Noncommunicable diseases and obesity are between the major health threat due to consumption of unhealthy foods and limited time spent on physical activities, a situation of particular concern among children. Since children spend most of their time at school, this study intends to investigate the effect of a school intervention program, which combines recreational football and nutrition education, on body composition, physical fitness, physical activity, blood pressure and heart rate, eating behaviours, nutritional knowledge, and psychological status in elementary school children.A total of 67 children, between 7 and 10 years old, were allocated into three groups: the Football Group (FG) which held 2 weekly sessions of 60 min of recreational football, the Nutrition and Football Group (NFG) which held 2 sessions per week of 60 min of recreational football plus 60 min of nutritional education and the Control Group (CG) which maintained its usual curriculum. The intervention lasted 12 weeks. All measurements were collected before and after the intervention.p < 0.05) improved BMI Z-score, rest heart rate, horizontal jump and shuttle test, physical activity level, and psychosocial health. The NFG group significantly decreased (p < 0.05) waist-to-height ratio and blood pressure, and significantly increased (p < 0.05) nutritional knowledge, fruit, and fish consumption. While FG significantly decreased (p < 0.05) the percentage of fat mass and significantly increased (p < 0.05) muscle mass and performance in the 20 m sprint.Intervention groups significantly (The results have shown to improve nutritional status, explosive strength, aerobic and neuromuscular fitness, as well as increase the level of physical activity. The nutritional education sessions contributed to increase nutritional knowledge and to improve the consumption of healthy food groups in a ludic-educational way. The “Football and Nutrition for Health” program was able to induce short-term improvements in several health markers, highlighting the role of the school curriculum in children's health. ObesityRegarding childhood obesity, although remains a major public health problem in the WHO European Region , 3, rateIn recent decades, changes in dietary patterns and physical activity behaviours have been identified as likely contributors to a rise in childhood obesity . PhysicaSince children are in a period with rapid physiological and behavioural change, it is a critical time for the formation and establishment of lasting adequate nutrition, physical activity, and healthy behaviours . Food prThe pilot project of “‘FIFA 11 for Health’ modified program for Europe was developed in Denmark and showed positive effects on blood pressure, fat percentage, social well-being, and health knowledge  . The conFor all describe reasons mentioned above, it was created the “Football and Nutrition for Health” project, inspired in “FIFA 11 for Health” modified program for Europe, that integrates the two major determinants of health in children: physical activity and nutrition as a non-pharmacological strategy to prevent and treat lifestyle diseases. Thus, the aim of this study was to understand the effects and differences of a recreational football intervention a sole and the combined effect of nutrition education and recreational football in body composition, blood pressure, heart rate, physical fitness, eating attitudes, nutritional knowledge, and perceived psychological status, after 12-week on schoolchildren aged 7–10 years old.2.2.1.The current study lasted 14 weeks: week 1 was used for the pre-tests; weeks 2 to 13 the intervention groups completed the “Football and Nutrition for Health” program, and in week 14, all groups completed post-testing with the same test battery as in week 1.n = 26, school I), Football and Nutrition Group  or Control Group . From those, 67 children completed both pre- and post- testing. The participant selection flow chart is presented in To be eligible for participation, boys and girls had to be between 7 and 10 years old and attend elementary schools in the municipality of Águeda. Children under the age of 7 were not included in the study, as well as those who were participating in any nutritional or weight loss programs. Moreover, individuals using any medication or having a pathology or clinical condition that could potentially influence the study outcomes or limit participation in physical activities were also excluded. BMI was not considered as an exclusion factor. A total of 72 Portuguese elementary school children, aged 7–10 years from three schools participated in this study and were cluster-randomized, with each school representing a cluster, into the Football Group  using the G*Power software version 3.1.9.6, considering an effect size 0.25, a statistical power of 0.95 at p < 0.05  performed 2 sessions per week of 60 min of recreational football and Nutrition and Football Group (NFG) performed 2 sessions per week of 60 min of recreational football plus 60 min of nutritional education, and Control Group (CG) continued their normal curriculum. It was incorporated physical activity based on recreational football practice and nutrition education in the school curriculum. The recreational football sessions were based on the structure, content, and implementation protocol for the “FIFA 11 for Health” program , 28 with2.3.2.3.1.‒2) and percentile were calculated  was determined with the average of three measurements on the left upper arm by an automatic blood pressure monitor . Heart r2.3.3.After a standardized warm-up, the children performed a physical test battery which consisted of the maximal horizontal jump length test , the 20 2.3.4.To estimate daily physical activity a tri-axial accelerometer  was used at baseline and at the end of the study. ActiLife Software v6.13.4 was used for data processing. Before each data collection, the ActiGraph was initialized according to the manufacturer's specifications . Data coAccelerometer data files were collected in 15-s epochs according to the respectively cut point chosen to record the spontaneous and intermittent activities of children more accurately , 42. The2.3.5.2.3.5.1.The collection and description of dietary intake was evaluated by a 24-hours recall using portion quantification methods with photography of home measurements  completed by the legal representatives . Detaile2.3.5.2.To complement the 24h recall, a food propensity questionnaire (FPQ) was applied. Parents (or the main caregiver) of children were asked to report the frequency of consumption of food and fluids items in the last month. Following the protocols proposed by IAN-AF studies , a food 2.3.5.3.The use of nutritional supplements was assessed through questions of propensity for habitual consumption, from a pre-defined list of different supplements, currently in use in the market, with the possibility of mentioning others not included in the initial list . The ref2.3.6.The questionnaire was developed with 6 questions related to the Portuguese food wheel concepts and based on the educational activities performed in the intervention program . The ins2.3.7.2.3.7.1.Body image was analysed with Collins' child figure drawing scale . This pi2.3.7.2.To assess health-related quality of life, the adaptation of the generic scale of the Pediatric Quality Life Inventory 4.0 (PedsQL 4.0), to the Portuguese population  was used2.4.Δ) between baseline and post-intervention was calculated for each variable. Effect size was calculated using eta-squared (η2). The McNemar test was used for categorical variables. Significance level was set at 0.05. Statistical analyses were conducted using SPSS version 27.0.Descriptive statistics  were calculated for the groups at baseline and after the intervention. None of the variables analysed showed significant deviations from normal distributions . Differences between groups at baseline and the effect of the football and nutritional intervention programmes relative to the CG was evaluated with a two-factorial repeated measures ANOVA. Percentage change  in 3 schools completed the program and both the preintervention and postintervention testing; of those, 23 were from the FG (age: 8.6 ± 0.8 years), 21 were from the NFG (age: 9.4 ± 0.3) and 23 from the CG (age: 8.6 ± 0.7 years).F(2.64) = 5.53, p = 0.006, η2 = 0.15], muscle mass  and fat-free mass  between intervention groups at baseline. On average, height of the FG is 7.35 ± 2.22 cm lower than the heigh of the NFG, muscle mass is 3.31 ± 1.05 kg lower than NFG and the fat-free mass of the FG is 4,41 ± 1.55 kg lower than the lean body mass of the NFG. Significant differences in diastolic blood pressure were found between intervention groups at baseline . On average the diastolic blood pressure of the FG is 7.22 ± 2.88 mmHg lower than NFG, but there is no difference between the intervention groups and the CG. Regarding physical fitness, a statistically significant difference on 20 m sprint test  between the intervention groups at baseline. On average, FG is 0.359 ± 0.125 s slower than NFG, but there is no difference between the intervention groups and the CG in 20 m sprint test at baseline.No significant differences were observed at baseline between groups in weight, BMI, waist circumference, WtHr, body fat mass, systolic bloop pressure and heart rate, horizontal thrust, shuttle run test, physical activity levels, body image, physical health dimension and quality of life. Whereas significant differences were found in height , and resting heart rate  and significantly increased in horizontal jump length , shuttle run , Index FAI , psychosocial health dimension , and moderate to vigorous physical activity (MVPA) (FG: F(1.64) = 10.13, p = 0.002, η2 = 0.14; NFG: F(1.64) = 7.87, p = 0.007, η2 = 0.11] over the 12-week intervention), while CG maintain constant values.Over the 12-week study period within-group improvements were observed in both intervention groups. The FG and NFG significantly decreased in BMI z-score , Shuttle run test improved 72 ± 21 m (16.7%) in FG and 198 ± 22 m (44.1%) in NFG, with differences between NFG and CG , Index FAI  reduce 0.26 ± 0.87 scores in FG and 0.24 ± 0.91 scores in NFG, psychosocial health dimension improved on average 6.58 ± 2.42 scores in FG (9.4%) and 14.07 ± 2.20 scores in NFG (20.5%), with differences between NFG and CG , and regarding MVPA, FG increase 8.00 ± 2.51 min/d (15.9%) the time spent in MVPA and NFG increase 7.38 ± 2.63 min/d (12.1%), with 39.1% and 57.1% of the children reaching the guideline of MVPA, respectively.In the intervention groups, BMI z-score reduced on average 0.19 ± 0.07 scores in FG (21.2%) and 0.21 ± 0.07 scores in NFG (30.5%), resting heart rate reduced on average 5.28 ± 2.12 bpm in FG (5.9%) and 7.62 ± 2.22 bpm in NFG (8.9%), horizontal jump length improved 0.08 ± 0.02 m (6.5%) in FG and 0.14 ± 0.02 m (11.1%) in NFG, with differences between NFG and CG , SBP , WC , and WtHr , and significantly increased in quality of life  while the FG and CG maintain constant values between baseline and post. These differences in NFG are the result of a reduction about 6.81 ± 1.77 mmHg in DBP (9.9%), 4.71 ± 1.94 mmHg in SBP (4.5%), 2.88 ± 0.56 cm in WC (4.3%), 0.03 ± 0.01 scores in WtHr (6.5%) and an improvement around 5.22 ± 1.99 scores in quality of life (7.3%).After the 12-week, the NFG significantly decreased in DBP , significantly increased in height , MM , and FFM , and significantly improved in 20 m sprint test , while the NFG and CG maintain constant values between baseline and post. In the FG, these significant differences are the result of a reduction on average about 3.20 ± 0.88% in body fat mass percentage (11.1%), and improvements around kg 1.60 ± 1.19 kg in muscle mass (11.6%), 2.80 ± 0.96 kg in fat-free mass (12.4%), 4.29 ± 1.28 cm in height (3.3%) and 20 m sprint test by reducing on average 146 ± 58 milliseconds (2.9%), with significant differences between FG and NFG .The FG, significantly decreased in % body fat mass , and in sodium intake , while CG maintain constant values. In the intervention groups, CHO