Scrophularia ningpoensis Hemsl., have wide clinical use with various biological effects. A new and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established with taxifolin as the internal standard (IS) in this study and was successfully used to study the pharmacokinetic profiles of four active components from S. ningpoensis Hemsl. in rats after sublingual intravenous administration. After protein precipitation with acetonitrile, the mobile phase (consisting of acetonitrile and 0.1% formic acid) was used to separate the analytes on an Acquity UPLC BEH C18 chromatography column  under gradient elution. The precursor-to-product ion transitions of 623.4 → 161.3 m/z for acteoside, 783.5 → 175.0 m/z for angoroside C, 493.3 → 345.2 m/z for harpagoside and 147.2 → 103.4 m/z for cinnamic acid were monitored by mass spectrometry with negative electrospray ionization in the multiple reaction monitoring (MRM) mode. The concentration range of 10–1,000 ng/ml could be detected by this method with a lower limit of quantification (LLOQ) of 10 ng/ml for each analyte. The intra- and inter-day precision (RSD%) of the method ranged from 2.6 to 9.9% and 2.7–11.5%, respectively. Meanwhile, the accuracy (RE%) was −9.6–10.7% in this developed method. The mean recoveries of four active components from S. ningpoensis Hemsl. were more than 76.7% with negligible matrix effects. The four active components from S. ningpoensis Hemsl. were stable under multiple storage and process conditions. A new, sensitive and simple analytical method had been established and was successfully applied to the pharmacokinetic profiles of four active components from S. ningpoensis Hemsl. in rats after sublingual intravenous administration.Acteoside, angoroside C, harpagoside, and cinnamic acid, which are the main bioactive ingredients of  Scrophularia ningpoensis Hemsl. has been used in traditional Chinese medicine (TCM) for a long time (S. ningpoensis Hemsl. has been documented in the Chinese Pharmacopoeia (2010 edition) for its ability to inhibit the proliferation of cancer cells  provided the reagents, including acteoside, angoroside C, harpagoside, and cinnamic acid (purity > 98%). Taxifolin (purity > 98%), acquired from the National Institute for the Control of Pharmaceutical and Biological ProdBucts , was used as an IS. Acetonitrile, methanol, and formic acid, obtained from Merck Company , were of HPLC grade. The water used in this study was produced by a Milli-Q Reagent water system .The analytes were separated on an ultra-performance liquid chromatography (UPLC) unit  through an Acquity BEH C18 column  and an inline 0.2 μm stainless steel frit filter (Waters Corp.). The mobile phase combining mobile phase A (acetonitrile) and mobile phase B (0.1% formic acid in water) was used under a gradient program: 0–0.4 min (10–10% A), 0.4–0.8 min (10–90% A), 0.8–2.0 min (90–90% A), and 2.0–2.1 min (90–10% A). The post time was set to 1.9 min. A 6 μL injection volume was added into the UPLC system, in which the flow rate, column temperature and autosampler temperature were 0.40 ml/min, 40°, and 10°C, respectively.S. ningpoensis Hemsl. were detected in negative ion monitoring mode by an XEVO TQD triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source. The precursor-to-product ion transitions were m/z 623.4 → 161.3 for acteoside, m/z 783.5 → 175.0 for angoroside C, m/z 493.3 → 345.2 for harpagoside, m/z 147.2 → 103.4 for cinnamic acid and m/z 303.4 → 125.1 for IS. The instrument control and data acquisition were conducted on Masslynx 4.1 software (Waters Corp.).The four active components from S. ningpoensis Hemsl. active components and IS. The calibration standard solutions were diluted with drug-free plasma at the following concentrations: 10, 20, 50, 100, 200, 300, 500, and 1,000 ng/ml. The QC samples were also prepared to 20, 400, and 800 ng/ml in the same way, and IS was 200 ng/ml in acetonitrile. The prepared standard working solutions were stored in a −80°C refrigerator before analysis.Stock solutions (1.00 mg/ml) were diluted with methanol to prepare the standard working solutions of the We prepared 200 µL of IS working solution in acetonitrile (200 ng/ml) and added to a 1.5 ml centrifuge tube after 100 µL of the plasma sample was thawed to room temperature. The tubes were vortex mixed for 1.0 min and spun in a centrifuge at 13,000 × g for 10 min. The supernatant (100 µL) was collected, and 6 µL was injected into the UPLC-MS/MS system for analysis.The plasma samples, QC samples, and calibration standard solutions were run to evaluate their specificity, recovery, matrix effect, accuracy and precision, and stability .By comparing different batches of blank plasma and corresponding spiked plasma chromatograms, the specificity of the method was studied to ensure that endogenous and other substances in the sample could not interfere with the analytes and IS analysis.The calibration curves were drawn by the peak area ratio of the analytes to IS vs. the nominal concentration of the analytes. The LLOQ was defined as the lowest concentration on the calibration curve where a signal-to-noise (S/N) was at least 10. The relative standard deviation (RSD%) and relative error (RE%) were calculated as a measure of precision and accuracy, respectively. The acceptance criteria for accuracy and precision of calibration curve data were 80–120% of the nominal concentrations and ± 20% of the nominal concentration at the LLOQ, respectively.To assess the intra- and inter-day accuracy and precision, three different concentrations of the QC samples  were detected on one day or on three consecutive days. The target RSD% and RE% were within ± 15%.We obtained and used three different processed QC samples to evaluate the extraction recovery and matrix effect of the analytes. We compared the peak areas of samples extracted from plasma (A), postextracted blank plasma spiked samples (B), and the corresponding pure reference standard solutions (C), where A/B was defined as the extraction recovery and B/C was considered as the matrix effect.To evaluate the stability of the analytes, we analyzed the QC samples at three different concentrations (n = 5) under different storage and process conditions .Male Sprague-Dawley rats (180–220 g) obtained from the Laboratory Animal Center of Wenzhou Medical University  were given free access to water and standard rat pellets. After the adaptation period, the rats were prepared for the study. We found a dose of 3.0 mg/kg of each substance in the mixture was suitable for investigating the pharmacokinetic trend in preliminary studies. All experimental procedures and protocols were performed under the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Wenzhou Medical University (wydw2018-0002) before the study began.S. ningpoensis Hemsl.The caged rats were fasted for 12 h before the experiment but were allowed water. Whole blood samples (0.3 ml) collected from the tail vein were placed into the corresponding heparinized polythene tubes at the following time points after sublingual intravenous administration of mixtures containing acteoside, angoroside C, harpagoside, and cinnamic acid (3.0 mg/kg): 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, and 6 h. We gavaged 2 ml of saline solution to the rats 2 h after the start of the experiment to reduce animal suffering. The plasma (100 μL) was separated into a 1.5 ml Eppendorf tube after the whole blood was centrifuged at 4,000 g for 8 min. The plasma concentration vs. time data were imported into DAS 2.0 software  to calculate the pharmacokinetic parameters of four active components from Sample extraction plays a key role in sensitivity and reliability and is an important factor in bioanalysis. Protein precipitation offered time-savings, simplicity, low matrix effects, and high recoveries in this research. We tried several organic solvents, including acetonitrile, methanol, and different ratios of methanol and acetonitrile, but we chose 200 µL acetonitrile as the precipitant due to its good performance.To obtain a better chromatogram behavior and ionization effect, we tried many chromatographic columns and mobile phases with different ratios (with or without ammonium formate or formic acid). We adopted a mobile phase consisting of acetonitrile and 0.1% formic acid. When compared to other columns, such as the Waters Acquity UPLC HSS C18 column , a Waters Acquity UPLC BEH C18 column  with gradient elution provided better peak symmetry, appropriate analysis time, and fewer matrix effects. The whole analysis time was 4.0 min under the selected conditions.The representative chromatograms of the analytes and IS had no obvious interference . The actThe method with an LLOQ of 10 ng/ml was linear over the concentration range of 10–1,000 ng/ml for the analytes. The coefficients of correlation for all the calibration curves were greater than 0.99.The results listed in S. ningpoensis Hemsl. in rats.The analytes were stable according to the FDA acceptance criteria under different