Intermittent fasting (IF) is associated with enormous metabolic alterations that underpin its diverse health effects. Changes in lipid metabolism, particularly ceramides, and other sphingolipids, are among the most notable of these alterations. This study investigated the lipidomic alterations associated with 29–30 days of Ramadan diurnal intermittent fasting (RIF) in metabolically healthy overweight and obese subjects. A prospective cohort of 57 overweight and obese adults , with an age range of 18–58 years was observed prior to and at the conclusion of Ramadan. At both time points, anthropometric, biochemical , and dietary intake measurements were taken. Using liquid chromatography-mass spectrometry, a lipidomic analysis of ceramides and other sphingolipids was conducted. Using paired sample t-tests, pre- and post-Ramadan anthropometric, biochemical, and dietary values were compared. RIF was associated with improved levels of lipid profile compartments and inflammatory markers. In addition, RIF was associated with a decrease in plasma sphingosine and sphinganine, which was accompanied by a decrease in sphingosine 1-phosphate and sphinganine 1-phosphate. In addition, RIF was associated with decreased C17, C22, and C24 sphingomyelin, but not C14, C16, C18, C20, and C24:1 sphingomyelin, as well as C20, C22, C24, and C24:1 dihydrosphingomyelin, but not C16 and C18 dihydrosphingomyelin. This study demonstrates that RIF is associated with improvements in plasma sphingosine, sphinganine sphingomyelin, and dihydrosphingomyelin lipid species, as well as improved lipid profile and inflammatory markers, which may confer short-term protection against cardiometabolic problems in patients with overweight/obesity. Lipidomics analysis is a novel advanced analytical technique that enables studying the lipid metabolism in human health and disease conditions2. Sphingosine and a fatty acid make up the family of lipid molecules known as ceramides (Cer), which are carried throughout the bloodstream by lipoproteins, especially low-density lipoproteins3. They serve as both prospective biomarkers of cardiometabolic disorders as well as pharmaceutical targets4. They are not only structural lipids but also multifunctional and bioactive molecules with significant involvement in many crucial cellular pathways, such as inflammatory processes4, diabetes, insulin resistance, fatty liver disease5, hypertension, and cerebro- and cardiovascular diseases (CVD)4. These obesity-related illnesses are caused in part by the abnormal buildup of toxic lipid metabolites in tissues that are not designed to store lipids 6. Particular ceramides have been associated with the risk of CVD, with positively associated species: phosphatidylcholine (PC) 16, PC 16:0/16:0; C18 Cer, Cer d; C24:1 Cer, Cer d; C16 Cer, Cer d and negatively associated species: C24, Per d; PC22:5, PC 16:0/22:5; PC 36:67.Lipids are integral components of the human body with versatile and vital physiological functions such as energy storage and cellular signaling and represent integral parts of cellular membranes and lipid particles such as exosomes and lipoproteins8. Lately, growing attention has been directed toward intermittent fasting (IF) (a dietary pattern that involves periods with very little or no food) as a suggested safe, disease-preventing, health-improving, and cost-effective regimen. Extensively detailed reviews recently unraveled that IF has a wide spectrum of positive effects on aging and neurodegenerative diseases, cardiometabolic dysfunction, and vascular problems. Therefore, IF continues to gain attention as a preventative and therapeutic intervention to counteract such chronic diseases9. Considering the drastic shift in lifestyle and dietary behaviors during the practice of IF, a wide range of physiological implications with distinctive metabolic switches are induced10. Several models of IF have been examined for their assumed protective effect in alleviating cardiometabolic risk factors11, including time-restricted eating/feeding12, periodic fasting, fasting-mimicking diet13, and alternate-day fasting14, with consistent positive effects reported.Fasting, the abstinence from drinking or eating for a specific time, is one of the most commonly observed dietary modifications by humans since ancient times, driven by several health behaviors, religious, and spiritual motives10. It is an obligatory practice imposed during the ninth month of the lunar calendar on adult Muslims. Intermittent fasting during Ramadan is observed by more than 1.5 billion Muslims throughout the world and is currently considered one of the most extensively studied forms of religious fasting15. Owing to its unique pattern of dry and diurnal fasting for 29–30 consecutive days from predawn to sunset16, the model of RIF has shown a plethora of beneficial anthropometric, metabolic, and inflammatory effects. These include improving body mass, dimensions, and composition18, alleviating metabolic syndrome components18, ameliorating inflammatory and oxidative stress markers20, normalizing glucose homeostasis21 and liver function markers22, and lessing cardiometabolic risk factors22 in healthy subjects.Among the types of IF, Ramadan intermittent fasting (RIF) is one of the most commonly observed religious forms of IF23, sleep quality and duration24, and physical activity25 in comparison with the non-fasting days; it is hypothesized that RIF will be accompanied by distinctive lipidomic fingerprints that distinguish it from non-fasting days. Recent studies using proteomic analysis demonstrated that the dawn-to-sunset model of RIF for thirty consecutive days results in distinctive fingerprint modifications in anticancer proteomic signature, and upregulates key regulatory proteins of the glucose and lipid metabolism, immune system, circadian clock, DNA repair, cytoskeleton remodeling and cognitive function in healthy people26. Further, distinct metabolomic changes have been reported upon the observance of RIF27. Such proteomic and metabolomic changes reported upon RIF are inevitably reflected in the lipidomic mapping in the human body, which requires further investigation using advanced lipidomics techniques. To understand the nature of metabolic changes accompanying fasting and feeding, it is of paramount significance to examine the lipidomic changes in these physiological states.During Ramadan fasting month, liberal eating is allowed during the night hours, without restriction on any of the allowed foods, with an eating window of 7–12 h (corresponding to 12–17 h of daily fasting based on the solar season that crosses with the lunar month of Ramadan). With the significant lifestyle and dietary changes associated with Ramadan month, including food groups and macronutrients28, and changes in circadian rhythm hormones29; IF is expected to be entailed with a list of lipidomic changes30. According to the available literature and the best of our knowledge, none of the published work had examined the lipidomics changes associated with the practice of RIF. Therefore, the current study was designed to investigate the lipidomic changes, expressed in terms of ceramides, sphingolipids, inflammatory markers, and lipid profiles associated with the practice of RIF in metabolically healthy people with overweight and obesity.Considering the extended fasting duration, the accompanying metabolic switches presented in the shift from glucogenic to ketogenic pathwayHijri lunar calendar of 1438 (from June 2016 to July 2016). Data were collected 1 week before Ramadan (baseline or pre-fasting) and at the end of Ramadan (after completing 29–30 days of the fasting month). During Ramadan, fasting people refrain from drinking, including water, and food, and do not smoke or engage in sexual activity from dawn to sunset. The daily fasting duration in this study was approximately 15 h. We compared the studied variables for each participant before and after or at the end of Ramadan, meaning each participant served as his control. Participants did not receive any recommendations for dietary, lifestyle, or physical activity changes at any stage during this study. The jurisprudence rules of Ramadan excuse females from commencing fasting during their menstruation; therefore, the fasting days for female participants were about 23–25 days. Since physical activity may interfere with the subjects’ biochemical measurement and body composition by the end of RIF, study subjects were instructed to keep on their habitual physical exercise levels before and during Ramadan. A flowchart of the recruitment and analysis steps is shown in Fig. This is a prospective cohort study executed during Ramadan month of the Arabic This observational study included a convenience sample of adults from the United Arab Emirates (UAE), including expatriates from Leavant countries , Sudan, and Egypt who were residing in Sharjah. Potential participants were invited to enroll in this study through personal communications, hospital bulletin, and social media. The announcement included the inclusion criteria . Those who were interested in the study contacted us and were asked to have a formal meeting at Sharjah University Hospital. This meeting involved informing participants about the study objectives and protocol, examining participants’ medical status and