intake reduced on average 47.9 ± 0.9 g in FG (16.9%) and 32.1 ± 23.6 g in NFG (7.9%), and sodium intake reduced on average 401.4 ± 34.8 mg (18%) in FG and 399.2 ± 337.9 mg (10.7%) in NFG.Between baseline and post, both intervention groups showed significant reduction in CHO intake  and in sodium intake  after the 12-week intervention.Significant differences between intervention groups and control group were found in CHO , while FG and CG maintain constant values, with between group differences , and significantly decrease the total energy intake  and consequently the energy intake/body weight  over the intervention. On average, NFG increase 2.58 ± 0.15 scores (24%) in the nutritional knowledge and decrease 149.6 ± 16.8 Kcal in the total energy intake (6.1%) and 5.0 ± 0.9 Kcal/Kg (7.8%).Over the intervention, the NFG was the only group that significantly increase in nutritional knowledge  and saturated fat intake , and decreased sugar intake  between baseline and post, while NFG and CG maintain constant. Differences between groups were found for sugar intake  over the intervention. On average, FG increase 11.8 ± 3.8 g of fat intake (29.9%) and 3.3 ± 0.1 g of saturated fat (24.1%), while decreased 23.8 ± 2.5 g of sugar (18.7%).The FG increased in total fat intake  and the frequency of fish consumption greater than or equal to 3 times a week  over the intervention.. In addition, we observed that children from NFG tend to increase the daily consumption of vegetables  and the daily intake of soup , with no significant difference. Children's food frequency questionnaire identifies the number of times a particular food or food group was consumed during the previous month, but not the total amount ingested. The analysis of the frequency of food and drink consumption reported by the children's guardians showed that only the NFG children significantly improved the daily fruit frequency consumption [Baseline = 28.6% vs. Post = 61.9%; 4.To the best of our knowledge, this study is the first to examine the effects of a 12-week football and nutrition intervention program on several health markers, nutritional status, and fitness profiles.The significant differences observed at baseline in muscle mass and fat-free mass between the intervention groups suggest that NFG was more active than FG before the intervention. Furthermore, differences in body composition, may be associated with an advanced maturity in the children.The present findings showed a significant reduction in BMI z-score in the FG and NFG children, in accordance with a 6-month intervention with obese boys . PreviouThe significantly increase of muscle mass and fat free mass in FG, with a greater change score, 11.6% and 13% respectively, is similar to reported in previous studies , 15, 22.In the present study, only the FG experienced significant reductions in body fat mass percentage, with no differences between groups. These findings are in accordance with other school-based physical activity interventions with 11-week , 15, 60,Nevertheless, we found a positive influence of school-based physical intervention on modulating the central-adiposity markers of the NFG, the only group that significantly decreased waist circumference (−4.2%) and WtHr (−6.2%), specific markers of upper body fat accumulation in children and able to predict cardiovascular disease risk factors , 61, 62.Paediatric obesity and lifestyle patterns have an important influence on the overall risk of cardiovascular diseases  and the The observed improvements concerning DBP and SBP in the NFG are similar to or even larger than reported in other comparable football intervention studies with 11 weeks , 15, 20.For adults, the risk of cardiovascular mortality and morbidity is reduced by 13% for every 5 mm Hg reduction in SBP . AlthougChildren with higher blood pressure are more likely to become hypertensive adults, and the observed decrease in resting blood pressure and resting heart rate in the intervention groups is an important finding, as in normotensive children, no clear link has been established between physical activity and blood pressure , 63. TheCardiovascular disease risk factors have been demonstrated to be associated with children level of physical activity and was highly associated with a lower level of fitness . MoreoveOver the “Football and Nutrition for Health” program, both intervention groups improved horizontal jump length and shuttle run test performance during the program with no differences between intervention groups. Improvements in explosive strength in the transition from childhood to adolescence are associated with positive changes in bone mineral density . AdditioShuttle run test improved in both intervention groups, revealing a better aerobic fitness after the 12-week intervention, being associated with a lower risk of cardiometabolic diseases, obesity, diabetes, and other health problems during the entire life cycle . Other sBoth intervention groups improved the performance in the 20 m sprint test, but only the FG had a significant difference between baseline and post intervention. Although, on average, the NFG was faster (4.53″) than FG (4.85″) to complete the 20 m sprint test in the post-intervention, the FG reduced they time in 2.5%, a good indicator of bone tissue health and is inversely related to risk factors for cardiometabolic diseases . ImproveThe most recent results from Portugal's 2021 Report Card on Physical activity for Children and Adolescents shows that less than 30% of children and adolescents achieve physical activity guidelines . In our Prior research indicates that engagement in physical activity has a noteworthy positive impact on the moods of children and adolescents . ConcernIn a comparable study, scores for the PedsQL questionnaire also identified significant improvements in the social and school dimension of well-being for the intervention group . LikewisIntervention groups also showed an improve in the Index FAI score, revealing a more consistent body image and weight status perception. Children with overweight were more likely to underestimate their actual weight status and in obesity prevention such underestimation may be a barrier for behavioural change . Our resThe positive influence of the “Football and Nutrition for Health” program on children's perceived psychological status underscores the significance of school-based initiatives in promoting mental health. It aligns with the goals outlined in the European Mental Health Action Plan, which advocates for impactful measures to enhance mental health and well-being. Specifically, this involves implementing comprehensive mental health promotion programs within schools .The implementation of interdisciplinary and playful educational health interventions within school environments has been shown to enhance knowledge of topics related to eating and nutrition. This approach proves effective in promoting healthier habits among children, as it fosters an improved understanding of dietary concepts through comprehensive education , 85.Differences between the baseline and post-intervention nutritional knowledge questionnaire in the NFG revealed significant increases in children's nutritional knowledge and significant differences between groups. NFG improved the nutritional knowledge in 2.6 scores (24%) after the 12 sessions. Likewise, in a 9-weeks school-based intervention the mean total nutrition knowledge increase by 1.1 scores . In a 5-Dietary records revealed differences in the total energy intake and in the ratio kcal/body weight from the baseline to post-intervention in NFG. Children from NFG decreased carbohydrate, and sodium intake. Children from FG decreased carbohydrate, sugar, and sodium intake, but increased total fat, and saturated fat. These changes observed in the intake of carbohydrates, fats and sodium remained within the reference values , 88. AltSurprisingly, the FPQ revealed that children from NFG had a low frequency of fruit, soup and vegetable consumption in the pre intervention compared with FG and CG. After realizing that the school is the environment where this consumption is most frequent , we noticed a difference in relation to other schools: the lack of a supervisor who encourages the consumption of these foods. At the end, the FPQ reported by the guardians, revealed an increase of fruit similar to FG frequency consumption. We believe that nutrition education classes, focusing on the role of fruits and vegetables in children's health and growth, influenced their consumption after the intervention.Similarly, MUN-SI program showed a positive behavioural change in the preference for including fruit and vegetables in home brought to school lunches (+2.3% in 2015/2016) and at breakfast (+16% in 2014/2015 and +11.9% in 2017/2018) . In ProgMoreover, we speculate that the School Fruit Scheme , a natioIn summary, the program revealed improvements in horizontal jump length, shuttle run performance, and MVPA, induced multiple effects on cardiorespiratory health, promoted a consistent body image and weight status perception (index FAI), and improved the psychosocial health status after 12-week. Furthermore, the intervention resulted in muscle mass and fat-free mass increases and reductions in BMI z-score, body fat mass percentage and rHR in the FG. While NFG decreased BMI z-score, waist circumference and WtHr, it improved DBP, SBP and rHR, increased fruit daily consumption and revealed an increase in nutritional knowledge. The “Football and Nutrition for Health" program confirmed the hypothesis that a combination of recreational football training and nutritional education classes are capable to induce short-term improvements in body composition, physical fitness, physical activity, eating habits, nutritional knowledge, and psychological status among children aged 7–10 years.5.The present study has limitations that should be acknowledged. The children were not randomly assigned to their respective groups. Furthermore, the dietary intake data relied on dietary records reported by parents, which may result in under-report or over-report of habitual nutritional intakes or changes in nutritional-intake patterns. Nevertheless, dietary records are widely used methods for the assessment of dietary intake valid in child populations. On the other hand, it is important to acknowledge the strengths of this study. It contributes to the existing knowledge by exploring the potential of a school-based intervention involving nutrition education and football, which, to the best of our knowledge, has not been previously undertaken. Moreover, the three groups were similar at baseline in the major characteristics. The utilization of accelerometers, an objective measure, further strengthened the design of our study. Overall, these strengths contribute to the robustness and credibility of our study's findings.6.This study highlights the role of the school-based programs as an important determinant of physical activity levels in elementary schoolchildren. Introducing nutritional education and recreational football in elementary schools could be a great and effective strategy to improve physical activity, physical fitness, body composition, nutritional habits, nutritional knowledge and to prevent noncommunicable diseases in boys and girls, constituting an effective tool in health education and promotion. The outcomes of this study hold considerable importance and interest for a diverse audience, encompassing parents, educators, teachers, school administrators, and policymakers. These results strongly advocate for the inclusion of active school programs within curricula, thereby fostering the promotion of children's health. These data reinforce and justify that priority should be given to the development of national action programs that encourage the adoption of healthier lifestyles and to the creation of structural and environmental conditions favourable to child health. Further studies are needed to assess whether this intervention can form part of a more complex behaviour change intervention to prevent childhood obesity.