storage and processing conditions  . In concS. ningpoensis Hemsl. in rats after sublingual intravenous administration of the mixture (3.0 mg/kg). The noncompartment model was used to calculate the pharmacokinetic profiles of acteoside, angoroside C, harpagoside, and cinnamic acid. The mean plasma concentration-time curves and the main pharmacokinetic parameters of the four active components are presented in We established a UPLC-MS/MS method with taxifolin as an IS and applied it to the pharmacokinetic profile study of four active components  from 1/2), and mean residence time (MRT). Among the four active components from S. ningpoensis Hemsl., harpagoside had the maximum CL, but its t1/2, MRT, and area under the curve (AUC) were minimal. Conversely, the cinnamic acid CL was minimal, but its t1/2 and AUC were the maximum. The acteoside and angoriside C pharmacokinetic parameters fell between the maximum and minimum. The apparent volume of distribution (Vd) can be roughly used to infer the distribution and combination of drugs in the body. Harpagoside had the largest Vd, while acteoside had the smallest Vd. When t1/2 and MRT are normally short, drug metabolism and excretion are relatively fast, and Vd should be relatively small in theory. Since the complete body is a complex system, the existence of these differences needs to be confirmed by further studies.Major TCM constituents are gradually metabolized and excreted after sublingual intravenous administration. The metabolism and excretion of the drug in the body are described by pharmacokinetic parameters such as clearance rate (CL), half-life  in rats after sublingual intravenous administration. This method was validated in a pharmacokinetic study. The sample was processed by acetonitrile for protein precipitation, and separation by chromatography took only 4.0 min. Clarifying the pharmacokinetic characteristics of the main TCM active ingredients is significant for accurately treating clinical diseases. It provides effective experimental basic data for clinical practice by studying the pharmacokinetics of the main bioactive components and identifying the mechanism of action. If the present study can be combined with pharmacodynamic research on the main active TCM ingredients, it can, not only avoid side effects caused by other ingredients in TCM, but can also provide scientific guidance for accurate treatment of clinical diseases with TCM.A simple, fast, and sensitive UPLC-MS/MS method was developed to simultaneously determine four active components from The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.The animal study was reviewed and approved by Wenzhou Medical University.SL: Writing original draft; Conceptualization; Data curation; Formal analysis; revise; LX: Investigation; Methodology; Resources; Supervision; revise; JC: Visualization; Writing original draft; CT: Project administration; Resources; Supervision; Writing review & editing; Validation; revise; R-AX: Project administration; Resources; Software; Supervision; Writing review & editing; Validation; revise.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.


Gyrodactylus konovalovi is an ectoparasite on the Amur minnow (Rhynchocypris lagowskii) that is widely distributed in the cold fresh waters of East Asia. In the present study, the phylogeography and demographic history of G. konovalovi and the distribution of its host in the Qinling Mountains are examined. A total of 79 individual parasites was sequenced for a 528 bp region of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene, and 25 haplotypes were obtained. The substitution rate (dN/dS) was 0.068 and indicated purifying selection. Haplotype diversity (h) and nucleotide diversity (π) varied widely in the Qinling Mountains. Phylogenetic trees based on Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP) methods and network analysis revealed that all haplotypes were consistently well‐supported in three different lineages, indicating a significant geographic distribution pattern. There was a significant positive correlation between genetic differentiation (Fst) and geographic distance. The results of mismatch distribution, neutrality test and Bayesian skyline plot analyses showed that whole populations underwent population contraction during the Pleistocene. Based on the molecular clock calibration, the most common ancestor was estimated to have emerged in the middle Pleistocene. Our study suggests for the first time that a clearly phylogeography of G. konovalovi was shaped by geological events and climate fluctuations, such as orogenesis, drainage capture changes, and vicariance, during the Pleistocene in the Qinling Mountains. Gyrodactylus konovalovi is an ectoparasite on East Asia minnow Rhynchocypris lagowskii that is widely distributed in cold freshwater. In the study, the phylogeographic pattern and demographic history of G. konovalovi along with the distribution of its host in the Qinling Mountains have been examined. PhylogeGyrodactylus von Nordmann, 1832; Paragyrodactylus Gvosdev et Martechov, 1953; Laminiscus Palsson et Beverley‐Burton, 1983) and 60 species of Gyrodactylidae Beneden et Hesse, 1864 were identified from freshwater fishes in China, including Cypriniformes, Gadiformes, Gasterosteiformes, Mugiliformes, Perciformes, Salmoniformes, and Siluriformes , which led to secondary infections and, ultimately, mortality  from a fishpond in central China  hosts the most diverse fauna of Gyrodactylus described on any fish species in the Palearctic region, including 17 species, whereas the Amur minnow R. lagowskii hosts three species of Gyrodactylus worldwide  gene from 79 individuals from eleven populations and its host distribution in the Qinling Mountains in central China, assessed the population genetic differentiation and demographic history and tested how the geological events or climate oscillation during the Pleistocene may have affected the current phylogeographic pattern of this parasite.In this study, we first examined the phylogeography and demographic history of the 22.1https://www.iucnredlist.org). None of the species (fish or parasite) sampled is endangered or protected in China  following the manufacturer's protocol. The ND5 gene sequences of the 79 individuals of G. konovalovi from the eleven populations were amplified by polymerase chain reaction (PCR) amplification using a newly specific primers designed based on the congeneric species Gyrodactylus brachymystacis and Gyrodactylus parvae  and the reverse primer gkND5‐R (5'‐ATTTGTAGATGATAGCAAG‐3') were used to amplify a segment of 528 bp. Each PCR amplification was performed in a total volume of 25 µl, containing 3.0 mM MgCl2, 10 mM Tris‐HCl (pH 8.3), 50 mM KCl, 0.25 mM of each dNTP, 1.25 U rTaq polymerase , 0.4 μM of each primer, 45 ng gDNA, tapped with Milli‐Q water. The following cycling conditions were applied: initial denaturation for 1 min at 93°C followed by 35 cycles of denaturation for 10 s at 92°C, annealing for 1.5 min at 50°C and extension for 2 min at 60°C with a final extension for 6 min at 72°C. All PCR fragments were initially purified with a PCR purification kit , subsequently subjected to electrophoresis in a 1% agarose gel and finally sequenced with PCR forward primer with an ABI Prism® 3,730 automated sequencer .Total genomic DNA of 2.42.4.1n) and synonymous (s) substitution rate (dN/dS) were calculated in MEGA v6.06.A total of 79 ND5 gene sequences were visually inspected and manually edited using BioEdit v7.0.9.0 Hall,  and thenH), haplotype diversity (h), and nucleotide diversity (π) were calculated in DnaSP v5.10.1 , maximum likelihood (ML), and maximum parsimony (MP) methods. For the BI and ML analyses, the TrN + I + G model was the most appropriate model of nucleotide substitution. The congeneric species 2.4.3Fst pairwise comparisons. The molecular variance was partitioned among groups (Fct), among populations within groups (Fsc), and within populations (Fst) in Arlequin v3.5.1.2  of the sample localities were analyzed to test for isolation by distance (IBD)  based on the haplotype sequences showed congruent topologies and were all divided into three lineages  and genetic diversity (π) across the samples were 0.955 ± 0.007 and 0.04984 ± 0.00458, respectively, indicating high level of genetic diversity. The h was highest in the YP population, whereas π was highest in the TTG population. The h and π were lowest in the XYB population, except for three populations , and the relationship between genetic diversity and the population latitude was uncorrelated  and G. truttae (0.1% to 0.5%) and approximating G. teuchis (0.1% to 11%) and G. corydori (0.1% to 6.2%)  against nonsynonymous substitutions in accordance with results from other gyrodactylids based on COI and ND5 genes, such as G. thymalli, G. salaris, G. teuchis, and G. arcuatus  was 0.068, indicating strong purifying selection with genes of relatively low nucleotide diversity  was very high within populations (Table Fst) and geographic distance   