eligibility, letting them sign the informed consent, and taking the pre-fasting baseline measurements. Participants had 100% compliance with the study requirements and all of them had completed the study in the two phases of the study. Inclusion criteria were healthy female or male adults (aged 18–60 years) who had not previously been diagnosed with any metabolic or chronic disease. A set of inclusion and exclusion criteria were applied in recruiting the study participants , while stature was measured by a portable stadiometer  without shoes. WC was measured at the umbilicus with participants in a standing position and breathing normally. BMI (kg/m2) was calculated accordingly. Diastolic blood pressure (DBP) and SBP were measured with participants in a sitting position using a digital upper arm monitor .Body weight (to the nearest 0.1 kg), height (to the nearest 0.1 cm), and waist circumference (WC) (to the nearest 0.1 cm), were measured using standardized protocolsA venous blood sample (10 ml) was drawn from all participants 8–10 h after starting the fast (approximately between 11 a.m. and 1 p.m.). Within three hours of blood collection, samples were centrifuged, separated, and frozen at – 80 °C. The plasma inflammatory markers tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-10 were quantified using enzyme-linked immunosorbent assay (ELISA) reagents .Utilizing the 24 h food recall technique, dietary intake was assessed. Dietitians gathered dietary data for three days (two weekdays and one weekend day) before and after or after Ramadan. Using two-dimensional food models, participants were able to recall and approximate the food portion sizes they consumed. Using Food Processor software , total caloric, macro-, and micronutrient intakes were determined.5. Briefly, The internal standard (IS) stock solution containing 17:0-sphingomyelin (d18:1/17:0) (2502 pmol/sample), dihydro-cer (d18:0/18:1) (5 pmol/sample), 16:0-ceramide (d18:1-d7/16:0) (6 pmol/sample), 18:0-ceramide (d18:1-d7/18:0) (2 pmol/sample), 24:0-ceramide (d18:1/24:0) (152 pmol/sample), 24:1-ceramide (d18:1/24:1) (20 pmol/sample), and 17:0-glucosylceramide (d18:1/17:0) (50 pmol/sample) was prepared in methanol. Serum samples were thawed on ice and vortexed before the lipid extraction. Samples were extracted in individual tubes, including a process blank. Serum (50 µL) was diluted in 133 µL of PBS and mixed with 225 µL of MeOH containing internal standards. The PBS-Serum-IS solution was then vortexed and 750 µL of methyl-tert-butyl-ether was added. We then incubated the sample on ice for 15 min, vortexing every 5 min. The solution was then centrifuged at 21,000 rcf and the top lipid-containing MTBE layer was transferred to a new tube. We then added an additional 750 µL of MTBE, incubated for 15 min on ice, vortexing every 5 min, and transferred the lipid-containing MTBE to the new tube. The lipid-containing MTBE was then dried down using a SpeedVac. The sample was then resuspended in 150 µL of HPLC grade isopropyl alcohol (IPA): acetonitrile (ACN): dd-H2O (8:2:2). We then sonicated and vortexed the tube to resuspend the solution. The samples were stored at 4°C preceding liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.Human plasma samples were used for lipid extractions as previously describedLipid extracts were separated on an Acquity UPLC CSH C18 1.7 μm 2.1 × 50 mm column, maintained at 60 °C. It was connected to an Agilent HiP 1290 Sampler and Agilent 1290 Infinity pump and equipped with an Agilent 1290 Flex Cube and Agilent 6490 triple quadrupole (QqQ) mass spectrometer. Quantification of sphingolipids was based on peak area ratios to the standards applied to the extracts. Lipid extracts are separated on an Acquity UPLC CSH C18 1.7 µm 2.1 × 50 mm column maintained at 60 °C connected to an Agilent HiP 1290 Sampler, Agilent 1290 Infinity pump, equipped with an Agilent 1290 Flex Cube and Agilent 6490 triple quadrupole (QqQ) mass spectrometer. Sphingolipids are detected using dynamic multiple reaction monitoring (dMRM) in positive ion mode. Source gas temperature is set to 210 °C, with a gas (N2) flow of 11 L/min and a nebulizer pressure of 30 psi. The sheath gas temperature is 400 °C, sheath gas (N2) flow of 12 L/min, capillary voltage is 4000 V, nozzle voltage is 500 V, high-pressure RF 190 V and low-pressure RF is 120 V. Injection volume is 2 µL and the samples are analyzed in a randomized order with the pooled QC sample injection eight times throughout the sample queue. Mobile phase A consists of ACN: H2O (60:40 v/v) in 10 mM ammonium formate and 0.1% formic acid, and mobile phase B consists of IPA: ACN: H2O (90:9:1 v/v) in 10 mM ammonium formate and 0.1% formic acid. The 5 chromatography gradient starts at 15% mobile phase B, increases to 30% B over 1 min, increases to 60% B from 1 to 2 min, increases to 80% B from 2 to 10 min, and increases to 99% B from 10 to 10.2 min where it’s held until 14 min. Post-time is 5 min and the flow rate is 0.35 mL/min throughout. Collision energies and cell accelerator voltages were optimized using sphingolipid standards with dMRM transitions as [M + H] +  → [m/z = 284.3] for dihydroceramides, [M + H] +  → [m/z = 287.3] for isotope-labeled dihydroceramides, [M-H2O + H] +  → [m/z = 264.2] for ceramides, [M − H2O + H] +  → [m/z = 267.2] for isotope-labeled ceramides and [M + H] +  → [M − H2O + H] + for all targets. Sphingolipids without available standards are identified based on HR-LC/MS, quasi-molecular ion, and characteristic product ions. Their retention times are either taken from HR-LC/MS data or inferred from the available sphingolipid standards. Results from LC-MS experiments are collected using the Agilent Mass Hunter Workstation and analyzed using the software package Agilent Mass Hunter Quant B.07.00. Sphingolipids are quantitated based on peak area ratios to the standards added to the extracts. Data processing was performed using Multiquanta™ Software for post-data acquisition and MarketView™ Software to perform statistical analysis.P < 0.05. Our Bioinformatics analysis was performed using SPSS version 17.0.Statistical analyses were performed using SPSS software version 26 . Continuous variables were described using mean ± standard deviation (SD), while categorical variables were described as the frequency of occurrence and percentage  statementA cohort of 57 adults  participated in this observational study. Supplemental Table P = 0.001) in body weight (86.73 ± 15.74 kg vs. 88.32 ± 16.24 kg), body mass index  (29.40 ± 4.94 vs. 29.89 ± 5.02), body fat percent (28.58 ± 7.34% vs. 29.51 ± 7.09%), fat mass (25.25 ± 9.40 kg vs. 26.51 ± 9.49 kg) at the end of RIF when compared to before Ramadan, respectively. In addition to that, a significant decrease (P < 0.05) was shown in WC and hip circumference (HC) , in comparison with before Ramadan .A comparison of some anthropometric measurements before Ramadan and at the end of Ramadan is shown in Supplementary Table P = 0.013) and an increase in HDL-C (P = 0.001)  and total DG . Taken together, these results suggest that RIF was associated with improved plasma TC sub-types, TG, and DG.To examine the impact of RIFon plasma lipids, we measured plasma TC, LDL-C, and HDL-C before and after RIF. Although RIF did not alter plasma TC, we found a significant decrease in LDL-C (01) Fig. A, suggesP = 0.001). Furthermore, we measured the plasma anti-inflammatory marker IL-10 and found that RIF was associated with a significant increase in plasma IL-10 levels . Together, these results display that RIF is associated with attenuated systemic low-grade inflammation.We evaluated the impact of RIF on plasma inflammatory markers IL-6 and TNF-α. We found that the RIF was associated with significant reductions in both IL-6 and TNF-α  in total protein intake at the end of Ramadan (89.93 ± 39.25 g/day) when compared to before Ramadan value (108.35 ± 36.61 g/day). However, there was a significant increase (P = 0.001) in total sugar intake at the end of Ramadan (107.73 ± 53.71 g/day) when compared to before Ramadan value (65.90 ± 31.33 g/day). A significant increase (P = 0.016) in intake was also marked in PUFA at the end of Ramadan (16.32 ± 16.81 g/day) when compared to before Ramadan value (10.47 ± 8.09 g/day).The changes in the intake of some macro- and micronutrients of the participants upon the observance of RIF are shown in Supplementary Table P = 0.001) was shown when comparing the dietary cholesterol intake at the end of Ramadan (272.56 ± 181.90 mg/day) when compared to before Ramadan (395.85 ± 176.89 mg/day). A significant increase (P = 0.006) was reported in vitamin C intake at the end of Ramadan (97.43 ± 66.71 mg/day) when compared to before Ramadan (73.50 ± 50.63 mg/day). Also, a significant increase was found when comparing the value obtained at the end of Ramadan with the before Ramadan value in omega 3 (P = 0.001) , lycopene (P = 0.048)  and in vitamin E intake (P = 0.042) . On the other hand, there was no significant change upon RIF in the intake of total calories, fat calories, total carbohydrate, total fat, saturated fat, water, MUFA, trans fat, alpha-carotene, β-carotene, omega-6, or selenium.A significant reduction (P ≤ 0.001) and sphinganine . Further, RIF was found to be associated with significant reductions in both plasma sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (Sa1P) . Collectively, these results demonstrated that the practice of RIF was associated with decreased levels of plasma sphingosine and sphinganine lipid species.Taking advantage of our large lipidomics dataset, we next assessed the changes in plasma sphingosine and sphinganine upon the observance of RIF. Our results revealed that RIF was associated with significant reductions in both plasma sphingosine , but not C14, C16, C18, C20, C24:1 SM , but not C16 and C18 DhSM   and ceramide species. RIF was found to be associated with reduced C17, C22, and C24 SM (P = 0.001). There was also a positive significant correlation between dihydroglucosylceramide (DhGC) and IL-10 before Ramadan , while there was a positive significant correlation between phytoceramides (Phyto) and IL-10 at the end of Ramadan . The DG was positively correlated with TNF-α before Ramadan , IL-10 at the end of Ramadan , and with TNF-α at the end of Ramadan . The TG is correlated positively with IL-10 before Ramadan  and at the end of Ramadan , and the TG also correlated positively with TNF-α before and at the end of Ramadan . The was no correlation between Cer, glucosylceramide (GC), DhSM, SM, and PC with any of the inflammatory markers both before and at the end of Ramadan. Supplementary Table The correlation between ceramides sphingolipids as independent variables and inflammatory markers as dependent variables, before and at the end of Ramadan, is shown in Supplementary Table 33.This study aimed to examine the extent to which the observance of RIF is associated with changes in plasma lipid profile, ceramides, sphingolipids, and inflammatory markers in overweight and obese subjects. The major novel findings of this study are as follows: (1) RIF is associated with reduced body mass and fat mass independent of changes in calorie intake, (2) this fasting regimen is associated with attenuated plasma TC, TG, and DG, (3) RIF is associated with reduces plasma sphingosine, sphinganine, S1P, and Sa1P, (4) RIF is associated with reduced plasma SM and DhSM, but not ceramide and DhCer species and (5) RIF is associated with attenuated inflammatory markers. To our knowledge, this is the first study demonstrating that practicing RIF is associated with reduced specific plasma sphingolipids, lipid profile, and systemic inflammation in overweight/obese subjects using lipidomic analysis. However, our observations do not reflect potential alterations in lipid flux. A better understanding of flux in these pathways will require future studies. Findings from this study support that the model of diurnal IF could be a strategy to combat the epidemic of overweight and obesity-associated impairments in lipid metabolism and systemic inflammation and the resultant cardiometabolic multimorbidity35. Our findings demonstrated that RIF was associated with significantly reduced LDL-C, TG, and DG levels while associated with a significant increase in HDL-C (P = 0.001) levels after 30 days of fasting in overweight and obese individuals. This indicates that fasting may contribute to the prevention of dyslipidemia and trigger weight loss by attenuating the lipid profile at the lipidomic level. These results are in agreement with the findings of a recent study that demonstrated that fasting during Ramadan was associated with improved cardiometabolic risk factors by significantly reducing serum TC (P < 0·0001) and TG (P = 0·0003) levels while significantly increasing HDL-C (P = 0.0001) after 25 days of fasting36. Furthermore, our findings are also supported by a previous study, which reported that TC, LDL-C, TG, and apoprotein A were significantly improved after Ramadan fasting in 84 patients with stable ischemic heart disease37. Papazoglou et al. also demonstrated that Ramadan fasting imposed significant reductions in LDL-C and TG levels in 30 patients with dyslipidemia38.Dyslipidemia is one of the major risk factors for the development of cardiometabolic diseases such as diabetes and atherosclerosis39. IL-6 and TNF-α are the most attributable pro-inflammatory cytokines that enhance atherosclerosis and metabolic dysregulation in obesity40. On the other hand, IL-10 is an anti-inflammatory cytokine used by immune cells. However, IL-10 derived from immune cells can suppress thermogenesis and energy expenditure in adipocytes that drive insulin resistance in obesity41. Herein we show that RIF is associated with significantly reduced pro-inflammatory cytokines (IL-6 and TNF-α), while IL-10 was increased significantly. Interestingly, the reduction in TNF-α is supported by a previous study, in which it was demonstrated that TNF-α was reduced after Ramadan fasting both at serum and mRNA levels36. Moreover, other studies conducted in Germany and the United Arab Emirates (UAE) showed that IL-6 and TNF-α were significantly reduced at the end of Ramadan, while IL-10 was increased but did not reach a significant level in the German study42, but it was significantly increased in the UAE study43.Abnormal regulation of fat metabolism may induce an inflammatory response during diet-induced obesity. Obesity-associated inflammation predisposes to the pathogenesis of atherosclerosis which triggers vascular inflammation in metabolic diseases44. Sphingolipid metabolism has a vital role in the regulation of inflammatory signaling pathways in the human body by working as the second messenger that propagates the inflammatory response45. The current study showed significant reductions in the lipid profile components including LDL-C, total TG, total DG, sphingosine, sphinganine, S1P, Sa1P, SM , and DhSMs . These improvements in the lipid profile components and sphingolipids at the end of the observance of RIF was concomitant with a significant reduction in the proinflammatory cytokines (IL-6 and TNF-α) and a significant increase in the anti-inflammatory cytokine IL-10. The observed reduction in these metabolic factors is consistent with the evident reductions in the cardiometabolic risk factors22, metabolic syndrome components18, and inflammatory and oxidative stress factors  upon Ramadan fasting, as evident in recent systematic reviews and meta-analyses22.Systemic low-grade inflammation is a common underlying factor in the development of obesity and the subsequent affluent cardiometabolic diseases that are now prevalent, including cardiovascular disease, type 2 diabetes, and non-alcoholic fatty liver disease45. In this present study, we demonstrate that IF during Ramadan is associated with attenuating sphingosine, sphinganine, sphingosine-1-phosphate, and sphinganine-1-phosphate. This treatment regimen also reduces the variety of SM  and DhSM  species. These findings suggest that one or more acid ceramidase enzymes are down, a possibility that requires future investigations. Fasting is commonly associated with free fatty acid oversupply46 which may inhibit acid ceramidases47. The reduction in these lipid species may have implications for elevated skeletal muscle blood flow48 and contribute to improvements in metabolic function47, both of which require further elucidation in the RIF model.Sphingosine can only be produced by the hydrolysis of ceramide, while sphingosine phosphorylation by sphingosine kinase produces S1P, which occurs at both the cytosol and plasma membrane. Ceramide can either be used for catabolism to generate sphingosine and sphingosine-1-phosphate or one of many complex sphingolipids with an additional head group18.It is noteworthy to remark that previous studies have revealed that the anthropometric and biochemical alterations resulting from Ramadan fasting can persist for up to one month following the conclusion of the fasting period49. The reported increased intake of simple sugars could be a response to the feeling of tiredness and inactivity at the end of the fasting hours. Further, eating simple sugars in the form of sweet palm dates is part of the prophetic guidance when Prophet Mohamed (Peace be Upon Him) encouraged fasting people to break their fasting with dates50.Lastly, the reported changes in dietary intake of sugars and other nutrients are consistent and repeatedly reported and mirror the reported changes in food groups and macronutrients by other research on Ramadan fastingThough the current work has several strengths such as being the first study with a sufficient sample size using high accuracy LC-MS machine on fresh blood samples, however, the current work entails several limitations that should be considered when discussing its results. The observational nature of the current work and the lack of experimentally controlled design make it difficult to infer causality. The use of the memory-based technique for assessing dietary intakes may impose some inaccuracies in dietary assessment results.Putting the previous findings together, the current study demonstrates that observance of RIF is associated with improvements in plasma sphingosine, sphinganine SM, and DhSM lipid species, along with improved lipid profile and inflammatory markers. It can be concluded, accordingly, that the model of RIF may impose short-term protection against cardiometabolic problems, and may be applied as a suggested cost-effective, health-improving, and cardiometabolic disease-preventing strategy for people with overweight and obesity.Supplementary Tables.