Manure is a potential substitute for chemical phosphate fertilizer, especially in intensive agriculture, such as greenhouse farming, but the associations between soil phosphorus (P) availability and the soil microbial community under manure application instead of chemical phosphate fertilizers are still rarely addressed. In this study, a field experiment in greenhouse farming with manure application instead of chemical phosphate fertilizers was established, including five treatments: a control with conventional fertilization and chemical phosphate fertilizer substitution treatments using manure as the sole P resource at 25% (0.25 Po), 50% (0.50 Po), 75% (0.75 Po), and 100% (1.00 Po) of the control. Except for 1.00 Po, all the treatments applied with manure harbored similar levels of available P (AP) as the control. Most of the bacterial taxa involved in P transformation were enriched in manure treatments. Treatments of 0.25 Po and 0.50 Po significantly enhanced bacterial inorganic P (Pi) dissolution capacity, while 0.25 Po decreased bacterial organic P (Po) mineralization capacity. In contrast, the 0.75 Po and 1.00 Po treatments significantly decreased the bacterial Pi dissolution capacity and increased the Po mineralization capacity. Further analysis revealed that the changes in the bacterial community were significantly correlated with soil pH, total carbon (TC), total nitrogen (TN), and AP. These results revealed the dosage effect of the impact of manure on soil P availability and microbial P transformation capacity and emphasized that an appropriate dosage of organic manure is important in practical production. As typical intensive agriculture, greenhouse farming is a potential approach to cope with food demand in future . GreenhoP is a macroelement for plants. To increase plant production, high amounts of P fertilizers are applied to the soil. As a result, approximately 80% of global phosphate rock is consumed as fertilizer . HoweverManure is a good substitute for mineral P fertilizers, as it is rich in P. A large amount of manure produced by livestock production largely changes the P cycle in agriculture . IntegraMicrobes are the primary drivers of P transformation in soil . The mic−1·year−1), P (90 kg P2O5·ha−1·year−1), and potassium  fertilizers and cattle manure (84 t ha−1·year−1). The manure-amended treatments were fertilized with the same amount of mineral N and K fertilizers as the control but with manure instead of mineral P fertilizers. The amount of manure applied to the 0.25 Po, 0.50 Po, 0.75 Po, and 1.00 Po treatments was 33.6, 67.2, 100.8, and 134.4 t ha−1·year−1, respectively. Each treatment contained three replicate plots. The size of each plot was 20 m2 (8 m × 2.5 m). The plant system in all treatments was tomato and muskmelon rotation. Except for the fertilization strategy, all the treatments were under the same management.The design of the experiment was detailed in a previous study . The fieSoil sampling and soil bacterial community measurement were performed according to the methods described in previous studies . In totaA FastDNA Spin Kit for Soil  was used for soil total DNA extraction. DNA was extracted from 0.5 g of fresh soil following the user guide.The primer set 515F/806R was used to amplify the V4 region of the bacterial 16S rRNA gene. The polymerase chain reactions (PCRs) were performed in a 50-μL system with a thermal cycle of initial denaturation at 95°C for 10 min; 30 cycles in a series of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min; and a final extension at 72°C for 10 min. The PCR system consisted of 25 μl PCR premix (TaKaRa Ex Taq), 1 μl forward primer (10 μM), 1 μl reverse primer (10 μM), 1 μl DNA template (20 ng), and 22 μl PCR grade water. After quality checks and purification, the PCR products were sequenced using an Illumina HiSeq 2,500 System.Bioinformatic analysis of the sequencing data was primarily performed using the VSEARCH package  accordinThe functional profiles of the bacterial community were predicted using PICRUSt2  Activity Assay Kit  following the user’s manual.The inorganic P dissolution potential of the soil microbial community was measured using an incubation method according to the previous study . In shorStatistical analysis was performed using R platform (version 4.0.2) according to the previous studies , 2023a. 4+‑N) and nitrate nitrogen (NO3‑N) in 0.25 Po and 0.50 Po were significantly lower than that in the control, while 0.75 Po and 1.00 Po contained similar levels of NH4+‑N and NO3‑N as the control.As shown in All the manure treatments contained higher AP than the control, and the AP content increased with a high amount of manure application, but only the increase in AP at 1.00 Po was statistically significant.Tomato yields did not differ significantly between the control and manure treatments , and musThe bacterial community in the studied soil was dominated by Proteobacteria, Acidobacteriota, Chloroflexi, Firmicutes, Bacteroidota, Planctomycetota, Actinobacteriota, Gemmatimonadota, Myxococcota, and Verrucomicrobiota, which accounted for 92.47% of the total reads . The varChao1 richness and Heip’s evenness were calculated to assess the impact of different treatments on bacterial diversity . The ChappaX and appA genes. In addition, the dosage effect of manure on these functional genes was also observed. For example, 0.25 Po, 0.50 Po, and 0.75 Po increased, but 1.00 Po decreased, the abundance of the ppaC gene. Po and 1.00 Po increased, but 0.50 Po and 0.75 Po decreased, the abundance of the phnW gene. When grouping the genes involved in inorganic P dissolution and organic P mineralization, the total abundance of genes involved in inorganic P dissolution varied little among treatments  repository, accession number PRJEB60932.JN and RS designed the experiment. LZ, XL, ZZ, KL, and FW performed laboratory measurement. LZ and RS performed data analysis. LZ, RS, and CZ wrote the study. All authors contributed to the article and approved the submitted version.This study was supported by the Key Science and Technology Project of Anhui Province (202103a06020012) and S&T Program of Hebei (21326904D).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This work explores the efficiency of supercritical fluid extraction (SFE) to recover minerals, pigments, and antioxidant compounds from the spirulina microalgae. Moreover, the fatty acids and phenolic profiles of the extracts obtained were also investigated, and the effect of the extracts on SH-SY5Y cell viability was tested. The extraction of phycocyanin was improved by SFE compared to conventional extraction, from 2.838 ± 0.081 mg/g dry matter (DM) (control) to 6.438 ± 0.411 mg/g DM (SFE). SFE treatment also improved chlorophyll a and carotenoid recoveries increasing from 5.612 ± 0.547 to 8.645 ± 0.857 mg/g DM and from 0.447 ± 0.096 to 0.651 ± 0.120 mg/g DM, respectively. Regarding minerals, the SFE improved Mg recovery with 77% more than the control extraction. Moreover, palmitoleic, stearic, γ-linolenic, eicosadienoic and eicosatrienoic acids recovery was improved by SFE. Phenolic profiles were identified via triple-TOF-LC-MS-MS. Considering heavy metals, a higher rate of Pb extraction was observed for the SFE extract, while no significant differences were observed for Hg between both extractions. Finally, SFE extract improved cell viability compared to the control extract. Thus, SFE constitutes an interesting tool to sustainably extract high-added-value compounds; however, potential contaminants such as Pb need to be controlled in the resulting extracts. In recent years, the interest in the consumption of fresh products rich in nutrients and bioactive compounds has increased due to their potential use and benefits in the medical and pharmaceutical industries . In thisSpirulina is a cyanobacteria that belongs to the blue microalgae and is widely dis-tributed in South America. Due to its composition and the health benefits associated with its consumption, this microalga has an interesting potential to become an important food in the future and to be used as an ingredient in the development of functional foods . It contConventional extraction techniques such as percolation, maceration, countercurrent extraction, and Soxhlet require the use of toxic organic solvents such as chloroform, methanol, acetone, and diethyl ether in large quantities and for long periods of time. Moreover, they could affect the biological activity of the extracted molecules ,9. The u2 under supercritical conditions. In this sense, the addition of a suitable polar co-solvent changes the polarity of SFE-CO2 and increases the solubility of nutrients and bioactive compounds [Supercritical fluid extraction (SFE) is a cost-effective extraction method that follows the principles of green chemistry. It enables the recovery of extracts with high extraction efficiency and selectivity by adjusting the parameters of temperature, pressure, and flow rate. SFE shows promise in applications in food processing and in the extraction of metals in the form of complexes and functional ingredients from natural sources. It can also be an interesting tool for the decontamination of hazardous substances . The maiompounds . This teompounds ,15,16.Finally, extracts obtained from microalgae are often rich in a variety of nutrients, vitamins, minerals, antioxidants, and other biologically active compounds that can promote cell growth, survival, and overall viability. By creating a nutrient-rich environment, microalgae extracts have the potential to improve cell viability and stimulate cell proliferation . MoreoveThe aim of this work is to investigate the recovery of total phenolic compounds (TPC), pigments, PC, and antioxidant active compounds from spirulina algae using SFE. The study compares the results obtained with SFE to those obtained with conventional stirred extraction (control). Additionally, the fatty acid profile of both SFE and conventional extracts is analyzed. The research also explores the potential of SFE in mineral recovery and monitors the presence of heavy metals in the extracted compounds. The primary objective is to provide an overview of the different compounds of nutritional interest extracted by each method. Furthermore, the study examines the effects of these extracts on cell viability over a 24-h exposure period to determine any potential beneficial effects on cells. Notably, the novelty of this research lies in the fact that there are currently no existing studies in the literature that evaluate the effects of spirulina extracts obtained through SFE on cell viability.2CO3), sodium hydrogen phosphate (Na2HPO4), and potassium dihydrogen phosphate (KH2PO4) were acquired from VWR . Nitric acid (HNO3) was obtained from Panreac . Methanol, absolute ethanol (EtOH) and hexane were purchased from VWR Chemicals . CO2 was obtained from Carburos Metálicos . EtOH 96° was obtained from Guinama . Folin–Ciocalteu reagent, potassium persulfate (K2S2O8), gallic acid, ABTS , fluorescein, 2,2-Azobis(2-amidinopropane) dihydrochloride (AAPH), and trolox  were purchased from Sigma Aldrich .Sodium carbonate (NaThe spirulina samples were obtained from Hainan Island (China). The microalga was grown in open flow-through ponds. No artificial light was used, and shade nets partially covered the culture ponds, allowing control of pigment production. At the time of harvest, all samples were dried with dry air at 60 °C. A shaking extraction process was performed following the protocol described by Sansone et al. . Briefly2 pump (PU-4387) capable of a flow rate between 5 and 40 mL/min, adjustable in increments of 0.01 mL/min, with