and geographic distance fit the IBD model in accordance with other studies  indicated highly significant genetic differentiation among groups assigned to three lineages 75.09%), among populations 13.33%) and within populations (11.57%). The genetic differentiation metric .33% and .09%, amo) Figure , suggest) Figure . Thus, b) Figure  and resu) Figure . The hos4.4G. corydori, G. teuchis, G. arcuatus, and G. gondae (Bueno‐Silva et al., G. thymall from the Glomma River in Norway was estimated as very recent several thousand years during the Holocene after the LGM (Pettersen et al., R. oxycephalus was affected by geological events and Pliocene climate fluctuations (Yu et al., A strict molecular clock was estimated for the haplotypes Figure . The tim4.5D and Fu's Fs values was positive and not significant (Table The neutrality test between Tajima's nt Table . Howevernt Table . The neunt Table . Therefont Table .M. gonialosae, G. arcuatus, G. gondae, and G. corydori (Bueno‐Silva et al., G. konovalovi underwent a sharp contraction from 0.25 Mya to 0.01 Mya during the late Pleistocene glacial epoch, which corresponds to the end of Lushan glaciation in accordance with F. taihangnica in the Qinling Mountains (Huang, G. konovalovi in the Qinling Mountains.Most of demographic analyses indicated that the expansion of lineage A and lineage C began simultaneously, 0.01 Mya after the LMG during the late Pleistocene, which corresponds to the end of Dali glaciation in the Qinling Mountains in accordance with findings in s Huang, . The vals Huang, . A slighs Huang, . Ancient5G. konovalovi in the Qinling Mountains. After the LGM, most lineages have experienced expansions of population size and range. The findings provide evidence that Pleistocene climatic changes profoundly affect this species distribution pattern and demographic history in the Qinling Mountains. Further studies employing extensive sampling across larger ranges and multiple molecular markers might provide more insight into the phylogeography and demographic history of G. konovalovi across the Qinling Mountains and test coevolutionary relationships between G. konovalovi and its host R. lagowskii at the population level and the magnifying glass hypothesis.The results of this study showed that the climate fluctuation and tectonic changes led to formed vicariance and drainage capture during the Pleistocene, which promoted lineages divergence and influenced the phylogeographic structure of None Declared.You P designed the research; Chen T, Chen J, Tang L, Chen X, Yan J, and You P performed the research; Chen T analyzed the data; Chen T and You P wrote the paper.


This study was designed to investigate blood glucose, total protein (TP), calcium (Ca), phosphorus (P), magnesium (Mg), iron (Fe), and triiodothyronine (T3) during late pregnancy and the PPP. Twenty-seven apparently healthy multiparous crossbred dairy cows (Friesian × Kenana) were included in this study. The cows were randomly allocated into three groups: group A (n = 10), cows with late pregnancy, group B (n = 7), cows in the PPP, and group C (n = 10), nonpregnant cows as control. One-way ANOVA was used to analyze the data. The results of this study showed that blood glucose was higher in late pregnancy and the PPP than in nonpregnant cows. The TP was significantly lower in late pregnant cows than during the PPP and in nonpregnant cows. Ca, P, and Mg were not significantly different between periods. Serum Fe and T3 were significantly lower during the PPP than that in late pregnant and nonpregnant cows. The results can provide indications of the nutritional status of dairy cows and a diagnostic tool to avoid the metabolic disorders that may occur during late pregnancy and the PPP.The late pregnancy (3 Cows develop serious metabolic and physiological changes during this critical period . The vasactivity , 3. For activity , 5. Glucactivity , and lowactivity , 8. Manyactivity . Animalsactivity , 11. Bloactivity . Most epactivity . Magnesiactivity . Hypomagactivity , 16. Paractivity ; therefoactivity . The actactivity . Furtheractivity , 21. T3 activity . This stMedicago sativa) and abu 70 (Sorghum bicolor) and concentrates. The cows were allowed to graze daily from 07 : 00 a.m. to 10 : 00 a.m. The Research Board (01/2020 on 01/03/2020), Faculty of Veterinary Medicine, University of Khartoum, approved this study. It was carried out in the University of Khartoum dairy farm during January to April 2020. Twenty-seven healthy multiparous crossbred dairy cows (Friesian × Kenana) were employed. The cows were under the semiclosed system and were milked manually twice a day in the morning (02 : 00 a.m.) and evening (02 : 00 p.m.). The age of cows between 4 and 8 years and their body condition scores (BCSs) vary between 2.5 and 3. The offered diet consists of roughages alfalfa (n = 10), cows with late pregnancy, group B (n = 7), cows in the postpartum period, and group C (n = 10), nonpregnant cows left as control. The blood parameters were assessed as mentioned above.This study was designed to investigate seven blood biochemical metabolites  during late pregnancy and the PPP. Twenty-seven multiparous apparently healthy crossbred dairy cows (Friesian × Kenana) were employed for this study. The cows were divided randomly into three groups: group A , then centrifuged at 2000 rpm for 10 min. Serum samples were separated and transferred to clean plastic vials and immediately frozen at −20°C for subsequent analyses.The assessment of plasma glucose, serum total protein, Ca, P, Mg, and Fe was done by using commercial kits  by using A25-Biosystem-Random Access Analyzer, and it is a fully automated analyzer. The blood glucose level was determined immediately after blood sampling. The serum level of TT3 was determined using ST AIA-PACK TT3  on TOSOH AIA System Analyzers.p < 0.05). SPSS was used for this purpose.Results were expressed as mean ± standard deviation (SD). The ANOVA test (one way) was used to investigate difference among the three animal groups. Significance level was considered at (p < 0.001) between the blood glucose concentration (mg/dl) during late pregnancy, postpartum period, and nonpregnancy. The serum total protein concentration (g/dl) was significantly different (p < 0.001) between these groups. There was no significant difference in serum calcium, phosphorous, and magnesium concentration during late pregnancy, postpartum period, and nonpregnant cows. The serum iron concentration (µg/dl) is significantly different (p < 0.001) between the cows in late pregnancy, PPP, and nonpregnancy. The serum total triiodothyronine concentration (ng/dl) was significantly different (p < 0.001) between these groups.a, b: mean values within the same row with different superscripts are significantly different. The mean difference is significant at the 0.05 level. ∗∗, p < 0.01; ∗∗∗, p < 0.001; NS: not significant.The findings of statistical analysis to our study are shown in n = 27).Blood glucose controls the reproduction as it is the main modulator of blood hormones and metabolites . It has This study revealed that the level of serum total protein  during the late pregnancy (6.42 ± 0.55) was lower than in postpartum (6.93 ± 0.98) or nonpregnancy periods (7.18 ± 0.40) . This ren = 27).Results showed that the serum Ca(mg/dl) of the cows is within normal range (2–10) , 30. ResResults showed that the level of serum P (mg/dl) during late pregnancy (6.44 ± 1.19) is insignificantly higher than the PPP (6.33 ± 1.38) and in nonpregnant cows (6.05 ± 1.57). This finding is consistent with the results of previous studies conducted in crossbred dairy cows , 32, 34.Results indicated that serum level of Mg (mg/dl) is within the normal range (2–2.4) , 30 and µg/dl) is lower during the PPP (124.76 ± 33.50) than in the late pregnancy (158.51 ± 36.50) and nonpregnant cows (142.93 ± 40.07); however, they were within normal range [Results showed that the level of serum Fe  are lower during the PPP (4.27 ± 0.85) than in late pregnancy (4.96 ± 0.64) and nonpregnant cows (5.58 ± 0.97). This result is in partial agreement with previous studies reported that there was a reduction of serum T3 and T4 in postcalving compared with the precalving periods , 39. ThiThis study concluded that the late stage of pregnancy and PPP have influenced blood biochemical metabolites and minerals. It was observed that during these periods, there are dynamic changes in the blood concentration of glucose, TP, Fe, and T3. The current result revealed that lower concentration of these parameters could be used to evaluate the cow's health status.Since the late pregnancy and the PPP are critical for the reproductive efficiency and production in dairy cows, the regular checkup of blood biochemical metabolites and minerals in these periods is very important. Accordingly, supplementation of a balanced ration and minerals are recommended. Moreover, during the late pregnancy, the lactating cows should be dried off from 4 to 6 weeks prior to calving date.