Results showed that Zn fertilization of wheat, irrespective of modes of application, significantly increased grain and biological yield, grain per spike, and 1,000 grains weight over control; however, its effect was more noticeable when applied as 7.5 kg ha−1 of soil Zn combined with foliar Zn at 2.5 kg ha−1. Zn application significantly increased the grain protein content from 9.40% in the control to a maximum of 11.83% at soil Zn of 10 kg ha−1. Similarly, Zn application improved Zn, phosphorus (P), and potassium (K) concentrations in wheat grains. Moreover, correlation analysis showed that the grain Zn concentration was positively correlated with the grain P concentration. The correlation between P concentration in wheat grains and 1,000 grain weight was not significant. A total of 1,000 grains weight was positively correlated with tillers per plant, grain yield, and biological yield. There were positive correlations between protein content, biological yield, grain yield, and tillers per plant. Therefore, soil-applied Zn + foliar application in alkaline soils with limited Zn availability is crucial for improving wheat yield and grain quality.Cultivation of high-yield varieties and unbalanced fertilization have induced micronutrient deficiency in soils worldwide. Zinc (Zn) is an essential nutrient for plant growth and its deficiency is most common in alkaline and calcareous soils. Therefore, this study aimed to evaluate the effect of Zn applied either alone or in combination with foliar application on the quality and production of wheat grown in alkaline soils. Zn was applied in the form of zinc sulfate (ZnSo Triticum aestivum L.) belongs to the family Poaceae and is a key cereal crop for the vast majority of people worldwide. For almost 36% of the global population (two billion people), wheat is the most favored staple food. Wheat accounts for nearly 55% of all carbohydrates and 20% of all calories consumed worldwide  to evaluate the effect of Zn application to soil, either alone or in combination with foliar spraying, on the quality and yield of wheat grown in alkaline soil; (ii) to determine the optimal rate of Zn application for maximum wheat yield and quality in alkaline soil; and (iii) to investigate the effect of Zn application on the protein content of wheat grains in alkaline soils.https://www.pmd.gov.pk/en/). During winter (November–May), the mean maximum and minimum temperatures were 20 and 39 °C, respectively. Physiochemical properties of the experimental site were measured according to the standard protocols, which has been mentioned in section 2.4. Soil pH was 8.1 and EC was 0.32 dSm−1, which showed that the soil was alkaline in nature. Soil texture was clay loam with 22%, 38%, and 40% silt, sand and clay, respectively. Soil organic matter, N, P and K content were 0.69%, 0.8 g kg−1, 1.5 mg kg−1, 55 mg kg−1, respectively. The soil AB-DTPA extractable Zn content was 0.58 mg kg−1, which showed that the soil was Zn deficient.The experimental site is located in Kohat region (33°30′22.4″N 71°26′06.0″E) of Khyber Pakhtunkhwa, Pakistan. The study site has a semi-arid and subtropical climate, with an average annual rainfall of 500–600 mm. Meteorological data were obtained from the Pakistan Meteorological Department website (2 (3 m ×3 m), and there were total of 33 plots. Wheat was sown at 120 kg ha−1 on November 22, 2020, and harvested on May 5, 2021. There were 11 treatments including; control (T1), soil Zn 5 kg ha−1 + 0 kg ha−1 foliar Zn (T2), soil Zn 3.75 + 1.25 foliar Zn (T3), soil Zn 2.5 + foliar Zn 2.5 (T4), soil Zn 1.25 + foliar Zn3.75 (T5), soil Zn 0 + foliar Zn 5 (T6), soil Zn 10 + foliar Zn 0 (T7), soil Zn 7.25 + foliar Zn2.50 (T8), soil Zn 5 + foliar Zn 5 (T9), soil Zn 2.5 + foliar Zn 7.5 (T10) and soil Zn 0 + foliar Zn 10 (T 11). Zn was applied in the form of zinc sulfate (ZnSO4) to the soil and as a foliar spray during the sowing and tillering stages, respectively. Each treatment received recommended dose of N-P-K at 120:90:60, in the form of urea, diammonium phosphate (DAP), and sulfate of potash (SOP), respectively, at the time of wheat sowing. All agronomic practices were timely done during wheat growth period. To measure wheat agronomic attributes at harvesting stage, five plants were randomly selected from each treatment and average of tillers per plant was calculated. 1,000 grains weight were randomly collected from each treatment using a grain counter machine and weighed using a weight balance. Grain yield was obtained by harvesting the four central rows of each plot, threshed using a mini wheat thresher, dried, and then weighed. Biological yield obtained by harvesting mature wheat from four central rows of each plot, then dried and weighed. Grain yield and biological yield were converted into kg ha−1 using the following formulas:A field experiment was carried out to assess the impact of soil and foliar applied zinc on the growth, yield, and quality of wheat crop during winter season 2020–2021. A Randomized Complete Block Design (RCBD) was used with three replications. The area of each plot was 9 mBiological yield (mg kg−1) Grain yield (mg kg−1) Soil samples were collected from 0–20 cm depth with the help of Auger from three different locations in each plot before wheat sowing. Visible plant residues and roots were removed by hand and stored in labeled plastic bags for laboratory analysis. Soil samples were air-dried and passed through a 2-mm sieve. Similarly, plant samples were collected randomly from each plot at wheat harvesting stage. The plant samples were cut into small pieces, air-dried, and stored for further analysis. Soil standard procedures were used to determine the soil pH  and elecP,  K and Zn (mg kg−1) In wheat grain, the Zn concentration was determined using the procedure of Zn (mg kg−1) 3 was added. After 24 h, 4 ml perchloric acid was added and heated on a hot plate until white fumes appeared. After cooling, the sample was diluted by adding 100 ml distilled water and filter through 42-wattman filtered paper in a 100 ml volumetric flask. For P determination, 1 ml filtrate in a 25 ml volumetric flask was taken and 5 ml ascorbic acid mix reagent (ammonium molybdate + antimony potassium tartrate) was added. The volume was adjusted to 25 ml and the sample was kept in the dark to develop a blue color. A visible UV–VIS spectrophotometer set at 880 nm was used to measure the P concentration in wheat grain.  A flame photometer was used to measure the K concentration in wheat grain. P and K content were determined by the following formulas:The total P and K concentration in wheat grain were determined using the method described by P (mg kg−1) K (mg kg−1) The protein content in the wheat grain was measured according to P < 0.05) in SPSS . Pearson’s correlations analysis was conducted using Microsoft excel 2016.All collected data were statistically analyzed by one-way ANOVA and means were compared using the Duncan test (−1) received soil + foliar Zn at rates of 7.5 kg ha−1 and 2.5 kg ha−1, respectively, followed by plot that received soil Zn at 5 kg ha−1 + 5 kg ha−1 foliar Zn with an average yield . The lowest grain yield  was observed in the control. Similarly, the highest biological wheat yield  was found in the plot where Zn was applied as soil and foliar at 7.5 kg ha−1 and 2.5 kg ha−1, respectively, followed by plot that received 5 kg ha−1 of Zn fertilizer in both soil and foliar, with an average biological wheat yield of 7,994 kg ha−1, whereas the lowest biological wheat yield was observed in control plot. The outcome of our experiment predicted that the number of tillers per plant in T11 (foliar Zn at 10 kg ha−1) was significantly different compared to the control (P < 0.05) as shown in n = 7.56) was found in T11, followed by the plot that were administered zinc through soil and foliar (5:5 kg ha−1) producing (n = 7.5) tillers per plant. The number of tillers per plant were higher in plots where the foliar ratio of Zn was higher than soil. The results showed that Zn addition increased the 1,000 grains weight but did not show a significant difference among treatments, except T1 (control) which showed the lowest 1,000 grains weight. Data on 1,000 grains weight ranged from 40.36 to 48.37 g. The maximum 1,000 grains weight was recorded in T8 (48.37 g) and T7 (47.47 g), followed by T6 (47.38 g), whereas the minimum (40.35 g) was recorded in T1.The results indicate that certain rates of zinc fertilizer application, whether soil applied or foliar, had a positive impact on both biological yield and grain yield