a maximum pressure of 50 MPa and a pulse extraction system; a cooling circuit (JULABO FL 1201) to cool the pump, offering a temperature range of −20 to 40 °C, a flow rate of up to 23 L/min, and a cooling power of 1.2 kW at 20 °C; an isocratic pump for organic modifiers  with a flow rate range of 0.001–10 mL/min, adjustable in 0.01 mL/min increments, and a peak pressure of 70 MPa; a thermostatic oven for glass reactions (CO-4065) with a temperature range of 4 to 90 °C; a thermostatic system employing Peltier and temperature transfer via air flow; a pressure regulator (BP-4340); and a 5 mL glass vessel for SFE . The extraction process lasted for 1 h, following a modified version of the method described by Mendes et al. [2:EtOH ratio of 90:10, with a flow rate of 16 mL/min.The supercritical fluid extraction (SFE) was conducted using a JASCO system . The system consisted of various components: an isocratic COs et al. . SpecifiThe measure of total antioxidant capacity was carried out by two different methods: Trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) tests. These assays were carried out following the protocol described by al Khawli et al. . The resThe content of chlorophyll a (Chla) and total carotenoids (Car) was calculated based on the procedure described by Lichtenthaler and Wellburn  with the2CO3 were added to a test tube, then 100 μL of standard or sample extract (appropriately diluted) and finally, 100 μL of Folin–Ciocalteu reagent were also added. Then, the samples were incubated for 60 min in darkness at room temperature measured at a wavelength of 765 nm.The total phenolic content (TPC) analysis was performed following the method described by Martí-Quijal et al. . BrieflyThe concentration of PC was determined following the protocol described by Patil et al. , measuri2O2 (35% v/v) and 1 mL of HNO3 (69% v/v) in a microwave system  (800 W and 180 °C for 15 min). Then, the samples were filtered and diluted to an appropriate volume using ultrapure water. Finally, the content of minerals  and heavy metals  was evaluated using an inductively coupled plasma spectrometer mass detector . For the quantification, a standard calibration curve was used. Results are expressed as µg/g DM of three independent experiments.The minerals analyzed were Mg, P, Ca, Fe, Zn and Se, as they are the more interesting minerals from a nutritional point of view, being essential for several biological processes . On the ® 590. A standard FAME Mix  was used to identify the fatty acids.The determination of lipid profile was carried out via gas chromatography coupled to flame ionization detector (GC-FID). Briefly, 1 mL of the extract resuspended at 20 mg/mL was mixed with 1 mL of KOH 2 N in methanol and 1 mL of hexane. After mixing them for 2 min, the hexane phase was taken and 2 µL were injected in a PerkinElmer Gas Chromatograph Clarus®, CPWAX 52CB silica capillary WCOT column with a dimension of 30 m of length, 0.25 mm internal diameter with a 0.25 μm thick cover (SUPELCO) SP-2560 Capillary GC Column was operated in split mode. The oven temperature program was established as 180 °C for 5 min, increased to 210 °C at a rate of 4 °C/min, and finally held at 250 °C for 20 min, increasing at a rate of 20 °C/min. Nitrogen was used as a carrier gas at a flow rate of 20 psi. The results were expressed as mg/g DM.The GC column SP-2560m/z. Prior to sample analysis, calibration of the equipment was performed using an external calibration system. The MS operated using an information-dependent acquisition (IDA) method, employing the survey scan type (TOF-MS) and the dependent scan type (product ion) with a collision energy of −50 V. The MS parameters were as follows: ion spray voltage of −4500 V, dust removal potential of 90 V, collision energy of −50 V, temperature of 400 °C with a curtain gas of 25 psi, ion source gas 1 at 50 psi, and ion source gas 2 at 50 psi. The IDA MS/MS was performed based on the following criteria: ions exceeding 100 CPS, ion tolerance of 50 MDa, collision energy set at 25 V, and dynamic background subtraction enabled.The phenolic profile was analyzed using triple-TOF-LC-MS-MS characterization, following the method described by Zhou et al. . In this2 at 37 °C, and 95% air atmosphere with constant humidity. The culture medium was refreshed every 2–3 days. To test the effects of various concentrations of spirulina extracts, the dry matter obtained after the extraction was resuspended in DMSO, and the solutions were added to the culture medium, ensuring that the final DMSO concentration did not exceed 1% (v/v). Control groups containing the same amount of solvent were included in each experiment.For cell viability analysis, SH-SY5Y cells , a type of human neuroblastoma cells, were cultured in DMEM Ham’s-F12 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. The cells were incubated under specific conditions: pH 7.4, 5% COTo assess cell viability, the MTT assay was conducted as described by Zingales et al. . In briet test was used to compare SFE and control samples. A p value of <0.05 was considered significant. All the experiments were carried out in triplicate. The results are presented as mean ± SD.The statistical analysis was performed using GraphPad Prism 9. Student’s Chlorophyll, Car, and polyphenols are bioactive compounds found in vegetables and algae that have been extensively studied for their antioxidant, anti-inflammatory and an-ticancer properties ,33. PC, The Chla, total Car, TPC, and PC values in both the control and SFE extracts are shown in As shown in 2) with a flow rate of 15 g/min of CO2 for 50 min and 26.70% EtOH 96%/water (v/v) as a co-solvent under 15 MPa and 60 °C. Despite the similarity in flow rate and extraction time, the pressure used by these authors was lower, and the percentage of EtOH was higher. These differences may account for our higher value of Car. The low yield in Car extraction may be attributed to pressures lower than 30 MPa, as suggested by Marzorati et al. . They fo2 flow rate of 1.2 Kg/h. Additionally, the results obtained through SFE correspond well with the values reported by Dejsungkranont et al. [SFE extraction also increased the recovery of phenolic compounds, resulting in an increase from 3.202 ± 0.547 mg/g DM using conventional extraction to 7.917 ± 0.406 mg/g DM with SFE. These findings align with those reported by Mallikarjun et al. , who obtt et al. , who recRegarding the value obtained for PC, a significant increase in the extraction yield of this protein was observed after using SFE, compared to the control extraction under stirring, improving from 2.838 ± 0.081 mg/g DM (control) to 6.438 ± 0.411 mg/g DM (SFE), which means an increase of 126.8%.Deniz el al.  used SFE2), SFE (with 10% EtOH as co-solvent), and pure EtOH at high pressure. The results were in agreement with the obtained in the present study, as they obtained a higher PC extraction when using SFE with 10% EtOH as co-solvent compared with pure EtOH. The PC extraction improved from 0.6  ±  0.2 up to 0.9 ± 0.3 (wt%), which means an increase of 150%.Pinto et al.  studied The results obtained regarding the antioxidant capacity  demonstrOn the other hand, the ORAC assay did not show significant differences between the control (189 ± 10.2 µmol TE/g DM) and the SFE (175 ± 8.60 µmol TE/g DM) samples.Spirulina maxima. However, this can be explained by the higher polyphenol values obtained by those authors, as discussed earlier, which correspond to a higher antioxidant capacity than what was found in our study.The results obtained in the present study for the TEAC assay were slightly lower compared to those obtained by Dejsungkranont et al. . They acMacrominerals are essential for a good health. Magnesium is a vital cofactor in over 300 enzyme systems and is essential for energy production, cell membrane transport, nerve impulse conduction, muscle contraction, and heart rhythm. Low Mg levels are associated with various chronic health conditions such as migraines, osteoporosis, Alzheimer’s disease, asthma, hypertension and insulin resistance . CalciumOn the other hand, microminerals  are also relevant for several functions in human body. In this sense, selenium’s nutritional benefits are achieved through 25 selenoproteins that contain selenocysteine. During low selenium supply, some selenoproteins are prioritized, including glutathione peroxidase. Selenoproteins play a vital role in human health. For example, Se supplementation can stimulate the immune system, including enhancing T cell proliferation and natural killer cell activity . Zinc is2 [Then, concerning the minerals, Mg, P, Ca, Fe, Zn and Se, the SFE recovered 77% more Mg than the conventional extraction, increasing from 49.33 ± 1.00 up to 91.50 ± 2.25 µg/g DM. In addition, Fe extraction was also improved through SFE, reaching 2.00 ± 0.08 µg/g DM for SFE extract while only 0.87 ± 0.04 µg/g DM for the conventional one. However, the conventional extraction obtained a higher yield for the recovery of P (52.98 ± 2.86 vs. 20.93 ± 2.25 µg/g DM) and Ca (52.27 ± 1.43 vs. 35.25 ± 2.25 µg/g DM) . This ca2 . NeverthIn this sense, Michalak et al.  also obtFor heavy metals , a higher Pb extraction was observed for the SFE extract (0.0953 ± 0.0030 µg/g DM) compared to control (0.0222 ± 0.0014 µg/g DM), while for Hg there were no significant differences between both extractions . NeitherCommission Regulation (EC) No 1881/2006  establisTo the best of our knowledge, no studies are available in the literature about heavy metals contents in spirulina SFE extracts, so the contents obtained in this study have been compared with the information available in raw spirulina. In this sense, Al-Harbi  studied Fatty acids are vital for human health, serving as building blocks for cell membranes and precursors for signaling molecules. Palmitoleic acid, a monounsaturated fatty acid, has been linked to lower inflammation levels and improved insulin sensitivity . LinoleiHowever, there was a reduction in linoleic acid content, with the control extraction having a higher concentration (2.200 ± 0.060 mg/g DM) than the SFE extraction (1.180 ± 0.042 mg/g DM).On the other hand, Conversely, the most significant increase occurs in γ -linolenic acid, which rises from 20.66% in the control extract to 36.49% in the SFE extract. It is also worth noting that long-chain polyunsaturated acids, such as eicosadienoic acid and eicosatrienoic acid, observing an increase in their proportion in the SFE extract compared to the control, with an increment from 0.25% and 0.17% to 0.33% and 0.21%, respectively. This implies that SFE can enhance the lipid profile in a way that promotes the extraction of unsaturated fatty acids.Finally, the only fatty acid that does not experience a significant modification regarding its proportion in the obtained extract is heptadecenoic acid, obtaining very similar values in both extracts.The results obtained in this study are similar to those described in the literature. In this regard, the values are within the same range as those obtained by Esquivel-Hernández et al. , with a Mendiola et al.  have alsIn Arthrospira platensis. On the other hand, Zhou et al. [Arthrospira platensis.The identification of hydroxybenzoic acid, 4-hydroxybenzaldehyde, and protocatechuic acid is in line with what has been described in the literature by Klejdus et al. , who ideu et al.  detectedu et al.  after exu et al.  also repp < 0.05) in the case of the 2000 μg/mL concentration, where the control extraction reduced cell viability to 64.3%, while the SFE extract was similar to control. This was likely due to the higher content of bioactive compounds, as previously