Sepsis-associated encephalopathy (SAE) usually manifests as sleep awakening cycle disturbance, cognitive impairment, delirium, and coma . ConsideThis study was conducted in the general intensive care unit (GICU) and included patients diagnosed with sepsis from November 2019 to January 2020 . PatientU 395.5, p < 0.001) and SAE recovery patients , we found the best critical value of ONSD for the detection of SAE in patients with sepsis was ≥ 5.5 mm, with a sensitivity of 80.4% and specificity of 83.5%  and the serum albumin level . Additionally, we found a correlation between ONSD and the bedside angle (0° 5.5 ± 0.5 mm versus 30° 5.1 ± 0.5 mm), even though only 18 patients were observed. In patients with SAE, the ONSD in deceased patients was slightly wider than that in surviving patients , but there was no statistical difference , and the median ONSD in each patient group was 5.1 (4.75–5.4) mm, 5.9 (5.6–6.25) mm, and 5.35 (5.075–5.5) mm, respectively (Table We found that ONSD may be a new diagnostic tool for SAE. There is not enough evidence to show that patients with wider ONSD values have a higher risk of death, and ONSD cannot be used as an indicator for predicting the prognosis of patients with SAE. There are many influencing factors of ONSD; thus, it is necessary to measure it with a unified standard.ONSD may not be accurate enough, since it is the estimated value of ICP and not the definite value. Moreover, there may be many potential factors affecting the ONSD, which need to be further explored.


Osteoporosis is the most common metabolic bone disease with complicated, multifactorial and heterogenic nature that has no known pathological cause. As the role of Diabetes Mellitus (DM) and thyroid dysfunctions in the prevalence of osteoporosis is not exactly known, therefore this study was designed to evaluate the probable association between osteoporosis with DM and thyroid dysfunctions in Iranian patients.In this cross-sectional study, 300 subjects out of the total number of patients referring to Gorgan bone densitometry centers (3000subjects) were selected via random sampling method in 2009. Individual characteristics, DM, thyroid dysfunctions and densitometry results were collected from densitometry records. Data analysis was carried out by SPSS version 16 software and by using Chi square and T-test. The level of significance in all tests was considered 0.05.The mean of T-score in lumbar and femoral areas of diabetic patients were -0.87 ± 1.08 and -1.94 ± 1.33 and in patient with thyroid dysfunctions was -0.80 ± 1.09 and -1.64 ± 1.24 respectively. The mean of BMD in lumbar and femoral areas of diabetic patients were 0.96 ± 0.19 and 0.75 ± 0.19 and in patient with thyroid dysfunctions were 0.96 ± 0.17 and 0.76 ±0.19 respectively. The mean of BMI in osteoporotic subjects in the lumbar and femoral areas were 25.94 ± 5.62 and 26.95 ± 5.20 respectively. The association between BMI and BMD in the lumbar and femoral areas were statistically significant, but the association between DM and thyroid dysfunctions with BMD and T-score in the femoral and lumbar areas was not statistically significant .The results of current study show that there is no association between DM and thyroid dysfunctions with osteoporosis. Osteoporosis is the most common metabolic bone disorder that is characterized by reduction of bone mass without alteration in the composition of bone, leading to fractures. The most obvious subclinical symptom of this disorder is the fracture in the vertebral and femoral neck bone . Osteopoth century . Four categories were defined for BMI: underweight (BMI < 18.5 kg/m2), normal weight (BMI = 18.5–25 kg/m2), overweight (BMI = 25–30 kg/m2), and obese (BMI > 30 kg/m2). In the analysis BMI was considered as a continuous and categorical variable. Thyroid disorders and DM were considered as binary variables and BMD were regarded as continuous and categorical variable .Among 3000 subjects referring to Gorgan densitometry centers (two centers) in 2009, 300 subjects were selected via random sampling method. The study was approved by ethic committee of Golestan University of Medical Sciences. BMD of the lumbar spine (L2 and L4) and proximal femur  was measured by Dual Energy X-Ray Absorptiometry (DXA) (Norland XR-46). According to the WHO criteria by using T-Score of lumbar and femoral BMD, osteoporosis (T-score less than -2.5 of standard deviation in DXA densitometry) and osteopenia (T-Score between -1 to -2.5 of standard deviation in densitometry) were defined . The demData were analyzed using independent T-test, ANOVA and Tukey Post hoc test, Chi square test and univariate and multiple linear regression. Analysis was done with SPSS version 16 software. A P-value less than 0.05 were considered as statistically significant.2 respectively. The mean BMD in the lumbar and femoral areas was 0.92 ± 0.19 g/cm² and 0.77 ± 0.16 g/cm² respectively. The mean BMI in osteoporotic, osteopenic, and normal subjects in the lumbar area was 25.94 ± 5.62 kg/m2, 27.71 ± 4.93 kg/m2 and 29.38 ± 5.49 kg/m2 respectively which was statistically significant . Post hoc test showed that this difference was statistically significant only between normal and osteoporotic cases . The association between BMI categories and BMD in lumbar and femoral areas was statistically significant   were women, 38 cases 12.7%) were diabetic, and 28 cases (9.3%) suffered from thyroid dysfunctions. The mean age and BMI of the participants was 52.7 ± 14.42 years and 28.14 ± 5.42 kg/m2.7% wereMean of T-score and BMD in participants according to DM and thyroid dysfunctions is presented in Table Among 38 diabetic subjects, 17 subjects (5.7%) were osteopenic, and 5 subjects (1.7%) were osteoporotic in the lumbar area. In the femoral area, 19 subjects (6.3%) were osteopenic and 14 subjects (4.7%) were osteoporotic which was not statistically significant in both areas (P > 0.05) (Table The association between thyroid dysfunctions and BMD in the femoral and lumbar areas is presented in Table The association between BMI, DM, and thyroid dysfunctions with BMD in linear regression model has been presented in Table In the present study, in lumbar and femoral areas, the mean BMI of osteoporotic subjects is lower than normal and osteopenic subjects and also underweight subject are more prone to osteoporosis. These findings are consistent with Amiri et al. study which showed that BMI is associated with osteoporosis and osteopenia .In the present study, no association was found between DM and BMD, which is consistent with Pajouhi et al. . In contThe results of BMD in type I and II DM subjects is different or even contradictory –11; so tThe results of current study show that underweight subjects are more prone to osteoporosis, but no association was found between DM and thyroid dysfunctions with osteoporosis.