of wheat . Regardl−1+ foliar 2.5 kg ha−1), followed by 0.46% in soil Zn + foliar application (1.25 kg ha−1 + 3.75 kg ha−1). The minimum Zn concentration in wheat grains was recorded in control. The effect of Zn supplementation either through soil or foliar application was non-significant on P grain concentration; however, plots that received Zn supplementation had the highest P concentration compared to the control. Zn fertilizer levels had a considerable impact on the potassium concentration of wheat grains. The potassium concentration in the wheat grain increased by 6.24% with foliar Zn at 5 kg ha−1(T6) compared to control. T6 was significantly different from all the treatments except T2 (soil-applied Zn at 5 kg ha(−1). The plot that received Zn only through the soil at a rate of 10 kg ha−1 had the lowest wheat grain K concentration.The findings regarding wheat grain Zn, K, and P concentrations affected by Zn addition at various levels through the soil and foliar are presented in 7 (soil Zn 10 kg ha−1) treatment showed a 25.88% increase over the control, followed by T8 (soil 7.50 kg ha−1 + foliar 2.50 kg ha−1), which increased grain protein content by 22%. T2-T10 showed an increase in protein content from 2.48% to 25.88% compared with control. The data showed that increasing soil Zn with decreasing foliar levels gradually enhanced the protein content in the grain.Zinc fertilization, whether applied through foliar or soil, significantly increased the protein content of wheat grains . There wCorrelation analysis showed that grain Zn concentration was positively correlated with P concentration in the wheat grains . The cor−1 and 2.5 kg ha−1, respectively, which greatly improved wheat grain production. These results highlight the significance of zinc availability in increasing wheat yield in alkaline soils. A published study reported that in alkaline soils of Pakistan, adding Zn application in soil at 5 kg ha−1 or two split doses as a foliar spray increased maize grain yield by 29% and 14%, respectively (−1) with both soil Zn + foliar application . Other investigators have also noted an increase in Zn concentration in wheat grains by Zn fertilization  in the current study had a significant effect on the K concentration of wheat grains compared to the other treatments, except T2 (soil-applied Zn at 5 kg ha−1). The increase in K content of wheat grains with soil Zn + foliar spray might be due to the zinc synergistic relationship with potassium  in wheat grain protein content compared to the control . Wheat grain potassium and phosphorus concentrations were enhanced by Zn addition to the crop regardless of the application method. The Zn application increased the wheat grain protein content from 9.40% in control to 11.83% at soil Zn of 10 kg ha−1. Thus, soil-applied Zn with foliar spray is recommended to increase wheat grain production and quality in alkaline soils. Further research could be conducted to optimize the timing, dosage, and method of zinc application to crops in order to maximize its efficiency and minimize any potential negative effects. Additionally, the use of Zn in agricultural practices could be expanded to increase the productivity and quality of crops other than wheat, particularly in regions with Zn-deficient soils.The application of Zn, whether through foliar spray or soil application was found to benefit both the yield and quality of wheat grown in alkaline soils. The highest grain yield, biological yield and 1,000 grain weight were found when soil Zn was applied at 7.5 kg ha10.7717/peerj.16179/supp-1Supplemental Information 1Click here for additional data file.


Betalains are known to possess ONOO− scavenging ability. Betanin, a betalain isolated from red beet, possesses antioxidant, anti-inflammatory, and antitumor activities; however, detailed studies of this isolated pigment have not been conducted, owing to its instability under physiological conditions. This study aimed to isolate highly purified betanin from red beetroots using an improved purification method involving deproteinization and citric acid co-precipitation and evaluated its antioxidant activities. The purified betanin thus obtained had a significantly lower isobetanin content than the commercially available betanin dyes. The antioxidant activity of purified betanin examined in the 2,2-diphenyl-1-picrylhydrazyl assay, the direct ONOO− reaction, ONOO−-dependent DNA damage, and lipid peroxidation reactions revealed that betanin possessed higher antioxidant capacity than general antioxidants such as ascorbic acid and quercetin. Furthermore, betanin showed indirect and direct cytoprotective effects against H2O2 and ONOO− cytotoxicity, respectively, in cultured mouse fibroblasts. To the best of our knowledge, this is the first study to demonstrate the cytoprotective effects of betanin against ONOO− toxicity. The highly purified betanin obtained in this study will aid in further exploring its physiological functions.Reactive oxygen species and reactive nitrogen species (RNS) are damaging for many biomolecules. Peroxynitrite (ONOO Similar to ROS, it is highly reactive and causes cellular dysfunction by oxidizing and nitrating DNA, proteins, and lipids, which are important cell components.Reactive oxygen species (ROS) and reactive nitrogen species (RNS) can damage many biomolecules including carbohydrates, lipids, and DNA ,2, there− scavenging ability and inhibit DNA damage and the nitrosation of tyrosine in vitro. Betanin, a type of betalain, was isolated from red beet. Red beets are a nutrient-rich superfood, possess anti-inflammatory activity, and exhibit hypotensive, vasodilatory, and hypermotility effects [− [Antioxidants effectively delay oxidative damage via several mechanisms, such as quenching free radicals directly and stimulating the biosynthesis of enzymatic and non-enzymatic antioxidants . Betalai effects ,7,8. The effects ,10,11; h effects . We prevfects [− . Moreove− toxicity was also examined using the NIH3T3 fibroblast cell line.Therefore, this study aimed to isolate highly purified betanin from red beetroots using an improved betanin purification method, involving deproteinization and citric acid co-precipitation, and evaluated its antioxidant activities . In addition, its cytoprotective activity against ONOOSince betanin is unstable and prone to decomposition during processing, refining, and storage, it is difficult to isolate high-purity betanin. Hence, commercial betanin dyes contain large amounts of stabilizers, such as dextrin. The amount of betanin per powder weight of both commercial and purified betanin was determined by measuring the absorbance at 538 nm. The findings revealed that the betanin content of the commercial betanin powder was very low, being less than 1% . In contHPLC analysis of commercial betanin and betanin purified using the method developed in this study revealed betanin and isobetanin peaks in a 1:1 ratio in commercial betanin A, wherea50 and Trolox equivalent antioxidant activity (TEAC) values  assay was expressed in terms of DPPH IC) values . Althoug− to determine their ONOO− scavenging activity. Betanin or quercetin were added in phosphate buffer (pH 7.2), and different concentrations of ONOO− were added. The absorbance was measured after 3 min  pUC19 plasmids with SIN-1 converted approximately 60–70% of pUC19 plasmids to single-strand-break open circular (OC) forms. In the presence of 0.5–2.0 mM betanin, the abundance of OC plasmids decreased, and that of SC plasmids increased in a betanin concentration-dependent manner (The protection conferred by betanin against DNA damage was investigated by incubating the pUC19 plasmid DNA in 0.1 mM 3-(4-morpholinyl) sydnonimine hydrochloride (SIN-1), an ONOOt manner A. Similat manner B. The pl− containing 0.6 nmol of thiobarbituric acid reactive substances (TBARSs) was used. A TBARS calibration curve was prepared using malondialdehyde (MDA) , the IC50 of betanin (36.0 µM) was considerably lower than those of the other antioxidants ascorbic acid, α-tocopherol, and Trolox, which were 239.8, 191.2, and 274.7 µM, respectively.A liposome solution without ONOOde (MDA) . The addntration . The ince values . The fin betanin . CompariA spectroscopic analysis of an NIH3T3 fibroblast cell culture supernatant with 25 µM betanin revealed changes in the absorption spectrum with time A. In par2O2 toxicity in NIH3T3 cells and the cytoprotective effect of betanin were examined. Betanin was found to be non-cytotoxic at concentrations below 25 μM in the 3 h, 6 h, and 3 h pretreatment + 6 h co-treatment groups  in the 3 h betanin pre-treatment and 3 h pre-treatment + 6 h co-treatment groups, compared to that of the group treated with H2O2 alone  and after a 3 h pretreatment  and NAD(P)H quinone oxidoreductase (NQO1)  A. Howeveand NQO1 B.