described, which help sustain cell viability at high concentrations compared to the control.The impact of the SFE spirulina extract on SH-SY5Y cell viability was evaluated and compared to the control extract . To the 50 at concentrations of 1700 ± 140 µg/mL (aqueous extract) and 1280 ± 220 µg/mL (methanolic extract). In human fibroblasts, the IC50 was reached at concentrations of 2340 ± 60 µg/mL and 2430 ± 40 µg/mL for the aqueous and methanolic extracts, respectively. These concentrations were considerably higher, indicating that our study aligns more closely with the behavior observed in fibroblasts. However, at the range of concentrations tested we did not obtain IC50, for either SFE or control extracts.Other researchers have examined the cytotoxicity of extracts from this microalga using different methods and solvents. Conversely to our results, Akbarizare et al.  found sl50 of the ethanolic spirulina extract after 24 h exposure to HeLa cells was 260.4 μg/mL, much lower than our values. However, this difference could be attributed to the intrinsic characteristics of HeLa cells, as in the same study, the authors also tested the extract on human dermal fibroblasts (HDFa cells), obtaining an IC50 value of 2065.7 µg/mL, which closely resembles the results of Akbarizare et al. and our results. This confirms that the low IC50 value obtained for HeLa cells may be specific to this particular cell line.In contrast, the results differ significantly from those obtained by Nurani et al.  for HeLaIn our study, the choice of the cell line was based on the evidence that they represent one of the most common in vitro model used for assessing the toxic effects of several substances .Therefore, it is essential to perform cytotoxicity studies considering the cell line used, the duration of exposure, and conducting a comprehensive characterization of the tested extract.In conclusion, this study demonstrates the effectiveness of SFE in efficiently extracting valuable compounds from spirulina microalgae. SFE outperforms conventional extraction methods, resulting in significant improvements in the recovery of phycocyanin, chlorophyll a, carotenoids, and magnesium. It also enhances the extraction of specific fatty acids such as palmitoleic, stearic, γ-linolenic, eicosadienoic, and eicosatrienoic acids. The phenolic profile of the SFE extracts is diverse and promising. However, careful monitoring of potential contaminants, particularly lead (Pb), is necessary. Notably, the SFE extracts show improved cell viability compared to the control extracts when tested on SH-SY5Y cells. Overall, SFE has proven to be a valuable and sustainable method for extracting high-value compounds from spirulina microalgae, with considerations for controlling potential contaminants in the extracts.


Researchers have developed the Dietary Inflammatory Index (DII) as a tool to quantify the inflammatory potential of a given diet. Higher DII scores indicated a more proinflammatory diet. While inflammation is known to have a strong impact on thyroid function, the precise nature of the association between DII scores and thyroid function has yet to be clarified. This study was conducted with the goal of exploring this relationship in a representative population of adults from the United States.For this study, we used data from the National Health and Nutrition Examination Survey (NHANES). Standardized questionnaires were used to collect demographic and dietary data from the participants, and laboratory tests were used to collect data on the participants' thyroid parameters and other relevant data. Linear regression models and smoothed curve fitting were used to assess the relationship between DII scores and thyroid function, with weighted data analyses and subgroup analyses being conducted as appropriate.β = .007; p = .027) and TT4  levels. Subgroup analyses showed a negative correlation between FT3 levels and DII scores in a population with high urinary iodine concentrations.In total, 7712 subjects were recruited from the NHANES 2007–2012 cohort. Their weighted mean age was 44.87 (0.47) years, mean DII score was 1.41 (0.05). Mean FT3 was 3.20 (0.01) pg/mL and mean TT4 was 7.81 (0.03) µg/dL. In adjusted analyses, higher DII values were related to increases in FT3 (These data indicate that the consumption of a more proinflammatory diet, as evidenced by elevated DII scores, is correlated with significant increases in FT3 and TT4 levels. However, for people with high urinary iodine concentrations, a more proinflammatory diet was associated with lower FT3 levels. Additional research will be vital to clarify the mechanistic basis for these findings. We explored the relationship between DII and thyroid function using data from the NHANENS database. Our study showed that consumption of a more proinflammatory diet, as evidenced by elevated DII scores, was associated with a significant increase in FT3 and TT4 levels. However, for people with high urinary iodine concentrations, a more proinflammatory diet was associated with lower FT3 levels. This index was initially formulated by Cavicchia et al.Thyroid hormone production and signaling activity play a key role in human growth and development, shaping physiological processes such as digestion, respiration, heart rate, and thermoregulation.These previous studies suggest that inflammation may be related to thyroid function. The specific nature of the link between the DII and thyroid functionality, however, remains to be clarified. Although in recent years, Liu et al. have shown a positive correlation between DII and TT4 levels in the male population, their study did not include a female population and did not exclude participants taking thyroid‐related medications.22.1www.cdc.gov/nchs/nhanes/). These analyses were performed as per the Declaration of Helsinki.The cross‐sectional NHANES collects a wealth of data pertaining to the demographic characteristics, health behaviors, and nutrition of individuals in the United States. Researchers can access NHANES data and related statistics online (This study used 2007–2012 NHANES data approved by the NCHS Research Ethics Review Board (ERB). Written informed consent was provided by all NHANES participants. For these analyses, 7712 subjects aged ≥18 years with complete thyroid function and dietary data used to calculate DII were selected for inclusion. See Figure 2.22.2.1The 24‐h dietary history interview was used to record dietary intake. Dietary intakes were collected and calculated using the USDA dietary survey tool, the Automated Multiple Pass Method (AMPM), which is a fully computerized recall method.2.2.2The thyroid parameters analyzed included levels of TSH, free triiodothyronine (FT3), free thyroxine (FT4), total triiodothyronine (TT3), total thyroxine (TT4), thyroglobulin (Tg), TgAb, and TPOAb. A third‐generation two‐site sandwich immunoassay with 0.34–5.6 IU/mL reference range was used to measure TSH levels. While the reference range for the two‐step enzyme immunoassay used to measure FT4 levels was 0.6–1.6 ng/dL, the reference range for the competitive binding immunoassay used to measure FT3 levels was 2.5–3.9 pg/mL. TT3 and TT4 levels were also determined using a competitive binding immunoassay with reference ranges of 5.0–12.0 µg/dL and 80–220 ng/dL, respectively. TPOAb and TgAb titers were also analyzed via a sequential two‐step sandwich immunoassay approach with respective reference range values of 0–9.0 IU/mL and 0–4.0 IU/mL. The Access Tg Assay is a single‐step sandwich assay. The NHANES Laboratory/Medical Technologist Procedures Manual provides comprehensive instructions for specimen collection and processing.2.2.32). We followed the classification used in a previous study of DII, which classified BMI for individuals older than 18 years as <25 , 25–29.9 (overweight), and ≥30 kg/m2 (obese).A standard questionnaire was used to assess participants’ age, race/ethnicity, poverty/income ratio (PIR), smoking history, gender, physical activity, and alcohol history. A mobile examination center (MEC) was used to assess body mass index (BMI). Considering these factors, the importance of iodine for thyroxine synthesis, the fact that iron deficiency impairs thyroid metabolism, and the correlation between thyroid function and renal function, we also included urinary iodine concentration (UIC), serum iron (Fe), and creatinine (Cre) in our study.2.3The Analytical Guidelines (NHANES:2007‐2012) were used to calculate and analyze weighted data. The NHANES data release file contains multiple sample weights, including interview weight (wtint2yr) or examination weight (wtmec2yr), and the most appropriate weight depends on the selected variables of interest. Because the MEC sample is a subset of the interview sample, this analysis was conducted using the combined MEC and examination weight. However, the thyroid examination sample from NHANES 2009–2012 (two survey cycles) is a subset of all participants who were interviewed for the MEC examination. Therefore, the subset weight (WTSA2YR) was used when analyzing data from these two survey cycles. The weight of the survey allows it to be extended to the civilian, noninstitutionalized US population.SDs) or percentages, as appropriate. Effect values are reported as β values with corresponding 95% confidence intervals (CIs). A p < .05 served as the cut‐off when defining statistical significance. All analyses were performed with R v4.2.2 using the “nhanesR” package (v0.9.4.2) and the “survey” package (v 4.1‐1).The relationship between DII and thyroid functionality was described with both unadjusted and adjusted linear regression models. Model 1 was unadjusted for covariates, Model 2 was adjusted for sex, age, and race/ethnicity of participants, and Model 3 was further adjusted for BMI, physical activity, alcohol consumption, smoking status, marital status, UIC, serum Cre, and serum Fe levels. Results were further analyzed using smooth curve fitting. In addition, subgroup analyses and tests for interaction were also performed for each covariate to investigate the independent effect of DII on thyroid function levels. Data are means with standard deviations (33.1SD) age of these subjects was 44.87 (0.47) years. DII scores ranged between −5.28 and 5.47 from least to most proinflammatory, with a weighted mean of 1.41 (0.05), while the weighted interquartile ranges of DII were −5.28 to 0.07, 0.08 to 1.64, 1.65 to 2.94, and 2.95 to 5.47, respectively. Study participants had no clinical thyroid problems and were not pregnant.A total of 7712 participants from the NHANES 2007–2012 dataset were incorporated into this analysis. Participant characteristics stratified according to DII quartiles are presented in Table 3.2β = .614, 95% CI: 0.209–1.019, p = .004; TT4: β = .101, 95% CI: 0.076–0.127, p < .001). In model 2, adjusted for sex, age, and race/ethnicity, DII scores were positively associated with FT3 and FT4, similar to the trends observed for TT3 and TT4. However, after further adjustment for other confounders , only FT3 and TT4 still remained positively associated with DII , while the same was not true for FT4 and TT3. When DII was used as a categorical variable in quartiles, TT4 levels were significantly higher in the highest quartile compared to the lowest quartile .Table Further analyses of the generalized additive model revealed that both FT3 and TT4 levels were also positively correlated with DII, and the association between TT4 and DII fit a J‐shaped curve Figure , whereas3.3p < .05 for the interaction effect . Written informed consent was provided by all NHANES participants.Supporting information.Click here for additional data file.


