0.5 mL (0.3127 mg·mL−1). The mixture was then incubated at room temperature for 30 min. The solution’s absorbance was noted at λ 450 nm. For blank, methanol was used. The calibration curve was drawn for Trolox by using the concentrations of Trolox between 2.5–100 µg·mL−1. The results were presented in mg Trolox per gram of dry extract (mg·TE·g−1 of DE).A reaction mixture was prepared by taking the equal volume (1:1:1) of CuClFRAP assay−1). The resulting mixture was incubated for 30 min at room temperature. The absorbance was determined at 593 nm. For blank, methanol was used. The calibration curve for Trolox was drawn to calculate the antioxidant potential. The results were exhibited in mg Trolox per gram of dry extract (mg·TE·g−1 of DE).A reaction mixture was made by taking acetate buffer , ferric chloride (20 mM), 2,4,6-tris(2-pyridyl)-s-triazine TPTZ (10 mM) in HCl (40 mM) (10:1:1). Then 2 mL of this reaction mixture was mixed with each extract solution of 30 µL  which correlate with the current results of antioxidant, urease and α-glucosidase activities of this plant. Moreover, bioactive compounds identified by GC-MS in all fractions of V. officinalis also validated the results of this study. The urease and α-glucosidase inhibition activities of V. officinalis were further justified by in silico molecular docking studies of GC-MS-identified ligands, Benzenepropanoic acid and 3,5-bis-4-hydroxy-, methyl ester, ar-Turmerone, Curlone, 3-pyrazolidinone, 4,4-dimethyl-1-phenyl with these enzymes. The biological and phytochemical potential of this plant demonstrated its importance for the ongoing process of further isolating bioactive compounds.The current study analyzed


Mechanical thrombectomy and systemic thrombolysis are important therapies for stroke patients. However, there is disagreement about the accompanying risk of acute symptomatic seizures.A retrospective analysis of patients with an acute ischaemic stroke caused by large vessel occlusion was performed. The patients were divided into four groups based on whether they received either mechanical thrombectomy (MT) or systemic thrombolysis . Propensity score matching was conducted for each group combination  using the covariates “NIHSS at admission”, “mRS prior to event” and “age”. The primary endpoint was defined as the occurrence of acute symptomatic seizures.A total of 987 patients met the inclusion criteria, of whom 208, 264, 169 and 346 belonged to groups 1, 2, 3 and 4, respectively. Propensity score matched groups consisted of 160:160, 143:143, 156:156, 144:144, 204:204 and 165:165 patients for the comparisons 1:3, 1:4, 2:3, 2:4, 1:2 and 3:4, respectively. Based on chi-squared tests, there was no significant difference in the frequency of acute symptomatic seizures between the groups. Subgroups varied in their frequency of acute symptomatic seizures, ranging from 2.8 to 3.8%, 2.8–4.4%, 3.6–3.8% and 4.9–6.3% in groups 1, 2, 3 and 4, respectively.There was no association between MT or ST and an increased risk of acute symptomatic seizures in patients with an acute ischaemic stroke caused by large vessel occlusion who were treated at a primary stroke centre.The online version contains supplementary material available at 10.1007/s00415-022-10968-5. Strokes are one of the most common causes of death or disability, affecting 1.1 million people per year in Europe alone , 3, whicIn this retrospective single-centre study, we used propensity score matching to evaluate the risk of acute symptomatic seizures in patients treated with mechanical recanalisation compared to patients treated with systemic thrombolysis or conservative treatment in a stroke unit.A retrospective analysis of stroke data from 2016 to 2020 at the University Hospital Frankfurt was performed using a matched case–control design. This analysis was approved by the local ethics committee of the Goethe University Frankfurt. Written informed consent of the patients was waived because the patient data were evaluated retrospectively. Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines were closely followed .Inclusion criteria were final diagnosis of acute ischaemic stroke according to ICD-10 criteria  caused by an occlusion of a large cerebral vessel. Ischaemic stroke was defined as a focal neurological deficit that persisted for more than 24 h without appropriate therapy  or was accompanied by evidence of irreversible cell damage by additional neuroimaging (MRI or CT). LVO was proven by either vascular imaging (CT or MR angiography) or by infarct demarcation that was only explainable by proximal vessel occlusion ; intracerebral haemorrhage was excluded by the respective neuroimaging. LVO was defined according to the literature as an occlusion of the internal carotid artery, M1 or M2 segment of the middle cerebral artery, A1 or A2 segment of the anterior cerebral artery, P1 or P2 segment of the posterior cerebral artery, the vertebral artery or the basilar artery .The collected data included age; gender; NIHSS at admission, 24 h after admission and at discharge; and modified Rankin Scale (mRS) prior to event and at discharge. In addition, the type of LVO and data from the mechanical thrombectomy, such as the time of vessel revascularisation and the outcome of mechanical thrombectomy according to the Thrombolysis in Cerebral Infarction (TICI)  classifiThe primary outcome was the occurrence of an acute symptomatic seizure in patients with an acute ischaemic stroke. An acute symptomatic seizure was defined as a clinically apparent epileptic seizure observed by medical personnel or a record of subclinical seizure patterns or nonconvulsive status epilepticus on electroencephalography within the first 7 days after the onset of ischaemic stroke. The onset of stroke was assumed to be either the patient- or third party-reported time of onset or, if this could not be assessed with certainty, the last asymptomatic contact. Time (given as full days) between symptom onset and acute symptomatic seizure was determined. If an acute symptomatic seizure occurred before intervention , this patient was evaluated in the subsequent intervention group.In addition, semiology of the seizure as well as the changes in electroencephalography (EEG) were assessed. Based on the semiology, the seizures were divided into four groups: (1) seizure with/without impaired awareness but without signs of non-convulsive status epilepticus (NCSE) in EEG, (2) seizure with impaired awareness and proven NCSE in EEG, (3) focal motor seizure, (4) generalized tonic–clonic seizure. The changes in EEG were divided into three groups: (1) no interictal or ictal discharges, (2) interictal discharges, (3) NCSE, (4) no EEG available.The patient groups were matched using propensity score matching. For this purpose, patients were divided into four groups: patients receiving mechanical recanalisation only (Group 1: MT+/ST−), patients receiving both mechanical recanalisation and systemic thrombolysis (Group 2: MT+/ST+), patients receiving systemic thrombolysis only (Group 3: MT−/ST+) and patients who received neither mechanical recanalisation nor systemic thrombolysis (Group 4: MT−/ST−). The analysis was performed by comparing the respective groups with each other: 1:3, 1:4, 2:3, 2:4, 1:2 and 3:4.http://www.rstudio.com/). Matching covariates were “age”, “NIHSS at admission” and “mRS score prior to event”. To reduce the number of unmatched patients, we defined different intervals for the covariates. For age, the intervals were defined as "age" , "age5" , and "age10" . For mRS, we used "mRS1"  or "mRS2" (mRS 0–1 combined). For NIHSS, "NIHSS1" , "NIHSS2"  and "NIHSS3"  were defined. Matching was accepted if a Wilcoxon test yielded a p value > 0.05 for the matching covariates. If more than one matching strategy fulfilled these criteria, the one with the most patients included was used. Final matching was performed with the following covariates: 1:3 , 1:4 , 2:3 , 2:4 , 1:2  and 3:4 . Covariables prior to matching are given in the Supplementary Table 1.Propensity score matching was performed using the MatchIt package by Ho et al.  in RStudU test for ordinal and numeric data or a chi-squared test for nominal data was used to determine intergroup differences. Results with a p value < 0.05 were determined to be statistically significant. Due to the multiple pairwise comparisons, a Bonferroni correction of p value was performed by multiplying it with the number of comparisons (= 6) made per variable.Analysis of descriptive statistics was conducted using SPSS . Ordinal scaled variables such as NIHSS at admission, NIHSS after 24 h, NIHSS at discharge, mRS prior to event and mRS at discharge were reported using medians (1st–3rd quartiles), whereas