- donor SIN-1 were determined. Cell viability was measured after 3 h of betanin treatment followed by SIN-1 treatment. The results showed that cell viability decreased in a concentration-dependent manner in the presence of SIN-1 alone at a concentration between 0.5 and 1.5 mM; cell viability decreased by approximately 50% after 6 h of treatment with 1.5 mM SIN-1 (p < 0.001) with betanin co-treatment compared to that measured after treatment with SIN-1 alone .p < 0.05, ** p < 0.01). In contrast, DCF fluorescence intensity significantly decreased in the presence of 25 µM quercetin (* p < 0.05), compared to that measured in the presence of SIN-1 alone, and a decreasing trend in fluorescence intensity was observed at 5 µM. In addition, phase-contrast observation of cell morphology revealed many cells with retracted pseudopodia in the SIN-1-alone treatment group  and SIN-1 for 6 h, cell-accumulated ROS/RNS were detected fluorescently using DCFH-DA. Cell morphology and DCF fluorescence were observed using a fluorescence microscope. DCF fluorescence was observed in cells treated with SIN-1 alone and was found to decrease on co-treatment with betanin and quercetin A,B. In pnt group B. A simunt group A. Thus, In this study, we were able to efficiently purify betanin in powdered form by deproteinization and citric acid co-precipitation. A HPLC analysis revealed that commercial betanin contained betanin and its isomer, isobetanin, in a 1:1 ratio, whereas betanin purified by our method had a much lower content (<10%) of isobetanin and low isomerization. In addition, the betanin powder purified in this study was stable for more than 2 years at 4 °C and in the dark, according to HPLC analysis. Betanin is usually unstable in solution and relatively stable in the solid state. The underlying reason for purified betanin being stable for more than 2 years is its crystallization. Commercial betanin is also thought to be stable for several years in the solid state, but it has not yet been confirmed whether it is stable after the removal of dextrin, which constitutes more than 95% of commercial betanin. 2O2. Furthermore, the coexistence of transition metal ions like copper and iron with ascorbic acid and other redox-active compounds may potentially accelerate the production of H2O2 and the degradation of ascorbic acid [2O2 in the cell culture medium D-MEM, which was also used in this experiment, due to polyphenols such as propyl gallate, (-)-epigallocatechin gallate, and quercetin [2O2 in cell culture medium, Long and colleagues recommend distinguishing between the direct intracellular effects of phenolic compounds and the effects resulting from H2O2 generated in the culture medium. They proposed investigating the behavior of added phenolic compounds, quantifying H2O2, and evaluating the impact of catalase supplementation [2O2 in cell culture media, such as D-MEM, similar to ascorbic acid and polyphenols, and it cannot be ruled out that betanin-dependent H2O2 generation might have influenced the stability of betanin under the cell culture conditions shown in 2O2 generation in the culture medium during betanin treatment and the analysis of the degradation products of betanin could provide new insights into the mechanisms of betanin instability.Further comprehensive investigation is needed to elucidate the stability of betanin. It is known that ascorbic acid reacts with dissolved oxygen in solution, leading to the generation of Hbic acid ,18. Addiuercetin . In consentation . In this2O2-induced cell death even after a short pretreatment of 3 h. Additionally, pretreatment with betanin for 24 h significantly induced Nrf2-regulated antioxidant gene expression, a result similar to that previously reported after a 72 h pretreatment [Betanin was shown to protect cells from Hreatment . Further2O2-induced DNA damage by betanin or a betalain-rich Cactus pear (Opuntia ficus-indica) fruit extract was reported in several human cell lines, such as human peripheral lymphocytes and the colon carcinoma cell line HT-29. In these studies, a 14–72 h betanin treatment showed protective effects against H2O2 toxicity [The inhibition of Htoxicity ,16,21,222O2 toxicity even after 3 h of pretreatment  were cultivated and harvested in open-air fields by contract farmers in Eniwa-shi, Hokkaido, Japan. Betanin was extracted and purified using the following method. Red beetroots (1 kg) were squeezed using a juicer, and 0.3% (w/v) citric acid  was added to the beetroot juice to adjust the acidic conditions (pH 2.5–3.0). For deproteination, 50% (w/v) saturated ammonium sulfate  was added. After incubation for >30 min at 4 °C, centrifugation was performed for 20 min at 8000 rpm and 4 °C. The supernatant (0.6 L) was poured directly onto 200 mL of resin  that had been pre-washed with methanol and placed in a Büchner funnel. After washing the resin with 1.5 L of a 0.3% citric acid solution, the selective elution of betanin with 0.6–0.7 L of a 10% EtOH-containing 0.3% citric acid solution was performed. The betanin-containing solution was evaporated under low pressure and re-dissolved in 6–7 mL of Milli-Q water (betanin concentration >10 mM). For co-precipitating betanin with citric acid, the betanin-containing solution was incubated for 1 week at 4 °C. The precipitate was filtered on filter paper, washed twice with 1 mL of 90–95% EtOH, and dried under low pressure to obtain a citric acid-free powder. The betanin concentration was calculated by measuring the absorbance at 538 nm using an ultraviolet–visible (UV–VIS) spectrophotometer , considering the molar extinction coefficient of betanin ε538 = 60,000 M−1·cm−1 [Red beetroots  with a pre-column  were used to evaluate the purification of betalains. Chromatography was performed using two mobile phases, i.e., (A) 1% aqueous formic acid, (B) 80% aqueous acetonitrile (B). A linear elution gradient was used starting with 2% B and increasing to 33% B in 30 min at a flow rate of 0.8 mL min −1 with an injection volume of 10 µL. The eluant was monitored at 538 nm.An HPLC system  and a reverse-phase column Puresil C− was synthesized using the method described by Patel and Darley-Usmar [− was immediately quenched with 1.5 M NaOH (30 mL). Excess hydrogen peroxide was removed by adding 1.3 g of manganese dioxide (MnO2), which was further removed by centrifugation (4000 rpm for 5 min at 4 °C). The solution was frozen at −80 °C; ONOO− tended to form a yellow top layer due to freeze fractionation, which was scraped for further studies . The concentration of ONOO− was determined by measuring the absorbance at 302 nm and using the molar extinction coefficient ε302 nm of 1670 M−1 cm−1 [ONOOey-Usmar . Ice-colM−1 cm−1 .The antioxidant capacity of betanin was estimated using the DPPH free-radical scavenging assay . BrieflyTo account for the background absorbance of betanin at 517 nm, a mixture of 20 µL of betanin at different concentrations and 80 µL of 0.1 M Tris-HCl buffer (pH 7.4) plus ethanol instead of the DPPH solution was used. The percentage of DPPH scavenging (betanin) was calculated from the following Equation (2):50 and TEAC were calculated to express the antioxidant capacity of the analytical samples, based on the method described by Shimamura et al. [50 value was calculated by interpolation by connecting the two points around the 50% inhibition. TEAC was estimated as follows (3):The DPPH ICa et al. . The DPP376 = 21.9 mM−1·cm−1 and ε265= 7.0 mM−1·cm−1, respectively. Betanin and ascorbic acid were dissolved in water, and quercetin, α-tocopherol , and Trolox  were dissolved in ethanol.The concentrations of quercetin  and ascorbic acid sodium salt  were estimated using the molar extinction coefficients ε− to determine their ONOO− scavenging activity. Betanin (10 μM) or quercetin (20 μM) was added to 0.25 M phosphate buffer (pH 7.4), and 5 μL of ONOO− solutions at different concentrations was added. The total reaction mixture was 1 mL. The absorbance spectra of the reaction mixtures were measured using a UV–VIS spectrophotometer  after 3 min of incubation. Five microliters of 0.5 M NaOH was used instead of ONOO− as a control.The analyzed compounds were directly reacted with ONOOv/v). To exclude the effect of acetone, acetone was also added to a final concentration of 0.1% to the catechin-free control and SIN-1 groups. After