This study assessed the CVD risk factors among Filipino women (FW) in Korea and compared them with FW in the Philippines and women in Korea (KW).A cohort of 504 women from the Filipino Women's Health and Diet Study (FiLWHEL) aged 20–57 years old were age-matched (1:1 ratio) with women from the 2013 National Nutrition Survey in the Philippines and the 2013–2015 Korean National Health and Nutrition Examination Survey. Anthropometric data, blood pressure (BP), lipid and glucose levels were compared across the four populations by calculating the odds ratio (OR)s and 95% confidence interval (CI)s using conditional logistic regression models.2 and waist circumference ≥ 88 cm, respectively. However, FW in Korea had the highest odds  of having hypertension compared to KW. FW in the Philippines had the highest odds of having dyslipidemia , but FW in Korea and KW had similar prevalence of dyslipidemia.Compared to KW, FW in Korea and FW in the Philippines were more than 2 and 3 times higher odds of having obesity for BMI ≥ 30 kg/mFW in Korea had higher prevalence of obesity and hypertension, with similar prevalence of dyslipidemia compared to KW in this sample. FW in the Philippines had higher prevalence of dyslipidemia compared to FW in Korea. Further prospective studies are warranted to examine the CVD risk factors among continental and native-born Filipino women.The online version contains supplementary material available at 10.1186/s12905-023-02218-1. Cardiovascular diseases (CVD) are primary causes of mortality and morbidity among women worldwide , 2. The Elevated blood pressure (BP), dyslipidemia and elevated blood glucose, unhealthy diet, overweight and obesity, physical inactivity, tobacco use, and harmful use of alcohol are well-established modifiable risk factors of CVD  but ageiMigrants from different cultural environments often find it hard to align with the cultural and traditional norms of the host country . SpecifiA study conducted by the National Institutes of Health on CVD risk factors among Asian Americans in the US revealed that migrants have a lower prevalence of CVD risk factors than the host population . HoweverMoreover, whether FW migrants in South Korea are susceptible to higher CVD risk factors than Korean women has not been reported. Understanding the CVD risk susceptibilities among migrant populations deserves attention and is required to design context-specific health promotion guidelines and policies to promote migrant health. In particular, the difference in disease prevalence of migrants in the country of origin and those in the host country may provide insights and perspectives that can help researchers and policymakers to understand the etiology of the disease and design the appropriate strategies.This study assessed and compared the CVD risk factors among FW migrants in Korea, FW in the Philippines, and KW.The current analysis included 504 migrant FW in Korea  matched for age  of the Philippines based on mutual agreement, whereas the KNHANES data were freely accessible online via Korea Disease Control and Prevention Agency (KDCA) . Second,Covariates associated with BP, lipids and glucose profiles included in this study were smoking, alcohol drinking, education level, occupation, BMI and waist circumference. Participants' smoking status was categorized into ever smoker and never smoker; ever smoker was defined as past or current smoker while never smoker was defined as never smoked at least 100 cigarettes in their entire lifetime. Alcohol drinkers were categorized into past or current drinker  or never drinkers . Participants' employment status was categorized as employed and unemployed . In addition, participants were asked about their educational attainment where elementary or middle or high school level were grouped into high school and below, and those who had at least college education were grouped into college and above.For the FiLWHEL, the Cobas 8000 C702-1/C703-I analyzer  was used to determine serum levels of TC, TG, and HDL-C by enzymatic method and glucose by hexokinase UV assay. For the NNS, the TC, TG, HDL-C and glucose were assessed by enzymatic colorimetric method using the Roche COBAS Integra and Hitachi 912 analyzer. For the KNHANES, the TC and TG were analyzed by enzymatic method, HDL-C by homogeneous enzymatic colorimetric method, and glucose by hexokinase UV assay on Hitachi Automatic Analyzer 7600/7600-210 . All blood samples were collected by phlebotomists or trained medical personnel after at least 8–12-h overnight fasting through a venipuncture procedure.The FiLWHEL coefficients of variation (CVs) were as follows: TC (1.43–2.24%), TG (1.85–2.10%), HDL-C (1.55–2.51%), and glucose (1.15–1.22%) . The blo2), and respondents were classified according to the World Health Organization cut-off points: underweight, < 18.5 kg/m2; normal, 18.5– < 23.0 (for Asian) and 23.0– < 25.0 kg/m2; overweight, 25.0– < 27.0 kg/m2; pre-obese, 27.0– < 30.0 kg/m2 and obese, ≥ 30.0 kg/m2 [BMI was calculated as weight (kg) divided by square of height  guidelines, systolic and diastolic blood pressures were classified as follows: normal, < 120/ < 80 mmHg; prehypertension, ≥ 120/ ≥ 80 mmHg or < 140/ < 90 mmHg; and hypertension, ≥ 120/ ≥ 90 mmHg .The 2005 American Heart Association/National Heart, Lung, and Blood Institute (AHA/NHLBI) criteria was used to categorize the blood lipids and glucose levels to determine dyslipidemia: high TC ≥ 200 mg/dL; low HDL-C < 50 mg/dL; high LDL-C ≥ 130 mg/dL; high TG ≥ 150 mg/dL, and elevated glucose ≥ 100 mg/dL .2) and waist circumference  as applicable. Tukey's posthoc test was used for multiple comparison analyses to assess significant differences in nutrient intake across the population. All statistical analyses were performed using the SAS version 9.4 software package , and two-tailed P values < 0.05 were statistically significant.Continuous variables were presented as means ± SD and compared across the three populations  using the analysis of variance (ANOVA), but categorical data were presented as n (%) and compared using Mantel-Haenzel test. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using conditional multivariate logistic regression models with the women from the KNHANES as the reference population. Women with missing BMI, waist circumference, BP, lipid and glucose data were excluded when we analyzed these variables as outcomes in the logistic regression models. The multivariate analyses were adjusted for energy intake , smoking status , alcohol intake , educational level , employment status , BMI  and had a higher waist circumference  compared to KW, (P < 0.0001). Additionally, 32.8% of FW in Korea had low educational attainment (high school or below) compared to 36.2% and 68.5% of the KW and FW in the Philippines, respectively (P < 0.0001). In contrast, unemployment was significantly (P < 0.0001) prevalent among FW in Korea (52.1%) compared to KW (39.5%). The prevalence of current smokers was significantly lower (P < 0.0001) among FW in Korea (1.2%) compared to FW in the Philippines (3.8%) and KW (5.5%). Similarly, the prevalence of current alcohol drinking was significantly lower (P < 0.0001) among FW in Korea (55.7%) compared to KW (81.7%).Table Table The prevalence of elevated TC was significantly higher among FW in the Philippines at 38.2%, whereas, there was seen similarity between KW at 28.0% and FW in Korea at 26.4%. Similarly, the mean TC levels for FW in the Philippines were 192.18 ± 41.53 mg/dL, 180.13 ± 35.91 mg/dL among FW in Korea and 184.49 ± 32.70 mg/dL among KW. Similar trends were observed for the means of LDL-C and TG, with the highest mean levels among FW in the Philippines. However, KW were observed to have slightly higher mean glucose levels compared to FW.FW in the Philippines had the lowest intakes for total energy, protein and fat percent energy, calcium, vitamins A and C, iron, thiamin, and riboflavin but the highest in carbohydrate percent energy intake. FW in the Philippines had lower intakes of protein, fat, and micronutrients compared with FW in Korea and KW. When we compared FW in Korea with KW, we found no significant difference in nutrient intakes except for the protein percent energy, calcium, iron, thiamin and riboflavin Table .Table 32) were 2.27 (95% CI 1.15–4.51) for FW in Korea and 3.49 (95% CI 1.59–7.64) for FW in the Philippines; the ORs for high waist circumference (≥ 88 cm) among FW in Korea and in the Philippines were 2.36 (95% CI 1.53–3.63) and 3.34 (95% CI 1.96–5.68), respectively, when compared with KW.We observed the higher prevalence of obesity among FW in KW and in the Philippines Table . ORs for2), the association became attenuated among FW in the Philippines and in Korea.The FW in Korea and Philippines had 5.51 (95% CI 3.18–9.56) and 2.38 (95% CI 1.19–4.79) higher odds of having hypertension, respectively, when compared to KW , low HDL-C < 50 mg/dL , high LDL-C ≥ 130 mg/dL  and high TG ≥ 150 mg/dL . However, FW in Korea showed similar lipid profiles to those of KW  to 41.9% (2017–2020) . In the The reasons why we observed unfavorable lipid profiles among FW in the Philippines compared to Korean are not clear but warrants further studies. It is possible that the transition to unhealthy lifestyle factors may be associated with unfavorable lipid profiles among Filipino women . In the kimchi) [We noticed that FW in Korea presented a higher prevalence of hypertension when compared to KW. A change in the food environment after migration might gradually change their dietary habit. Our study showed that FW in the Philippines had the lowest mean percent energy from fat, but after migrating, the mean percent energy from fat of FW in Korea was higher than KW. Koreans have one of the highest salt intake