age was given as mean ± standard deviation (SD). For statistical testing of the intergroup frequency of acute symptomatic seizures , a chi-squared test was performed. When comparing patient characteristics between the respective groups, a nonparametric Mann–Whitney From 2016 to 2020, a total of 987 patients met the inclusion criteria for this study. There were 208, 264, 169 and 346 patients in the MT+/ST−, MT+/ST+, MT−/ST+ and MT−/ST− groups, respectively  were observed between the matched groups Table . Only Map = 1.0, p* (* = corrected for multiple testing) = 1.0; MT+/ST− vs MT−/ST−, Phi = 0.08, p = 0.156, p* = 1.0; MT+/ST+ vs MT−/ST+, Phi = 0.02, p = 0.76, p* = 1.0; MT+/ST+ vs MT−/ST−, Phi = 0.05, p = 0.36, p* = 1.0; MT+/ST− vs MT+/ST+, Phi = 0.025, p = 0.61, p* = 1.0; MT−/ST+ vs MT−/ST−, Phi = 0.04, p = 0.428, p* = 1.0. The frequency of acute symptomatic seizures among the groups created by each matching varied, ranging between 2.8 and 3.8% after mechanical recanalisation without systemic thrombolysis  and between 2.8 and 4.4% after mechanical recanalisation with systemic thrombolysis . In patients without mechanical recanalisation but with systemic thrombolysis , the risk of acute symptomatic seizures was 3.6–3.8%, whereas without mechanical thrombectomy or thrombolysis , the risk was 4.9–6.3%  and with NCSE 2) were found in 1.6% and 0.5%, 1.0% and 2.4%, 0.6% and 0.0%, as well as 2.2% and 0.4% in groups 1, 2, 3, and 4, respectively. Focal motor seizure (3) was reported in 0.0%, 0.5%, 1.2%, and 1.3%, whereas GTCS (4) was found in 1.0%, 0.0%, 1.8%, and 1.3% in groups 1, 2, 3, and 4, respectively. In subgroup analysis of patients with GTCS, chi-square test did not show significant differences between the respective groups. Among patients with acute symptomatic seizures, no interictal discharges (1) were found in 33.3% (2/6 = results of EEG/number of acute symptomatic seizures within group), 12.5% (1/8), 66.6% (4/6) and 50% (6/12) in patients of group 1, 2, 3 and 4, respectively. Interictal discharges (2) or NCSE (3) were found in 50% (3/6) and 16.6% (1/6), 12.5% (1/8) and 62.5% (5/8), 33.3% (2/6) and 0.0% (0/6), and 25% (3/12) and 8.3% (1/12) in groups 1, 2, 3, and 4, respectively  and the fact that the control group was randomly selected rather than matched limited the statistical robustness of the results [However, some studies have suggested an increased risk of acute symptomatic seizures after systemic thrombolysis . Neuroto results . Further results , 27. Sim results . By inclRegarding post-stroke epilepsy within 2 years, which is pathophysiologically distinct from acute symptomatic seizures, Naylor et al. showed an increased risk in patients with anterior circulation cerebral infarction and systemic thrombolysis or mechanical recanalisation (adjusted OR 3.4–5.5) . HoweverThere were some significant differences between the treatment groups regarding the location of the LVO. In particular, the groups of patients who received mechanical thrombectomy had a significantly higher proportion of M1 occlusions compared with the MT−/ST+ or MT−/ST− groups. This is due to the fact that M1 occlusion is mostly associated with a severe neurological deficit and a large penumbra volume . In addiThis study had several limitations. Although this study evaluated one of the largest monocentric cohorts so far, the limited number of acute symptomatic seizures might increase the risk of overlooking a potential difference between the groups. Furthermore, matching regarding the underlying vessel occlusion or success of recanalisation was not possible due to the limited number of available cases. The final infarct size was approximated by stroke severity recorded by NIHSS and not radiologically determined. Studies that include larger number of patients and therefore allow for a matching based on the final infarct size and vessel occlusion would be necessary. In addition, systematic comparison between hospitals with and without stroke unit care would allow to assess the impact of consistent prevention of reperfusion syndrome. Propensity score matching has been described as a method for quasi-randomization of observational studies, however it bears the risk of underestimating latent covariates since matching was only based on measured variables such as age, NIHSS and mRS. An example of a latent variable would be cerebral