incubation, DNA was purified using the FastGene Gel/PCR extraction kit . The DNA was collected and analyzed using 1% agarose/TBE gels. After electrophoresis, the gels were stained with 0.5 μg mL−1 of ethidium bromide for 30 min and then photographed under UV illumination. Signal intensity was calculated using the ImageJ software [DNA cleavage was analyzed by detecting the conversion of the SC form of the pUC19 plasmid DNA  to the OC and linear forms. DNA (50 ng) was incubated with 0.1 mM SIN-1  ,27 in thsoftware .w/v) concentration, and the mixture was vortexed for 5 s and then stirred for 10 min using an ultrasonic homogenizer  to obtain uniform liposomes.The commonly used TBARS assay was employed to measure lipid peroxidation. One gram of egg lecithin  was dissolved in 10 mL of chloroform, and chloroform was removed using an evaporator; phosphate buffer  was added to reach a 5%  liposome solution  was first dispensed into 1.5 mL microtubes, and droplets of the ONOO− solution were attached to the wall of the microtubes without touching the liposome solution. The lipid peroxidation reaction was then initiated by agitating quickly using a vortex shaker for 5 s. After 5 min of incubation at room temperature, 0.05% butylated hydroxytoluene  and a thiobarbituric acid (TBA) reagent consisting of 15% (w/v) trichloroacetic acid  and 0.375% (w/v) TBA  were added, and the mixture was incubated at 95 °C for 30 min, then quenched with cold water and centrifuged at 12,000 rpm for 15 min. The supernatant was centrifuged at 12,000 rpm for 15 min, and the absorbance at 532 nm was measured using a UV–VIS spectrophotometer  to quantify the lipid peroxides.Since the lifetime of ONOOAlthough TBARSs have a maximum absorption at 532 nm, a compound with an absorption at 538 nm, such as betanin, would interfere with the TBARS measurement. Therefore, to account for the sample (betanin)-derived 538 nm absorption in the TBARS measurement, the 532 nm absorbance of the sample-containing liposome suspension was used as the sample background.The relative lipid peroxidation (%) was calculated from the following Equations (4)–(7):50 value is the concentration of the antioxidant sample that could inhibit 1 mM ONOO--induced lipid peroxidation by 50%. The TBARS value was determined from a graph by plotting relative lipid peroxidation% against sample concentration, as shown in The TBARS IC2 and 95% air at 37 °C.The mouse embryonic fibroblasts NIH3T3  were maintained in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum  and penicillin (100 units/mL)/streptomycin (100 µg/mL)  in a humidified atmosphere with 5% COv/v). NIH3T3 cells were treated with 5 µM and 25 µM betanin and quercetin for 3 h or 24 h. The medium was then replaced with fresh medium containing 0.3 mM H2O2  or 1.5 mM the ONOO− generator SIN-1 with or without betanin or quercetin. In the quantification of gene expression experiments, NIH3T3 cells were treated with betanin or quercetin for 3 h or 24 h, and the total RNA was extracted. The degradation of betanin under cell culture conditions was also determined. Purified betanin (25 μM) was incubated under the cell culture conditions described above  for 24 h, its the ultraviolet–visible spectrum was determined over time using a cuvette with a 1 cm pathlength. The absorbance was also measured using betanin-free D-MEM medium as a control.A stock solution of betanin (1 mM) was prepared in distilled water. After sterilization using a syringe filter , the concentration was measured prior to use. Additionally, a stock solution of quercetin (25 mM)  was prepared in dimethyl sulfoxide , and its concentration was measured after filter sterilization. Quercetin was added to the culture medium so that the final concentration of DMSO was 0.1% (3 cells/well). After culturing for 24 h, the cells were treated as described in TM rainbow, Tecan, Männedorf, Switzerland). Cell viability was expressed as a percentage of that of the control, and the experiments were performed in triplicate.Cell viability was measured using the 3--2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, NIH3T3 cells ware seeded in 96-well plates (at a density of 3.0 or 8.0 × 105 cells/well). After culturing for 24 h, the cells were treated for 3 h or 24 h with betanin or quercetin at different concentrations. After the medium was removed, total RNA was isolated with the Isogen Ⅱ  reagent. Complementary DNA (cDNA) templates were generated using the PrimeScriptTM RT reagent kit with gDNA Eraser . Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using TB Green Premix Ex Taq  in a Thermal Cycler Dice Real Time System Ⅱ . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was employed as an endogenous control for RT-qPCR. The fold change of the mRNA expression was calculated using the standard curve method. The primer sequences used are listed in NIH3T3 cells were seeded into the wells of 6-well plates (at a density of 2 × 103 cells/well). After culturing for 24 h, the cells were treated as described in NIH3T3 cells were seeded into the wells of 96-well plates (at a density of 3.0 or 8.0 × 10p < 0.05.The data are presented as mean ± standard error of the mean or standard deviation, as applicable, and were statistically analyzed using one-way analysis of variance, followed by Dunnett’s test or Tukey’s test. All statistical analyses were performed using R ver. 3.6.0. . The sig− reaction, ONOO−-dependent DNA damage, and the lipid peroxidation reaction, and the results showed that betanin possessed higher antioxidant capacity than general antioxidants such as ascorbic acid and quercetin. Furthermore, betanin showed indirect and direct cytoprotective effects against H2O2 and ONOO− cytotoxicity, respectively. The isolation of highly purified betanin in this study will enable more detailed studies on its physiological functions in the future and may potentially contribute to elucidating the food functions of betanin, betanin-containing red beets, and other similar foods.In conclusion, we improved the betanin purification method and established a purification method to produce betanin powder with long-term stability. The purified betanin obtained by the improved purification method had a significantly lower isobetanin content than the commercially available betanin dyes. The antioxidant activity of purified betanin was examined using the DPPH assay, the direct ONOO


Subacute rumen acidosis (SARA) will cause an increase in endotoxin, which will have a negative effect on the bovine rumen epithelial cells (BREC). Flavonoids are effective in treating inflammation caused by endotoxin. Quercetin is a vital flavonoid widely occurring in fruits and vegetables and has received significant interest as a prospective anti-inflammatory antioxidant. Nonetheless, quercetin’s protective machinery against such damage to BREC induced by lipopolysaccharide (LPS) remains unclear. A combined quercetin and LPS-induced BREC inflammation model was utilized to elucidate the effect of quercetin protecting BREC from LPS-induced injury. After treating BREC with different doses of LPS  for 6 h or 24 h, the mRNA expression of inflammatory factors was detected. Our experimental results show the establishment of the BREC inflammation model via mRNA high expression of pro-inflammatory cytokines in BREC following 6 h treatment with 1 µg/mL LPS. The promotive effect of 80 μg/mL quercetin on BREC growth via the cell counting kit-8 (CCK8) assay was observed. The expression of pro-inflammatory cytokines and chemokines, notably tumor necrosis factor α (TNF-α), Interleukin 1β (IL-1β), IL-6, CC-motif chemokine ligand 2 (CCL2), CCL20, CCL28, and CXC motif chemokine 9 (CXCL9), etc., was significantly reduced by quercetin supplementation. We also analyzed the mRNA detection of related pathways by qRT-PCR. Our validation studies demonstrated that quercetin markedly curbed the mRNA expression of the toll-like receptor 4 (TLR4) and myeloid differentiation primary response protein (MyD88) and the nuclear factor-κB (NF-κB) in LPS-treated BREC. In addition, western blot result outcomes confirmed, as expected, that LPS significantly activated phosphorylation of p44/42 extracellular regulated protein kinases (ERK1/2) and NF-κB. Unexpectedly, this effect was reversed by adding quercetin. To complement western blot results, we assessed p-ERK1/2 and p-p65 protein expression using immunofluorescence, which gave consistent results. Therefore, quercetin’s capacity to bar the TLR4-mediated NF-κB and MAPK signaling pathways may be the cause of its anti-inflammatory effects