TCRP4.0 analysis demonstrated a statistically significant reduction of astigmatism with superior incisions (P < 0.001), and the combined mean polar values for SIA changed significantly in the temporal (Hotelling T2 = 1.977), superotemporal (Hotelling T2 = 0.544), superonsal (Hotelling T2 = 1.066), and superior incision groups (Hotelling T2 = 1.134) . The posterior axis alignment should be considered in cataract surgery with CCI, and the SIA is affected by axis rotation, and incision orientation.Clear corneal incision (CCI) is a commonly used surgical approach in cataract surgery. In this prospective study, we evaluated the effect of CCI site on surgically induced astigmatism (SIA) and other postoperative astigmatic changes. CCIs were constructed based on the steep meridian of the total corneal refractive power in the 4.0-mm-zone (TCRP However, corneal surgically-induced astigmatism (SIA) still occurs, and studies have reported that the severity of SIA depends on the length, width, and site of the incision6.For an increasing number of younger patients diagnosed with cataract or presbyopia, refractive surgery is becoming an option to correct refractive errors. It not only improves visual acuity, but also vision quality. Since 1990, sutureless clear corneal incisions (CCI) have become the standard option in cataract surgery to minimize postoperative astigmatism and promote rapid visual rehabilitation10. The Pentacam is a diagnostic tool that uses a slit illumination system and works according to the Scheimpflug principle; it can directly measure both anterior and posterior corneal curvatures11.Today, precise estimation of corneal power and astigmatism is critical for anterior segment refractive surgery. Until now, corneal power and astigmatism have been measured using only the anterior corneal curvature, which is measured by methods such as manual keratometry, auto-keratometry, and simulated keratometry (Sim-K). All of these neglect the contribution of posterior corneal astigmatism. Therefore, only a few studies have described posterior corneal shape changes after cataract surgeryet al.12 demonstrated that the parameters for differentiating keratoconus from healthy eyes included pachymetry, corneal curvature, and anterior and posterior corneal axis alignments. To date, the most accurate method for determining corneal astigmatism axis alignment remains unclear. However, posterior axis alignment is most likely vertical. Mean posterior corneal astigmatism values ranging from −0.26 to −0.78 D have been reported16. Despite numerous studies, there is still significant confusion regarding the best method to evaluate SIA of the cornea19. Abulafia et al. recently provided a new definition for SIA of the cornea (SIACornea)20.Shajari 4.0) steep axis after cataract surgery.The aim of the current prospective study was to determine the effects of the site of CCI on the total corneal refractive power (TCRP) at 4.0-mm zone (TCRPCornea differed significantly with age (P = 0.001). There were no statistically significant differences in any preoperative demographic data between the four groups   . In univariate analysis the AKP(+0) decreased significantly in all incision groups, and AKP(+45)—which is a representation of torsional force twisting in the astigmatic direction—was not significant after 2 months , and significant reduction of astigmatism in Sim-KAnt. on the site of superotemporal, superonasal, and superior directions. There were no statistically significant differences in Kmean between preoperative and postoperative in TCRP4.0, TCRP3.0, and Sim-KAnt. , with R-square (R2) = 0.95, adjusted R2 = 0.95, and standard error = 0.34 D. For steepest keratometry (K2), 97.6% of the cases were within the 95% confidence intervals of different values of K2, with R2 = 0.93, adjusted R2 = 0.93, and standard error = 0.44 D. For the average of keratometry (Kmean), 95.3% of the cases were within the 95% confidence intervals of different values of Kmean, with R2 = 0.96, adjusted R2 = 0.96 and standard error = 0.30 D. For the angle of astigmatism, 91.5% of the cases were within the 95% confidence intervals of different values, with R2 = 0.05, adjusted R2 = 0.05, and standard error = 50.6 degrees  in the temporal group, 39 of 49 eyes (79.6%) in the superotemporal group, 46 of 61 eyes (75.4%) in the superonasal group, and 40 of 53 eyes (75.5%) in the superior group  is widely used in optometry and ophthalmology5. The Pentacam biometric device provides precise values for corneal power and topometric indices, both in normal and in keratoconic eyes28. In the current study, Scheimpflug tomography was used to analyze anterior and posterior curvatures based on a ray tracing technique combined with a rotating Scheimpflug camera.Keratometry has traditionally been the favored method for evaluating astigmatic changes; however, it cannot be used to evaluate the peripheral cornea or irregularities of the central cornea11. In our study, patients in the 60–69 years age group were more likely to undergo superior incision, while patients in the 70–79 years age group were more likely to undergo temporal incision. In principle, when the steep meridian on the anterior surface is aligned vertically, it creates with-the-rule (WTR) astigmatism; in contrast, vertical alignment on the posterior surface creates against-the-rule (ATR) astigmatism, which has negative power and partially compensates for anterior astigmatism.According to a previous study, the percentage of eyes with vertical alignment of the steep meridian decreased with increasing age (from 64% in patients aged 60–69 years to 48.1% in those aged 70–79 years), and the percentage of patients with horizontal alignment increased from 17.5% to 31.8% for the anterior corneal surfaces; however, almost cases of eyes in vertical alignment on the posterior corneal surfaceet al.4 reported that steep meridian incision in CCI surgery was associated with significantly reduced keratometric astigmatism at the temporal, superotemporal, and superior locations. Astigmatic polar value of net astigmatism (AKP)19 analysis was used in that study; the changes in AKP(+0) were −0.26 ± 0.53 D in a temporal group, −0.39 ± 0.45 D in a superotemporal group, and −0.46 ± 0.63 D in a superior group. Moreover, there were significant decreases in astigmatism values in AKP(+0), but no significant changes in AKP(+45). In the analysis of the effects of CCI in each steep meridian axis group in the current study, superior incisions were more effective than the others (P < 0.001). In the present study, the changes in AKP(+0) were −0.96 ± 0.76 D in the temporal group, −1.06 ± 1.79 D in the superotemporal group, −0.73 ± 0.96 D in the superonasal group, and −0.87 ± 1.05 D in the superior group, and all of these changes were statistically significant (TCRP4.0). Moreover, the changes of TCRP3.0 in AKP(+0) were −1.37 ± 0.79 D, −1.02 ± 1.01 D, −1.16 ± 0.87 D, and −1.20 ± 1.10 D, respectively; the changes of Sim-KAnt. in AKP(+0) were −0.80 ± 0.61 D, −0.90 ± 1.05 D, −0.76 ± 0.79 D, and −1.11 ± 1.08 D, respectively. Notably, however, torsional force twisting in the astigmatic direction, represented by AKP(+45) values was not significant after 2 months. These results were similar to the results of previously reported studies involving similar groups4, but the changes in SIACornea AKP(+0) polar values were greater in our study. Moreover, analysis the reduction of astigmatism, there were statistically significant in Sim-KAnt. on the incision site of superotemporal, superonasal, and superior directions, and significant reduction of astigmatism in TCRP4.0 was investigated on the incision site of superior direction .In 2012, Rho 29 was used to investigate the effect of each incision site on SIACornea. Scatterplots of the astigmatic correction, SIACornea/Predicted SIACornea, depicted a greater bias toward unity and zero in each group. The values were widely and more evenly distributed, suggesting a lower potential for intended induced astigmatism. With regard to corneal anatomy, the distance from the cornea to the limbus is shorter in the horizontal direction than the vertical direction30. Moreover, as the strength of corneal fibers is much stronger for short distances from the central cornea, it is reasonable to suggest that there are changes in the morphology of the cornea and astigmatism after surgery33. Hence, the SIACornea is different depending on the location of CCI. In current study, we investigated the mean and standard deviation values of SIACornea in each location of CCI were 0.27 ± 0.19 D, 0.34 ± 0.35 D, 0.38 ± 0.35 D, and 0.40 ± 0.37 D, respectively (P = 0.310). Slade et al.34 reported that the image-guided surgical planning system was effective for accurate correction of preexisting astigmatism in cataract surgery, especially when combined with femtosecond laser-assisted cataract surgery. Byun et al.35 studied similar groups and suggested that anterior keratometry does not accurately reflect total corneal astigmatism. They also reported that the correction index was higher with ATR astigmatism than with WTR astigmatism. Accordingly, the refractive cylinder was higher than the keratometric astigmatism in the ATR group and the oblique group, and lower in eyes with WTR astigmatism. However, the similar correction