seizure propensity and the extent of cortical damage. The use of NIHSS can mitigate this to some extent. Other variables such as infarct volume on cerebral imaging or hemorrhagic transformation can be included and improve this estimate and can incorporate the effect of latent variables into the statistical analysis through statistical models . BecauseWe did not identify an association between mechanical thrombectomy or systemic thrombolysis and an increased risk of acute symptomatic seizures in patients with an acute stroke resulting from an LVO who were treated at a primary stroke centre.Supplementary file1 (DOCX 15 KB)Below is the link to the electronic supplementary material.


However, the applicability of using low specific activity (LSA) 99Mo asks for sorbents with high sorption capacity. Herein, this study aims to evaluate the sorption behavior of LSA 99Mo towards several CeO2 nano-sorbents developed in our laboratory. These nanomaterials were prepared by wet chemical precipitation (CP) and hydrothermal (HT) approaches. Then, they were characterized using XRD, BET, FE-SEM, and zeta potential measurements. Additionally, we evaluated the sorption profile of carrier-added (CA) 99Mo onto each material under different experimental parameters. These parameters include pH, initial concentration of molybdate solution, contact time, and temperature. Furthermore, the maximum sorption capacities were evaluated. The results reveal that out of the synthesized CeO2 nanoparticles (NPs) materials, the sorption capacity of HT-1 and CP-2 reach 192 ± 10 and 184 ± 12 mg Mo·g–1, respectively. For both materials, the sorption kinetics and isotherm data agree with the Elovich and Freundlich models, respectively. Moreover, the diffusion study demonstrates that the sorption processes can be described by pore diffusion (for HT-synthesis route 1) and film diffusion (for CP-synthesis route 2). Furthermore, the thermodynamic parameters indicate that the Mo sorption onto both materials is a spontaneous and endothermic process. Consequently, it appears that HT-1 and CP-2 have favorable sorption profiles and high sorption capacities for CA-99Mo. Therefore, they are potential candidates for producing a 99Mo/99mTc radionuclide generator by using LSA 99Mo.  The production of these generators includes different technologies, for instance, sublimation, electrochemical, solvent extraction, supported liquid membrane (SLM), and column chromatographic approaches [99mTc onsite [99Mo on a sorbent for decay so that the daughter 99mTc can then be easily separated in high purity by using an isotonic saline solution at desired time intervals [There is an increasing interest in using 99mTc T1/ = 6.01 hindustry ,4. This proaches ,6,7,8,9.c onsite . The identervals ,12. 99mTc parent, 99Mo, can be produced in nuclear reactors either by fission of 235U or by direct neutron irradiation of natural Mo or enriched 98Mo targets [99mTc generators on the market because it provides 99Mo with high specific activity (>104 Ci/g Mo). On the other hand, this route has some disadvantages. The main ones are the prohibitive production costs, the generation of large amounts of radioactive waste, and the increasing proliferation risks [99Mo of low specific activity (LSA) (~5–10 Ci/g Mo) [2O3), the material used, is limited to only 2–20 mg.g–1 for molybdate (99MoO42−) [The  targets ,13. On ton risks ,15,16. TCi/g Mo) ,18,19,2099Mo. These new sorbents might make it possible to produce 99Mo/99mTc generators by using LSA 99Mo.Recently, nanotechnology has shown that it has the potential to develop the properties of different materials. These newly developed materials provide attractive solutions in multidisciplinary sectors, such as biology, chemistry, and medical sciences . Nanostr99Mo on several CeO2 nanosorbents developed in our laboratory. To achieve this goal, we report the preparation of CeO2 NPs using wet chemical precipitation and hydrothermal methods. Moreover, we investigated the sorption efficiency of the prepared materials for Mo under different experimental conditions such as pH, the initial concentration of the molybdate solution, contact time, and temperature. In addition, the sorption kinetics, equilibrium isotherm, and thermodynamics were evaluated.In this paper, our main objective is to evaluate the sorption profile of LSA 3)3·6H2O, sodium