on LPS-induced inflammatory reactions in BREC. According to these results, quercetin may be utilized as an anti-inflammatory medication to alleviate inflammation brought on by high-grain feed, and it also lays out a conceptual foundation regarding the development and utilization of quercetin in the later stage. Subacute rumen acidosis (SARA) is a metabolic ailment that easily occurs in high-yield cows. The disturbance of gastrointestinal microflora initiated via a high-concentrate diet is linked with a swift drop in rumen pH and causes subacute rumen acidosis . When thLately, more focus has been placed steadily on the utilization of flavonoids in the feed plants of livestock . There aIn short, quercetin may be a potential therapeutic strategy to alleviate SARA. Therefore, this study investigated the inhibitory effect of quercetin on LPS-induced inflammation of BREC. This lays the foundational knowledge for further exploration and refinement of quercetin-centered functional feed.p = 0.737, p < 0.01, p < 0.01), as shown in p = 0.065).To evaluate the cytotoxicity of LPS on BREC after different concentrations of LPS treatment, BREC viability was analyzed via CCK-8 cell viability assay. It was noted that the addition of different concentrations of LPS had a non-significant outcome on the cell activity of BREC in the cases of 6 h and 24 h incubation  the TAOC, SOD, CAT, and GSH-Px levels in BREC activated by LPS. The outcomes signified that quercetin hampered the LPS-induced oxidation of BREC.This section probed quercetin’s capacity to protect BREC from oxidative stress initiated by LPS. Compared with the control group, the group treated with LPS had lower levels of TAOC, SOD, CAT, and GSH-Px. . The results demonstrated that quercetin prevented BREC pro-inflammation brought on by LPS.To investigate the inhibitory effect of quercetin on mRNA expression of pro-inflammation cytokine, the levels were evaluated via qRT-PCR. According to the results in p < 0.01, p < 0.05). According to these outcomes, quercetin decreased BREC’s immune responses induced by LPS.When compared to unstimulated cells, the BREC with LPS treatment dramatically increased the mRNA expression of the genes for CCL2, CCL20, CCL28, CXCL14, CXCL2, CXCL5, CXCL8, and CXCL9 (p < 0.01). According to these data, quercetin’s protective effect was closely linked to the TLR4 signaling pathway.Using qRT-PCR, the expression levels of TLR4, TLR2, CD14, MYD88, IRAK1, IRF3, and NF-κB mRNA were measured to determine if quercetin’s protective effect was linked to the TLR4 signaling pathway. TLR4, CD14, MD2, MYD88, IRF3, and NF-κB mRNA levels increased significantly following LPS treatment but decreased significantly in the treatment group receiving 80 μg/mL quercetin amidst 1 μg/mL LPS  . SARA ocFor Experiment 1, authors probed the effects of diverse concentrations/dosage of LPS and stimulation time on the cell viability and pro-inflammatory factors of BREC. These results show that the treatment with the addition of diverse dosages of LPS for 6 h or 24 h has no significant effect on cell viability. Thus, our interpretation of this result is that stimulus concentrations lower than 10 µg/mL with LPS do not have a cytotoxic effect on BREC. Our findings are in line with those that have been previously published . During Unlike monogastric animals, the rumen of dairy cows is inhabited by a variety of microorganisms, including rumen protozoa, rumen bacteria, and anaerobic fungi, as well as a few bacteriophages . The abiQuercetin has been reported to possess antioxidant and anti-inflammatory characteristics ,33. AddiLPS and TLR4 together have been found to activate inflammatory pathways (NF-κB) and cause the generation of ROS, which controls the expression and activity of antioxidant enzymes . In thisWe focused on quercetin’s impact on LPS-treated BREC to investigate its anti-inflammatory activities. A pattern-recognition receptor for LPS is TLR4 . TLR4 reThis research has demonstrated that quercetin reduces BREC’s oxidative stress caused by LPS in vitro. Through the interference of the NF-κB signaling pathway, quercetin can lessen the inflammatory response. In summary, by means of the TLR4-NF-κB signaling pathways, treatment with 80 µg/mL quercetin can protect BREC’s normal physiological function from LPS-induced oxidative stress and inflammatory response. This research will establish a theoretical foundation for investigating the pharmacodynamic mechanisms of quercetin and its potential applications in the deterrence and management of SARA in dairy cows.2).BREC was procured from Yangzhou University’s Institute of Animal Culture Collection and Application in China, and the development of immortalized BREC was based on findings from an earlier investigation . By usin3 cells/well). Post 12 h cells were incubated for 6 h or 12 h with 0, 1, 5, or 10 μg/mL LPS (n = 5). The cells were subsequently exposed to 10 L of CCK-8 reagent, and they were incubated for a further three hours at 37 °C with 5% CO2. The enzyme labeling instrument  was used to quantify the OD values at 450 nm.According to Wang’s description , the imp2 after being sown in 6-well plates with 1 × 106 cells per well. Three experimental groups were produced using DMEM/F12 medium: (1) Control; (2) DMEM/F12 medium + LPS  for 6 h; and (3) DMEM/F12 medium + LPS  during 24 h collection of BREC for pro-inflammatory factors  mRNA expression.The BREC was cultivated at 37 °C with 5% CO5 cells/mL on 96-well culture plates. The media was taken out of the culture after 12 h. Quercetin  was used to stimulate the BREC for 24 h. CCK8 was then added to each experimental group  and cultured for 3 h at 37 °C with 5% CO2. Utilizing a microplate reader , the OD values were measured at 450 nm.The impact of various quercetin concentrations on the proliferative activity and toxicity analyses of BREC was identified using the CCK-8 kit. The procedure was the same as that used in Experiment 1. BREC were added and adjusted to 1 × 106 cells/mL on 6-well culture plates. The BREC were separated into the following 4 groups, namely (1) DMEM/F12 medium for the control group; (2) 1 µg/mL LPS for LPS group; (3) 80 µg/mL quercetin for Q group; and (4) 1 µg/mL LPS + 80 µg/mL quercetin for LPS + Q group), cultured for 24 h, and then a fresh medium was used. The cells from each group were harvested independently after 6 h of incubation.BREC were added and adjusted to 1 × 10® Premix Ex Taq ™ II kit  was used for real-time PCR detection. β-actin and GAPDH are already known to be suitable for BREC and were estimated by the 2−ΔΔCT method [This was completed per the operation instructions of the RNA extraction kit . CDNA was obtained using a reverse transcription kit . SYBR T method . Similar6 cells/mL. The cell suspension was inoculated into a 6-well plate. When cells proliferated to approximately 80–90%, LPS and quercetin were added. The cells were collected after 6 h. According to the manufacturer’s instructions, we used the antioxidant related Assay Kit  to measure the levels of TAOC, MDA, GSH-PX, SOD, and CAT in cell samples.After the digestion of BREC, cell suspension density was to be adjusted to 1 × 10TMII, Code No.154534, New York, NY, USA) and adjusted to 2 × 103 cells/mL. Cell proliferation was approximately 80–90% and treated with 1 µg/mL LPS and 80 µg/mL quercetin. Next, the slides were washed thrice using PBS for 3 min each time, fixed at room temperature for 30 min with 4% paraformaldehyde, rinsed thrice with PBS, and then re-rinsed with ethylenediaminetetracetic acid disodium  for antigen retrieval. PBS containing 3% horse serum was introduced drop by drop onto the slides and blocked at room temperature for 1 h. The blocking solution was drawn out, followed by primary antibody addition and incubation all night at 4 °C. The selection of the first antibody refers to Wang’s experiment [BREC was added to an 8-well chamber slide  was utilized to compute protein concentrations in cell samples for total protein extraction. Protein samples were prepared according to Wang’s method . Followip > 0.05), while values with different letter values show differences that are significant (p < 0.05).The data outcomes were presented as means ± standard error (SEM). For scenarios involving two or more variables, group mean comparisons were conducted using two-way ANOVA, followed by either Tukey’s or Sidak’s multiple comparison tests. The obtained experimental data were examined utilizing the SPSS Statistics program. Values with the same letter do not show any differences that are significant (