worldwide, almost half of which is derived from traditional diets such as salted vegetable (kimchi) . Excessikimchi) . Exposurkimchi) . Althougkimchi) , but suckimchi) .2, 60.4%) [2 to ≥ 30 kg/m2, including diabetes  and hypertension , at a higher TG , and at a lower HDL-C level  [Unhealthy dietary habits and the lifestyle of Filipinos may be important in the significant transitions of CVD events in the last decade. A recent review reported several risk factors of CVD prevalent among Filipino immigrants in the US. The study had indicated that Filipinos tend to develop an acculturated Westernized diet, which is associated with a higher risk for CVDs, hypertension, type 2 diabetes, and metabolic syndrome . The pre, 60.4%) , abdomin, 60.4%) , high ca, 60.4%) , 51, and, 60.4%) . Additioctively) .To the best of our knowledge, our study is the first to compare the susceptibility of CVD risk factors among Filipino and Korean women of diverse backgrounds matched by age (1:1 ratio). Studies conducted on Filipino immigrants in South Korea are sparse and out-of-date. Thus, the literature is limited to support the account regarding the Filipino immigrants’ health status. Another strength of our study is the multivariate adjustment for potential confounders. Some limitations of our study warrant consideration in the interpretation of the results. First, the cross-sectional design of our study delimits the inference of causality. Second, the FiLWHEL study has a relatively small sample size based on convenience sampling which may not represent the general population. Third, we did not adjust for physical activity even though it is a known risk factor of CVD because of the different questionnaires used in collecting physical activity information across the studies. However, adjustment of BMI or waist circumference may take into account the adjustment for physical activity levels to some degree.The prevalences of high TC, low HDL-C, high LDL-C and high TG were higher among FW in the Philippines than FW in Korea and KW. FW in Korea had higher prevalences of obesity and hypertension than KW. Still, there was a similarity between the lipid profiles of FW in Korea and those of KW.Additional file 1. Fig. S1: Flow chart of age-matched population of FiLWHEL, NNS, and KNHANES participants.


The compound belongs to the triclinic system P − 1 space group, with dimensions of a = 9.2304 (6) Å, b = 11.1780 (8) Å, c = 11.3006 (6) Å, α = 107.146 (5)°, β = 93.701 (5)°, γ = 110.435 (6)°, Z = 2 and V = 1025.61 (12) Å3. The crystal structure showed that C–H···N and C–H···O hydrogen bond linkages, forming infinite double chains along the b-axis direction, and enclosing R22(14) and R22(16) ring motifs. The Hirshfeld surface analysis revealed that H…H (44.1%) and H…C/C…H (15.3%) interactions made the most significant contribution. The newly synthesized (Z)-4-bromo-N-(4-butyl-3 (quinolin-3-yl)thiazol-2(3H)-ylidene)benzamide, in comparison to oleanolic acid, exhibited more strong potential against elastase with an inhibition value of 1.21 µM. Additionally, the derivative was evaluated using molecular docking and molecular dynamics simulation studies, which showed that the quinoline based iminothiazoline derivative has the potential to be a novel inhibitor of elastase enzyme. Both theoretical and experimental findings suggested that this compound could have a number of biological activities.A new compound, CThe online version contains supplementary material available at 10.1186/s13065-023-00985-4. The presence of a quinoline moiety  can both be produced by and be found in the skin itself. ROS are produced as a natural byproduct of regular metabolic processes; they are an essential component of normal cellular function; nevertheless, they rarely cause significant damage due to the presence of intracellular mechanisms that mitigate the effects of their toxicity . AntioxiA potential elastase inhibitor with good antioxidant potential was explored using quinoline-based iminothiazoline analogue. Experimental results were validated by computational approaches, including density functional theory calculation, molecular docking, and molecular dynamics studies. The study suggested that the designed molecule has potential for further exploration at the molecular level, leading to the synthesis of drug-like molecules with greater potential and safety profiles. This approach may provide an efficient way to protect against skin aging by inhibiting elastase enzyme activity.A novel iminothiazoline analogue with a quinoline base was synthesized using a multi-step chemical process. The initial reaction involved the combination of pentanoyl chloride 1 with potassium thiocyanate in a dry acetone solvent, followed by the addition of 3-aminoquinoline 3 to generate acyl thiourea 4. The purified acyl thiourea 4 was then mixed with p-bromophenacylbromide 5 to produce (Z)-4-bromo-N-(4-butyl-3-(quinolin-3-yl)thiazol-2(3H)-ylidene)benzamide 6, as demonstrated in Scheme p-bromophenacylbromide, and the acidic N-1 proton transfers to a base to form the isothiourea intermediate 7. This intermediate undergoes rearrangement of alkyl and aryl groups, facilitated by the removal of a proton from N-3. The rearranged intermediate 8 has a negative charge on N-3, which then attacks the carbonyl group. The final step involves dehydration, resulting in the formation of the product. The spectral characterization of the compound is given in the Additional file Scheme The x-ray analysis revealed that the analyzed compound contains a bicyclic system consisting thiazole ring. The thiazole ring is not visible in the molecular structure due to its conformation, while the pyridine ring forms a part of the bicyclic system. The molecular structure, depicted in Fig. 22(14) and R22(16) ring motifs  Å and Cg3…Cg3ii = 3.4714(12) Å , and a weak C–H···π interaction .Intermolecular C–H···O and C–H···N hydrogen bonds were linking the titled molecule. The compound was crystallized in triclinic system according to X-ray diffraction data. The dimensions and volume of the unit cell were as follows; a = 9.2304 (6) Å, b = 11.1780 (8) Å, c = 11.3006 (6) Å and V = 1025.61 (12) , and cystic fibrosis. Therefore, the development of inhibitors that can selectively target and regulate elastase activity has become an area of great interest in the pharmaceutical industry.As a result of the findings, it was demonstrated that compound 6 and the quercetin could dock into the active pocket of the elastase protein. Compared to the standard quercetin, which showed a docking value of − 7.2 kcal/mol, the crystal displayed a higher docking score of − 7.4 kcal/mol. This suggests that the crystal has a greater ability to suppress the elastase enzyme's activity. The docking outcomes also showed that the crystal exhibited a number of non-covalent contacts, such as hydrogen bonds and hydrophobic interactions, with the active site residues of the elastase protein. These interactions might help explain why the crystal has a higher affinity for the elastase protein. In conclusion, the molecular docking experiments gave important information on the binding affinity and possible inhibitory activity of the produced crystal against the elastase protein, and they may help create new inhibitors for the treatment of disorders associated with elastase. The molecular interactions between the synthetic crystal and the standard against the elastase enzyme are shown in Table The crystal that was created (6) was docked against the elastase enzyme utilizing molecular docking studies, and the results showed that the crystal had a docking score of − 7.4 kcal/mol. In the process of analyzing the results of the docking, it was found that the crystal established hydrogen bonds with the Gln34 residue of the elastase enzyme. The bond length of these bonds was 3.1 Å. In addition to this, the crystal was found to have interactions with hydrophobic residues such as Asn76, Glu80, Thr75, Asn74, Ser37, Tyr38, and Leu73. The formation of hydrogen bonds and the presence of hydrophobic contacts in the crystal-elastase complex are both indicators that the molecule that was synthesized has the potential to serve as a powerful inhibitor of the elastase enzyme. Figure The standard compound, quercetin, was also subjected to molecular docking against the elastase enzyme, and it exhibited a docking score of − 7.2 kcal/mol. The hydrogen bonding residues for quercetin were Asn188, Asn132, Ser162, and Trp159, with respective bond lengths of 3.14, 2.71, 2.84, and 3.22 Å. The hydrophobic residues involved in the binding were Gly187, Gly184, Pro161, Trp171, Val163, and Asn133. These findings suggest that quercetin produce stable interactions but synthesized crystal (6) had outperformed the standard in terms of binding affinity. The findings of in-silico investigations were also supported by in-vitro studies. So it can be deduced that crystal (6) may also be a potential inhibitor of elastase, as it displayed strong binding affinity and hydrogen bonding interactions with key residues in the active pocket of the enzyme. Figure Molecular dynamics (MD) simulations are an important tool for investigating the thermodynamic properties of biological systems in physiological conditions. The stability of complex (protein–ligand) was evaluated by using the Desmond software. The best docked conformation was used as the starting structure, and the simulation was run for 100 ns. The deep understanding of the complex behavior was evaluated by root mean square deviation; RMSD, rot mean square fluctuation; RMSF, contact and ligand interaction profile. The data obtained from these matrices enabled us to examine the complex's structural stability and dynamic behavior over time, providing valuable insights into the