hydroxide, ethanol absolute, and nitric acid were purchased from Merck, Darmstadt, Germany.All chemicals were of analytical grade purity and were used without further purification. Milli-Q water was used for the preparation of solutions and washings. Cerium nitrate hexahydrate Ce with 5 mL 1 M NaOH solution after ~7 d from the calibration date. The total 99Mo radioactivity was measured with a Capintec Radioisotopes Calibrator . The 99Mo eluate solution was passed through 0.45 micro-Millipore filter to retain any alumina particles. Then, the 99Mo solution was diluted with HNO3 solution to the desired pH value.99Mo. A pH-meter with a microprocessor  was used to measure the pH values. A thermostated shaking water bath  was used for all batch equilibrium studies. The X-ray powder diffraction (XRD) patterns were performed via a D8 ADVANCE diffractometer  with a Cu-Kα radiation source (λ = 0.1542 nm) operating at 40 kV, 40 mA, and a scanning range of 2θ = 10–80° at room temperature. The Brunauer–Emmett–Teller (BET) surface area of the synthesized materials was measured using nitrogen sorption isotherms at −195 °C on a model Gemini VII, ASAP 2020 . Before the analysis, the sample was degassed for 12 h at 110 °C. Zeta potential (ζ) measurements were studied using a Zeta-sizer  for isoelectric point (pHIEP) measurements. A JSM-7100F (JEOL) was used to conduct field emission scanning electron microscopy (FE-SEM) measurements.Radiometric identifications and measurements were carried out by using a multichannel analyzer (MCA), Inspector 2000 model, Canberra Series, Mirion Technologies, Inc., Meriden, CT, USA, coupled with a high-purity germanium detector (HPGe). Samples of constant geometry were counted at a low dead time (<5%). The radioactivity levels were determined by quantifying the 740 keV photo peaks corresponding to 2 NPs were prepared by a wet chemical method via the precipitation of cerium nitrate in an alkaline medium. In a typical procedure, 2 g of Ce(NO3)3·6H2O were dissolved in a mixture of Milli-Q water and ethanol absolute with a ratio of (1:4 v/v). Then, this solution was added drop-wise to a freshly prepared 0.4 M sodium hydroxide solution (pH~13) under continuous stirring, and the pH value was controlled at about 11. The mixture was stirred until a yellow suspension of CeO2 NPs was formed after approximately 30 min. After that, the formed precipitate was washed several times with de-ionized water and ethanol absolute so that the soluble salts and fine impurities could be removed. Subsequently, the washed precipitate was centrifuged at 4000 rpm for 15 min and dried in a drying oven at about 50–70 °C for 12 h. The resulting sample is denoted as CP .CeO2 NPs were synthesized according to the same procedure as described in v/v). First, the formed NPs were transferred into a Teflon-lined stainless steel autoclave. Then, the autoclave was sealed and maintained at 150 °C for 12 h. Subsequently, the formed precipitate was washed several times with de-ionized water and ethanol absolute, centrifuged, and dried at about 70 °C for 12 h. The dried material is called HT .Hydrothermally modified CeO2-CP and CeO2-HT NPs were heated at different temperatures: 200 °C and 400 °C with a heating rate of 10 °C/min for 2 h  to 300 mg of each sorbent. The uptake behavior was investigated at different pH values, which ranged from 1 to 12. The initial pH of the 99Mo solution was adjusted by using either HNO3 or NaOH. The mixtures were shaken in a thermostatic shaker at 25 ± 1 °C for 24 h. After that, the liquid phases were separated, centrifuged, and then 1 mL aliquot was withdrawn for radiometric measurements. In these analyses, we measured the total solutions radioactivity before and after equilibration with an HPGe counter by using an appropriate gamma-ray peak (740 keV). Additionally, both initial and equilibrium pH values were measured. Finally, the uptake percent of each material was calculated as a function of solution pH by using the following equation:o and Ae are the initial and equilibrium of 99Mo radioactivity expressed in counts/min.The influence of medium pH on carrier-added 99Mo on the prepared CeO2 NPs, we determined the uptake percent of 99MoO42− ions (~pH 3) at different time intervals. Typically, 300 mg of each sorbent were suspended in 30 mL of 99MoO42− solution (50 mg·L−1 in 0.001 M HNO3) and shaken in thermostated shaker bath at 25 ± 1 °C for different time intervals (5 min–50 h). Then, 1 mL of the supernatant was separated and measured. The sorption of Mo on each material was followed with time until the equilibrium was established. Finally, we calculated the Mo capacity (o (count/min) is the initial activity, and 99Mo activity at a time (t), −1) is the initial 99MoO42– ion concentration,To estimate the sorption rate of carrier-added Furthermore, for a comprehensive understanding of the reaction kinetics and diffusion mechanisms, we used the obtained results and applied different sorption kinetic and diffusion models. 