index was investigated in each group in the present study, and superior outcomes were found in the ATR and WTR groups.In the present study, the Alpins methodet al.36 reported that SIA was influenced by the size and location of the incision, and Denoyer et al.37 proposed that corneal biomechanical properties should be taken into account when making the incision in refractive surgery. In the current study, SIACornea was influenced by the steep meridian axis location, irrespective of the incision width. Furthermore, there was a statistically significant difference between preoperative and postoperative Kmean (TCRP4.0) values (a difference of 0.16 ± 0.30 D), but there were no statistically significant differences in TCRP3.0 and Sim-KAnt. .Alió et al.38, the flattening effect at the incision meridian should be used for toric IOL calculation. In a previous study, Ferreira et al.39 compared flattening effects after CCI in a femtosecond laser group and a manual group, which involves making the side port at 90 to 110 degrees from the main incision. They reported flattening effects of −0.11 D in the femtosecond laser group and −0.13 D in the manual group, and additionally, with regard to superior oblique incisions, the values were −0.21 D in the femtosecond laser group and −0.34 D in the manual group. Interestingly, in the present study, the flattening effects were −0.04 ± 0.26 D in the temporal group, −0.06 ± 0.27 D in the superotemporal group, 0.03 ± 0.38 D in the superonasal group, and −0.03 ± 0.33 D in the superior group.As recommended by Alpins 8 recently reported the effects of CCI on the TCRP3.0 steep meridian axis. The procedure they utilized involved the use of a 1-mm side port, 70 degrees to the left of the main incision, including angles of incisions that were 40 degrees larger than those in the groups in the current study, at each location. According to their results, total corneal astigmatism (TCA – 3 mm apex/zone data from the Scheimpflug) was associated with significant changes in AKP(+0) and AKP(+45) 2 months postoperatively. The AKP(+0) means and standard deviations were −0.22 ± 0.20 D in the WTR microcoaxial group and −0.20 ± 0.21 D in the ATR microcoaxial group, and the respective AKP(+45) values were 0.12 ± 0.10 D and 0.11 ± 0.10 D. However, there were no statistically significant changes in keratometric astigmatism. Nevertheless, their study indicated that steep meridian incisions may have a significant torsional effect and that TCA is an effective tool for analysis of corneal astigmatic changes after cataract surgery. However, in our study, the postoperative AKP(+0) of Sim-KAnt. were −0.04 ± 0.47 D, 0.04 ± 0.59 D, 0.05 ± 0.63 D, and −0.01 ± 0.95 D, respectively, and it was much lower than the results of the previous study.Park and KimCornea37. Dry eye syndrome remains one of the postoperative symptoms experienced by some patients42, and is associated with ocular soreness, pain, and a burning sensation, among other things. In an analysis reported by Kasetsuwan et al.40, only 9.8% of eyes had dry eye symptoms after 3 months following cataract surgery; in the present study, patients with severe dry eyes were excluded. In addition, only Pentacam measurements labeled with the quality specification “OK” were accepted as valid in the current study. These factors could not affect the postoperative evaluation of SIACornea. In the context of study limitations, notably optimized corneal incision sites would improve ocular aberration results27. Further studies are required for comparative evaluation of their effects on SIACornea and corneal higher-order aberrations (HOAs).The present study has several limitations. It has been reported that incision width and corneal hysteresis influence SIA4.0 steep meridian is effective in reducing SIACornea changes, and has a slight flattening effect after surgery. The VERION Image-Guided System is effective in guiding the surgeon to achieve accurate CCI during cataract surgery. The current study is potentially meaningful in the context of clinical research because all phacoemulsification procedures were performed by one experienced surgeon. The above-described surgical option may be used more frequently in cataract surgery.In conclusion, unsutured CCI on the TCRPThis prospective study included 212 eyes from 200 patients who underwent phacoemulsification. A single, experienced surgeon (C. K. J.) performed all the procedures at the Seoul St. Mary’s Hospital Eye Center between January 2016 and August 2017. Informed consent was obtained from all of the patients before the commencement of the study. The protocol adhered to the tenets of the Declaration of Helsinki, and St. Mary’s Hospital’s institutional ethics committee approved the study.43. TCRP, Sim-K and its steep meridian were measured using the Scheimpflug analysis system, Pentacam HR . To ensure sufficient quality, only measurements assigned the quality specification “OK” were accepted as valid. The site of the CCI just in the quadrant of the steep meridian was determined according to patients’ TCRP4.0 steep meridian axes.All patients underwent a routine comprehensive ophthalmic examination preoperatively. Refractive power and corneal curvature were measured using an auto-keratometer . Ophthalmoscopy was performed to evaluate the fundus under a dilated pupil. Cataract nuclear density was graded according to the Lens Opacities Classification System III2, and no viral or bacterial infection. Exclusion criteria were a history of previous ocular surgery, surgical complications, or any corneal or macular pathology, and poor fixation. Cases involving severe dry eye were also excluded.The inclusion criteria were a diagnosis of cataract with regular corneal astigmatism ranging from 0.50–3.50 D (as determined via an auto-keratometer), a postoperative follow-up period of at least 2 months, a corneal endothelial cell count >2000 per mmCornea. Patients were divided into four groups based on the incision site: temporal, superotemporal, superonasal , and superior4.All phacoemulsification procedures were performed under topical anesthesia (proparacaine 0.5%). The VERION Image-Guided System  was used to determine the steep meridian axis orientation and digital markers for the microscopy Fig. . A 2.2-mCornea19 is the same as that used for calculating prediction error by Holladay et al.44. In general, the goal of refractive surgery is to neutralize the refractive error such that the final postoperative refraction is plano. When this is achieved, the Predicted SIACornea and the actual SIACornea are equal. When the actual SIACornea does not match the Predicted SIACornea, a postoperative refractive error is induced. In cataract surgery with CCI, the intended outcome is postoperative astigmatism of 0.50 D. The differences between preoperative and postoperative polar values were calculated and compared. For example, the preoperative and postoperative astigmatic polar values (AKP) for the preoperative net cylinder A@a and the postoperative net cylinder B@b were defined as follows19:The formula for calculating SIA2 correlation coefficient, and correlation coefficients  were used to assess the associations between pairs of variables. Hotelling trace test was used for comparison of intraindividual changes, and univariate analysis was used to examine changes in respective polar values in each group. P values < 0.05 were considered statistically significant.Descriptive and theoretical analyses were performed using SPSS for Windows, version 18.0 . Data normality was confirmed using the Shapiro-Wilk test. The Wilcoxon Rank-Sum test was used for nonparametric analyses. Comparative evaluation of preoperative and 2-month postoperative keratometric parameters was performed. The Kruskal-Wallis H test was used for multiple comparisons among the four groups. Scatterplots, the R


The highest values of antioxidant activity were found in the non-encapsulated beet powder, followed by the powder encapsulated with maltodextrin and, to a lesser extent, the powder encapsulated with inulin. The glass transition temperature was 61.63 °C for M and 27.59 °C for I. However, for B it was less than 18.34 °C, which makes handling difficult. Encapsulation of beet extract with maltodextrin and inulin by lyophilization turned out to be an efficient method to increase solubility and diminish hygroscopicity.Betalains are powerful antioxidants contained in beets. These are divided into betacyanins (red-violet) and betaxanthins (yellow-orange), and they can be used as natural colorants in the food industry. The effects of freeze-drying pure beet juice (B) and the encapsulation of beet juice with a dextrose equivalent (DE) 10 maltodextrin (M) and agave inulin (I) as carrier agents were evaluated. The powders showed significant differences ( Beta vulgaris rubra) is a member of the Chenopodiaceae family [The beet  [Betalains have low stability to various factors, such as high temperatures , alkaling) . Due to g) . Among tg) ,54. Howep < 0.05) in all variables analyzed: water absorption index (WAI), water solubility index (WSI), glass transition temperature (Tg), total betalains (TB), betacyanins (BC), betaxanthins (BX), total polyphenols (TP), antioxidant activity (AA) ) (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH)) and total