protein–ligand interaction.Figure This study employed RMSF analysis to generate the fluctuation profiles for all residues, revealing optimal fluctuations for the majority of amino acid residues. However, it was observed that residues Thr147, Gly148, Gly240, Ile241, and Met242 exhibited slightly higher fluctuations for more than 1 angstrom, whereas the remaining residues were significantly more stable during MD simulations. These amino acid residues belong to terminal regions, which are less rigid and tend to be more flexible. In addition, the amino acids in contact with the ligand were significantly stable, particularly the amino acid residues ranging from 20 to 50 that were in contact with the ligand and exhibited RMSF values of less than 1 angstrom, which demonstrate the significant stability pattern. The average RMSF value for the entire protein was found to be 1.19 Å, which is well within an acceptable limit. In addition, the RMSF data for liganded protein was also retrieved which revealed that ligand bound protein got more stable with average RMSF value of 0.81 Å. In addition, fluctuations of each residues was also got stable after binding of lignad with targeted protein. Figure The stability of protein ligand complex and binding of ligand with active site residues was further validated by taking the snapshot of MD simulation trajectory at different intervals and superposition these snapshots with respect to reference frame taken at 0 ns. It was notable that ligand remained attached to active site and didn’t exhibited flipping at any interval i.e., during initiation, middle and end of the trajectory. However slight rearrangements of ligand did observed during initiation and terminal stages of simulated trajectory. Figure Ligand contact profile is an important analytical metric to determine the efficiency of protein–ligand interactions. It was notable that strong interactions were observed with active site residents. Especially, Tyr39, Arg65, Leu73, Leu151, and Pro150 were engaged in hydrophobic interactions for more than 10% of the simulation time, and the interaction fraction was 50% for Tyr39. In addition, water bridges and hydrogen bonding were also stabilizing the protein ligand complex. Figure Figure The radius of gyration (Rg) is a critical analytical metric that helps determine the center of mass and compactness of proteins. A low Rg value indicates that the protein is highly compact with fewer structural changes, while a high Rg value suggests poor stability and more structural variations in the protein. Here, the protein showed very few structural changes with Rg ranges between 16.6 and 16.9 Å, indicating that the protein's mass was evenly distributed around a single point and remained compact throughout the simulated trajectory. Figure The MD simulation studies were conducted under constant pressure and temperature conditions using different barostat and thermostat for maintenance of pressure and temperature. It was observed that system was equilibrated constant pressure of 1.01 bar pressure and at constant temperature of 300 K. The electrostatic energy and equilibrated temperature and pressure is illustrated in Fig. In order to make the acyl thiourea product, a solution of potassium thiocyanate (1 mmol) was combined with dry acetone (15 mL) in a round-bottom flask that had a reflux condenser attached to it. This step was carried out in a reflux apparatus. After this, the mixture was swirled for a total of five minutes. After that, a 1 mmol solution of pentanoyl chloride that had been dissolved in acetone was added to the reaction mixture in a dropwise manner. In order to produce the isothiocyanate, the mixture was subjected to heating at a temperature that produced reflux for three to four hours. Following the step of cooling, an acetone solution containing one millimole of 3-aminoquinoline was added slowly, and the temperature was kept at 60 °C for 12–14 h in order to generate the acyl thiourea product. After this step, the reaction mixture was cooled using water that had been chilled with ice, and the result was separated using filtration. In order to obtain the finished product, the crude product was recrystallized in ethanol in order to undergo purification.p-bromophenacyl bromide solution containing one millimole of p-bromophenacyl bromide was added to it over the course of 30 min. Following the use of TLC to validate that the reaction was successful, the mixture was filtered, and a rotary evaporator was utilized to remove the solvent. As can be seen in Fig. To synthesize quinolinyl-iminothiazoline 6, acyl thiourea (1 mmol) was mixed with dry dichloromethane (15 mL) and triethyl amine (1 mmol) in a round bottom flask. After heating the combination to 50 °C for 24 h in a nitrogen environment, a f = 0.45 ; FT-IR (ATR) in cm−1, 3059 , 2957 , 2928, 2858 , 1738 (C=O), 1589 ; 1H-NMR ; in δ (ppm), 8.93 , 8.32 , 8.23 , 7.91 , 7.82 , 7.70 , 7.41 , 6.45 , 2.36 , 1.53 , 1.2 , 0.90 ; 13C-NMR  in δ (ppm), 172.5 (C=O), 171.0 , 149.2, 148.1, 137.3, 134.6, 133.8,130.9, 129.8, 129.4, 128.5, 127.5, 127.0, 126.9, 126.2, 105.0 , 31.2, 22.5, 19.2, 13.2. Anal. Calc. for C23H20BrN3OS: C, 59.23; H, 4.32; N, 9.01; Found: C, 59.21; H, 4.31; N, 9.00.Brown crystalline; M.P = 173 °C; yield = 75%; Rhttp://www.ccdc.cam.ac.uk).The crystallographic data for compound (I) was collected using Mo Kα radiation (λ = 0.71073 Å) on the Rigaku Oxford Diffraction diffractometer. The data was processed using the SHELX program packages SHELXL  and SHEL23H20BrN3OS was obtained through a series of experiments. The chemical formula of the compound was determined to be C23H20BrN3OS with a molecular weight of 466.39. The crystal system was found to be triclinic with the space group P − 1, and the temperature used for the experiment was 173 K. The crystal dimensions were measured and found to be 9.2304 (6) Å, 11.1780 (8) Å and 11.3006 (6) Å for a, b, and c, respectively. The angles α, β, and γ were 107.146 (5)°, 93.701 (5)° and 110.435 (6)°, respectively, with a volume of 1025.61 (12) Å3 and a Z value of 2. The diffraction data were collected using the Rigaku Oxford Diffraction diffractometer and the empirical absorption correction was carried out using the CrysAlis PRO 1.171.38.46 software. The scaling algorithm used for absorption correction was SCALE3 ABSPACK. The Tmin and Tmax were 0.842 and 1.000, respectively, and a total of 12,759 reflections were measured, out of which 6744 were independent and observed with I > 2σ(I). The Rint value was found to be 0.030 and (sin θ/λ)max was 0.761 Å−1. In terms of refinement, the H-atom parameters were constrained and the Δρmax and Δρmin were 0.59 and − 0.54 e Å−3, respectively. The R[F2 > 2σ(F2)], wR(F2), and S values were 0.042, 0.090, and 1.02, respectively, with a total of 263 parameters used. Overall, the crystal data provides a detailed insight into the structural properties of the titled compound and lays the foundation for further exploration and study of its properties.The crystal data for the titled compound Ci and de were determined, and Dnorm was used to estimate the normal contact distance, which was illustrated by the colors red, white, and blue. The white surface displayed contacts at distances that were equivalent to the sum of the van der Waals radii [The Crystal Explorer 17.5 software , 48 was ls radii , but thels radii . The stals radii , 52. Thils radii . In accols radii .The optimization of the molecular structure of the synthesized compound was conducted using Density Functional Theory (DFT) calculations . SpecifiN-succinyl-Ala-Ala-Ala-p-nitroanilide) to assess inhibition. The detailed protocol is given in our previously published article [We used a modified approach to assess elastase inhibition , 60. Ela article  and alsohttps://www.rcsb.org/) [Molecular docking is an efficient approach to study these interactions between ligands and proteins . In our sb.org/) . The retsb.org/) , 66. To sb.org/)  and LigPsb.org/) . Additiosb.org/) .Fig. 21ITo analyze the interaction between protein and ligand, Desmond software was used where a 100 ns simulation of protein–ligand complex was carried out using the TIP3P solvent model. The optimized potential for liquid simulations (OPLS3) forcefield  was emplIn conclusion, the current study demonstrated the successful synthesis and characterization of a structurally varied molecule known as quinolinyl iminothiazoline. This compound demonstrated significant activity against the elastase enzyme. The synthesized derivative had substantial activity in an in vitro elastase inhibition assay, with an IC50 value of 1.21 µM. This value indicated that the synthesized derivative was more potent than the standard, which was oleanolic acid, which had an IC50 value of 13.45 µM. The kinetic analyses provided additional confirmation that the chemical possesses remarkable binding energies as well as a competitive method of inhibition. The in silico studies including x-ray analysis, HS analysis, crystal void analysis, DFTs, molecular docking, and MD simulations supported the experimental findings by demonstrating the potential molecular interactions and stability of the protein–ligand complex. Furthermore, the x-ray analysis provided insights into the structural properties of the compound, revealing planar rings positioned at dihedral angles, and the molecules in the compound forming infinite double chains through hydrogen bonds and π-π interactions, creating a three-dimensional architecture. These findings altogether indicate that the synthesized compound holds strong potential as a lead compound for the development of a new drug against elastase enzyme.Additional file 1. Table S1 Experimental details. Spectroscopic characterization data. Elastase inhibition assay.