99MoO42– ion concentration from 50 to 5000 mg·L−1 while keeping the sorbent dose constant. Herein, we mixed 30 mL of 99Mo solution (pH ~ 3) with 300 mg of each material. The other parameters, such as equilibrium time and temperature, were maintained at 24 h and 25 ± 1 °C, respectively. Subsequently, the aqueous phases were pipetted, centrifuged, and measured. The equilibrium adsorption capacity (Qe) in mg·g−1 was calculated using Equation (2). The obtained results were evaluated using sorption isotherm models.Sorption isotherm studies were conducted by varying the initial 2), average percentage error (APE), and root mean square error (RMSE). These functions can be calculated according to the following equations:In order to evaluate sorption isotherm models, three error distribution functions were used. These functions are Chi-square , the standard entropy change (ΔS°), and the Gibbs free energy change (ΔG°).2 NPs, different parameters were studied using the batch contact method. These parameters include solution pH, contact time, initial ion concentration (mi), and temperature. The impact of each parameter on the Mo sorption was studied separately. Meanwhile, the other factors were kept constant. In order to evaluate the sorption profile of the molybdate on the synthesized CeO2 nanoparticles prepared via wet chemical precipitation and hydrothermal methods are shown in 2 nanoparticles have face centered cubic structure with the lattice parameters a = b = c = 5.411 Å and α = β = γ = 90°. The diffraction peaks were found at 28.56°, 33.08°, 47.47°, 56.36°, 59.08°, 69.40°, and 76.70° [2. The average crystallite size (D) of CeO2 nanoparticles is calculated by using the Debye–Scherrer equation: 2 nanoparticles were evaluated and listed in The XRD patterns of CeO2 nanoparticles using a nitrogen adsorption isotherm  measurements listed in 2 NPs morphology. 2 NPs prepared at different temperatures. As shown in the SEM images, the hydrothermally prepared CeO2 NPs exhibited spherical morphology with almost uniform characteristics. There is no obvious change in the morphology upon changing the temperature from 50 °C to 400 °C. The CeO2 NPs prepared by the wet chemical precipitation method showed a rough morphology compared with the hydrothermally modified analogous especially at a low temperature (50 °C). Upon increasing the temperature, the rough shape was regulated and semi-spherical NPs appeared as shown for CP-2 and CP-3. Additionally, as the morphology of the nanoparticles is a key characteristic factor for their performance, we investigated the effect of the preparation method with different temperatures on the CeO2 NPs for carrier-added 99Mo sorption from aqueous solutions. The pH of the Mo solution is the key factor that controls the sorption process, as it has a profound impact on determining the ionic state of the functional groups that exist on the sorbent surface. Furthermore, it affects the dissociation, complexation, and/or ionization of the adsorbed ions [99Mo uptake, the sorption behavior of each material was evaluated at a wide range of pH values (1–12). The results are displayed in Batch experiments were conducted to verify the usefulness of the synthesized CeObed ions . In ordeIEP is reached at ~pH 7. Consequently, the CeO2 surface carries a positive charge at low pH (pH < 7). The change in the surface charge of the solid phase is because the active sites on the surface are hydrated and covered by amphoteric hydroxyl groups. Hence, based on the pH of the medium, these hydroxyl groups can undergo different reactions with either H+ or OH− to develop positive or negative charges on the surface. Herein, at pH < 7, they are protonated, and the CeO2 surface develops a positive charge as follows:Since the external solution pH governs the sorbents’ surface acid-base properties, we attempted to clarify the isoelectric point (ζ) of each material. 