protein concentration (TPC). The powders  presenteOpuntia stricta), using a combination of maltodextrin and gelatin (range yields between 7.76–14.85 g/100 g). Meanwhile, Obón et al. (2009) [The yield of the powders was found in a range from 35,772 to 65,329 g/100 g, with the highest percentage presented in the powder encapsulated with dextrose equivalent (DE) 10 maltodextrin (M) , while the powder encapsulated with agave inulin (I) indicated 61,678 g/100 g. The non-encapsulated powder (B) had a yield of 35,772 g/100 g, which was the lowest yield. The yield values found in this study are greater than the values found by Castro-Muñoz Saenz et al., 2009  in the e. (2009)  reported. (2009)  mentione. (2009) ,48, and . (2009) .In the WAI, percentages of 44.01 ± 1.000, 27.09 ± 6.096 and 30.68 ± 3.427 were obtained for B, M and I, respectively. No significant differences were found for I and M values. Higher WAI observed on B may be due to the fact that the hydrophilic functional groups of polyphenols, betalains, betacyanins and betaxanthins are free and can form hydrogen bonds with water molecules. Betacyanins pigments quickly absorb humidity from the environment due its hydrophilic groups and the encapsulation with maltodextrin protects the pigment by allowing the drying process and also helps to reduce hygroscopicity by increasing their stability . Moreovep < 0.05) compared to B and I. Water holding capacity is usually defined as the amount of water retained or absorbed by a specific compound after acquiring the state of equilibrium. High water holding capacity is directly related with the presence of hydrophilic regions in the chemical structure [The WSI values were 8.95 ± 0.023%, 9.05 ± 0.016% and 9.37 ± 0.014% for B, I and M, respectively. This indicates that when exposed to water, the powder encapsulated with M presents greater solubility  between the powders, with values of 18.34 °C for B, 27.59 °C for I, and 61.63 °C for M. This property is important since it is related to the stability of the product [g values indicate a high hygroscopicity of the powder [g values obtained in this investigation were lower than those reported in other studies, in which the Tg was in the range of 32.27 to 387.10 °C [g, largely confirming the stability of betalains [g values between 98.96 and 105.86 °C when using the spray drying method [g of 90 °C [g in the range of 32.27 to 38.11 °C [g of 54.7 °C was reported [The T product . Low Tg e powder , with B e powder . In gene87.10 °C ,47,66,67etalains ,47,66,67® by spray drying [The average betalain content of beets is approximately 130 mg/100 g ,69; howey drying . Also, vy drying . The higy drying ,71. Variy drying . The encThe content of TP  was founp < 0.05) between the powders using both techniques. The highest AA was for the beet powder without carrier agent (B) with an ABTS value of 0.90 ± 0.001 mM TE/100 g and 0.44 ± 0.001 mM TE/100 g using DPPH. The beet extract encapsulated with maltodextrin (M) was ranked second with values of 0.74 ± 0.001 mM TE/100 g in the ABTS technique and using DPPH with 0.29 ± 0.001 mM TE/100 g, while the extract encapsulated with inulin (I) presented the lowest antioxidant activity with a value of 0.64 ± 0.001 mM TE/100 g and 0.20 ± 0.001 mM TE/100 g, with ABTS and DPPH, respectively. The values obtained in this research are higher than those previously reported in yellow pitaya powders encapsulated with maltodextrin by spray drying (0.07 mM TE/100 g for ABTS and 0.09 mM TE/100 g using DPPH) [The AA of the beet powders  was maintained in a range of 0.65 to 0.91 mM TE/100 g using the ABTS methodology and DPPH from 0.21 ± 0.001 to 0.45 ± 0.001 mM TE/100 g, with significant differences (ng DPPH) . In pricng DPPH) .p < 0.05) were found in the TPC. The values were 5.974 ± 0.001 for beet extract without encapsulation (B), 3.524 ± 0.001 for the beet extract encapsulated with maltodextrin (M), and 3.655 ± 0.001 for the beet extract encapsulated with inulin (I).Significant differences (p < 0.0002). This may be because polyphenols are powerful antioxidants [p < 0.0003). This could be due to the presence of bethalamic acid since it acts as an antioxidant agent. Similar results have been reported in red prickly pear extract [p < 0.0001). This could be attributed to the fact that sugars and proteins generate products with high antioxidant activity [p < 0.0001). This could be because betalains are the main polyphenols present in beets [g , which may be due to the powders beginning to hydrate at relatively low temperatures [Among the most important correlations, a positive correlation was found between AA and TP  and agave inulin, both from Sigma-AldrichThe 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Trolox, ammonium salt, potassium persulfate, Folin-Ciocalteu, sodium carbonate, gallic acid, trichloroacetic acid, citrate, and phosphate standard reagent were purchased from Sigma-Aldrich . High-performance liquid chromatography (HPLC)-grade methanol and HPLC-grade water were purchased from J. T. Baker . Deionized water was obtained using a deionizer . All other reagents used were analytical grade.The beet juice was extracted with a domestic extractor . The encapsulating agents  were added following the technique used by Antigo et al., 2017  in an am−1), W2 = weight (g) of the collected product, and W1 = weight (g) of mass in the feed.The lyophilization yield was calculated using the technique described by Castro-Muñoz et al., 2015 , by detev/v). The dispersions were homogenized for 15 s (Vortex-Ultra-Turrax IKA T18 basic) with a dispersion tool . The mixture was then centrifuged at 3000× g for 10 min . Supernatants were filtered using a 0.42 µm pore polyethylene filter  and stored frozen (−20 ± 3 °C) until analysis.The extracts used to determine the content of total polyphenols and antioxidant activity were obtained according to Pitalua et al., 2010  with somg for 10 min . The supernatant was decanted into a capsule at a constant weight; the new weight was recorded (capsule + supernatant). Then it was dried at 85 °C for 24 h and subsequently weighed again . The WAI was calculated as the weight of the sediment obtained after removal of the supernatant per unit weight of the original solids on a dry basis. And WSI was the percentage of dry matter recovered after evaporation of the supernatant obtained from WAI. Each determination was carried out in triplicate, and an average value was obtained for each sample.The WAI and WSI were determined according to a method previously reported by Anderson et al., 1970  with somg was determined following the method described by Ahmed et al., 2010 [g generated in the fourth heating cycle. Each determination was made in duplicate, and an average value was obtained for each sample.The Tl., 2010  by diffeg  for 10 min at 25 °C. The supernatant was filtered through a 0.45 µm pore polyethylene filter . Samples were kept at −20 °C until analysis.The betalains extraction was carried out according to Güneşer, 2016 . AliquotThe photometric quantification of total betalains was determined following the method described by Ruíz-Gutiérrez et al., 2014 . The aquThe quantification of BC and BX were calculated separately, and these two results were added to determine the BT content. These measurements were carried out in triplicate, and the results obtained were expressed in mg/100 g of powder.The standard curve for TP was performed according to what was reported by Xu and Chang, 2007 . For theThe TP was determined following the Folin-Ciocalteu spectrophotometric method reported by Singleton and Rossi, 1965  with som2 = 0.99). The dilutions used were 1.20, 1.00, 0.80, 0.60, 0.40, 0.20, 0.10, 0.08, 0.05, and 0.03 mM.The standard curve for ABTS and DPPH was performed according to what was reported by Thaipong et al., 2006 . A stockAA by ABTS methodologyIt was carried out as established by Thaipong et al., 2006 . A solutAA by DPPH methodologyThe antioxidant capacity was determined by the DPPH  method established by Thaipong et al., 2006  with sliThe calibration curve corresponding to the TPC was performed according to the method described by Bradford 1976 . Seven sThe TPC was determined according to the method described by Bradford, 1976 . The proPowders characterization results (measured only once in time) were analyzed through an analysis of variance (ANOVA) procedure through the following modelg, which indicates good stability.The highest values for all the variables were found in the unencapsulated beet powder (B); however, as this has a high hygroscopicity, its handling is difficult. The advantages of using maltodextrin and inulin as encapsulating agents are the high solubility, mild taste, and low hygroscopicity of the obtained powders. The powder encapsulated with maltodextrin (M) presented higher values of the bioactivities analyzed, and also, the powder had a higher TDespite the fact that the total betalain values in the encapsulated powders were lower than those reported in other studies, the encapsulation process by lyophilization turned out to be efficient, suggesting that encapsulated beet powders are promising natural pigments that can be used in a variety of products. In addition, due to its antioxidant activity, these beet powders could have beneficial effects on consumer health.