42−). It polymerizes in an acidic medium to polymolybdate (Mo7O246−) with higher molybdenum content per unit charge [Furthermore, the data in t charge . Therefo2 was studied at different time intervals, and the results are represented in The progress of Mo sorption onto the synthesized CeOFurthermore, the results revealed that compared to materials prepared via the wet chemical method, the hydrothermally modified samples introduce a faster reaction rate. However, heated materials at 400 °C (CP-3 and HT-3) exhibit a relatively slow sorption rate. This behavior may be explained because the thermal treatment of nanomaterials decreases the number of active surface sites, which reduces the sorbent reactivity .Based on the earlier experimental and surface morphological evaluations, the hydrothermal modification improved the sorption profile of HT-1 considerably. Moreover, except for CP-2, the thermal treatment does not enhance the uptake capability of the heated materials. On the contrary, it adversely diminishes the sorption rate for the ones heated at 400 °C. Therefore, CP-2 and HT-1 were selected for subsequent evaluation studies. 99Mo onto CP-2 and HT-1 by using three different kinetic models, namely, Lagergren-first-order (Equation (8)) [−1) are the amount of adsorbed molybdenum at a time (t) and equilibrium, respectively, −1) is the Lagergren-rate constant, −1) is the desorption constant representing the degree of surface coverage.To design an effective sorption process, determining kinetic parameters is crucial . Kineticion (8)) , pseudo-ion (8)) , and Eloion (8)) ,37. Thest) values versus contact time (t). The calculated kinetic parameters are presented in 2) of the three applied models  than Lagergren-first-order model (R2 = 0.989 and 0.990) and the pseudo-second-order model (R2 = 0.991 and 0.990) for CP-2 and HT-1, respectively. Accordingly, we suggest that the Elovich model is the most appropriate to explain our sorption kinetics data. This finding may indicate that the prepared CeO2 matrices possess heterogeneous surfaces, and the uptake of carrier-added 99Mo develops multi-adsorption layers [d models , it can n layers ,37.−1) are the amount of adsorbed molybdenum at a time (t) and equilibrium, respectively. Ci (mg·L−1) is the initial Mo concentration, W (g) is the sorbent mass, and V (L) is the solution volume. −1), 1/2), and Generally, the uptake of adsorbate ions on the sorbent material occurs through : (i) the2) are tabulated in 2) of each model. For CP-2, the application of the McKay model resulted in a higher value (R2 = 0.992) than both Weber and Morris model (R2 = 0.977) and the Bangham model (R2 = 0.967). These findings indicate that the sorption of Mo on CP-2 might occur through an external film diffusion mechanism. For HT-1, the correlation coefficient values of the three models were: McKay model (R2 = 0.968), Weber and Morris model (R2 = 0.946), and Bangham model (R2 = 0.980). Therefore, it can be concluded that the pore diffusion model might control Mo sorption on HT-1.The rate constants can be determined from the slope of the linear plots of e) and the uptake percent of Mo onto CP-2 and HT-1 at constant temperature (25 ± 1 °C). The figure shows that on increasing the initial Mo concentration, the uptake percent decreases, and on the contrary, the sorption capacity (Qe) increases. These findings are attributed to the increased mass driving force at higher molybdate concentrations [Sorption isotherms describe how the adsorbed ions are dispersed between liquid and solid phases when the adsorption process reaches an equilibrium state at a constant temperature ,42. Constrations ,46,47.e (mg·g−1) is the equilibrium sorption capacity, Ce (mg·L−1) is the equilibrium concentration of molybdate ions, Qm (mg·g−1) is the maximum capacity. KF (mg1-n·Ln/g), KL (L·mg−1), and −1) are the Freundlich, Langmuir, and Temkin constants, respectively. The parameters n and B reflect the heterogeneity of adsorption sites and the heat of adsorption, respectively. −1·K−1), and T (K) is the absolute temperature. Based on the sorbents’ behavior and the mode of interaction, the equilibrium isotherm data shown in e) values versus (Ce). The calculated equilibrium isotherm parameters and their corresponding correlation coefficient (R2) are given in 2) values, the Freundlich isotherm best describes the equilibrium data. It has a higher correlation coefficient  than Langmuir  and Temkin  for CP-2 and HT-1, respectively. This result is confirmed by the lower values of the error functions (X2), (APE), and (RMSE) for the Freundlich isotherm compared to the Langmuir and Temkin models (n models . Further99Mo with CP-2 and HT-1 at 25 ± 1 °C until no further uptake occurred. The maximum sorption capacity was determined according to the following equation:−1) is the maximum (saturation) sorption capacity, −1) is the initial concentration, Furthermore, the maximum sorption capacity for Mo was evaluated under static conditions. We conducted repeated equilibrations of carrier-added −1, respectively. These values might be attributed to the high surface area of CP-2 and HT-1. In addition, the sorption behavior of each sorbent allows the formation of multi-adsorption layers on their surfaces, which is confirmed by kinetic and equilibrium isotherm studies. It is pertinent to point out that the sorption capacities of CeO2 nanosorbents we developed are much higher than the sorption capacity of conventional alumina (2–20 mg Mo·g−1) [99Mo/99mTc generators by using LSA 99Mo.The results show that the sorption capacities of CP-2 and HT-1 reach 184 ± 12 and 192 ± 10 mg Mo·g99Mo uptake is observed on HT-1. Meanwhile, the sorption capacity of CP-2 increases noticeably. These findings indicate that the sorption process is endothermic in nature.The effect of temperature on molybdenum uptake onto CP-2 and HT-1 was studied in the temperature range (298–343 K). With increasing the reaction temperature, a slight increase in carrier-added d (mg·mL−1) is the distribution coefficient, R is the universal gas constant (0.008314 kJ·mol−1·K−1), and T (K) is the absolute temperature. In order to understand the thermodynamic behavior of the sorption process, we investigated the values of the enthalpy change (ΔH°), the entropy change (ΔS°), and the free energy change (ΔG°) in our experimental temperature window. These parameters were calculated by using Vant’ Hoff equations , as folld versus the reciprocal absolute temperature (1/T). The values of ∆H° and ∆S° can be determined from the slope and intercept, respectively. The calculated values of ΔH°, ΔS°, and ∆G° are listed in 99Mo on several CeO2 nanosorbents developed in our laboratory. First, these materials were synthesized by using simple wet-chemical precipitation (CP) and hydrothermal (HT) methods. Then, they were heated at different temperatures: 200 °C and 400 °C. Eventually, an assessment of the carrier-added 99Mo uptake behavior was carried out under different experimental conditions. We found that the hydrothermal modification improves the morphological and sorption profile of HT-1 considerably. Except for CP-2, the thermal treatment did not enhance the sorption capability of the heated materials. In contrast, it diminished the Mo sorption for the ones heated at 400 °C. In addition, sorption kinetics data of carrier-added 99Mo Mo onto CP-2 and HT-1 were most favorably described by the Elovich model. The diffusion data showed that film and pore diffusion models controlled the sorption process for CP-2 and HT-1, respectively. Moreover, out of the investigated isotherm models, the Freundlich model was the most appropriate to describe the equilibrium isotherms of carrier-added 99Mo on both sorbents. Furthermore, we studied the influence of reaction temperature on Mo uptake, and the results confirmed the spontaneous and endothermic nature of the sorption processes. Finally, we evaluated the maximum sorption capacity of CP-2 and HT-1 towards carrier-added 99Mo under static sorption conditions. Their capacity reached 184 ± 12 and 192 ± 10 mg Mo.g–1, respectively. From the outcome of our investigation, it is possible to conclude that HT-1 and CP-2 have favorable sorption profiles and appreciable uptake capacities for carrier-added 99Mo. Consequently, they have the potential for producing a 99mTc radionuclide generator by using LSA 99Mo. Clearly, the next stage of our research will investigate the feasibility of using these two sorbent materials to develop a useful 99Mo/99mTc generator.Our main goal was to evaluate the sorption profile of LSA