Currently, the data on the relationship between obesity and gastroesophageal reflux disease (GERD) in Asian populations are scarce.The aim of this study is to investigate the prevalence of reflux esophagitis (RE) among obese Japanese patients in each body mass index (BMI) range group. In addition, we aim to investigate the risk factors for RE in obese Japanese patients. The present retrospective cohort study included 674 obese Japanese patients who underwent bariatric surgery between January 2003 and April 2016. The patients were stratified into five groups based on BMI range.2. The prevalence of RE among each of the groups was as follows: Group 1 (BMI 30–34.9) = 20.7%; Group 2 (BMI 35–39.9) = 24.0%; Group 3 (BMI 40–44.9) = 25.2%; Group 4 (BMI 45–49.9) = 26.7%; and Group 5 (BMI ≥50) = 24.8%. Overall, the prevalence of RE was 24.2% in our study. Furthermore, no significant difference in BMI was noted between the RE and non‐RE groups . According to the multivariate logistic regression model, gender, Helicobacter pylori infection status, GERD‐related symptoms, and hiatal hernia were significantly correlated with RE.The mean BMI was 42.7 ± 9.24 kg/mOur study shows that the prevalence of RE in severely obese Japanese patients was significantly higher than the average prevalence of RE in Japan. However, the prevalence of RE did not increase with BMI in our cohort. Our study is the first to report the prevalence of RE in severely obese Japanese patients. Although it has certain limitations, it can still provide valuable reference data in epidemiology. Gastroesophageal reflux disease (GERD) is a disease characterized by a series of troublesome symptoms with complications that arise from the reflux of gastric contents into the esophagus.2, respectively. Consequently, it appears that a higher BMI is proportional to the occurrence of RE. Currently, there is insufficient data regarding the relationship between obesity and GERD or RE in other Asian countries. In particular, such a relationship in severely obese Japanese patients has not been reported despite the growing severely obese population in Japan.Obesity is a known risk factor for GERD or reflux esophagitis (RE). In earlier studies, several pathophysiological mechanisms linking the two conditions were reported.Here, we aim to investigate the prevalence of RE in obese Japanese patients who have undergone bariatric surgery. To the best of our knowledge, this is the first study on this topic.2 with obesity‐related comorbidities), which was approved by the Institutional Review Board. Each patient was preoperatively screened and evaluated by our multidisciplinary team.Helicobacter pylori (H. pylori) infection, GERD‐related symptoms, Barrett's esophagus (BE), and hiatal hernia. Abdominal computed tomography scans were routinely performed to assess visceral/subcutaneous fat measured on one cross‐sectional scan obtained at the level of the umbilicus.This retrospective cohort study included 674 consecutive obese Japanese patients  who underwent bariatric surgery between January 2003 and April 2016 at Yotsuya Medical Cube, Tokyo, Japan. All patients provided written informed consent. The inclusion criteria for laparoscopic bariatric surgery were based on the Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) Guidelines for Clinical Application of Laparoscopic Bariatric Surgery . The severity of RE observed on gastroscopy was graded from A to D and was based on the Los Angeles classification system.Z‐line and proximal margins of the gastric mucosal folds, which were observed with considerable air insufflation during inspiration. The distance was measured using the centimeter markings during the upper GI endoscopy.In our cohort, all the patients underwent gastroscopy before bariatric surgery. Regarding the status of 2), Group 2 (35–39.9 kg/m2), Group 3 (BMI 40–44.9 kg/m2), Group 4 (BMI 45–49.9 kg/m2), and Group 5 (≥50 kg/m2). Then, the prevalence of RE in each group was determined. The associations between the visceral fat ratio and BMI were analyzed using Spearman's correlation coefficient. In addition, it was the researcher's aim to investigate the risk factors for RE that were associated with obese Japanese patients.The patients were divided into five groups according to their BMI: Group 1  value <0.05 was considered statistically significant. A correlation analysis was also performed using Spearman's correlation coefficient. The study by Guilford (1956) was used to interpret and compare the correlation coefficients. The correlation coefficients he described are discussed below. Here, <0.20: “slight almost negligible relationships”; 0.20–0.40: “low correlation”; 0.40–0.70: “moderate correlation”; 0.70–0.90: “high correlation, marked relationship”; and > 0.90: “very high correlation, very dependable relationship.”Statistical analysis was performed using JMP® 11 . The results were expressed as the mean ± standard deviation and percentage. Moreover, the grouped data were expressed as the median (range), and nonparametric methods were used. A univariate analysis was performed using the chi‐square test or Fisher's exact test to identify the associations between variables. Furthermore, Cox proportional hazards regression was performed to analyze gender, the status of 2, 117.7 ± 29.4 kg, and 41.2 ± 10.3 years, respectively. On examination, H. pylori infection was found in 29 patients (4.3%). The mean visceral fat ratio was 0.39 ± 0.19. GERD‐related symptoms were also noted in 41% of patients. Among the 674 patients, Grades A, B, C, and D were present in 114 cases (16.9%), 37 cases (5.5%), 11 cases (1.5%), and 1 case (0.2%), respectively. In all, the prevalence of RE was 24.2% in our study. Approximately 40% of patients who underwent surgery at our institution had hiatal hernia, and 1.6% had BE. Prior to surgery, 8.9% of patients had already taken PPI medication. Other patient characteristics are shown in Table n = 27/130); Group 2, 24.0% (n = 43/179); Group 3, 25.2% (n = 35/139); Group 4, 26.7% (n = 27/101); and Group 5, 24.8% (n = 31/125)   . The proportion of patients who experienced GERD‐related symptoms and hiatal hernia was significantly higher in the RE group compared with the non‐RE group . On the contrary, the proportion of patients with H. pylori infection was significantly lower in the RE group (P < 0.02) . Technically, endoscopy and 24‐h pH monitoring impedance testing are required to properly diagnose NERD. However, pH monitoring and impedance testing are practically difficult to conduct in all severely obese patients who have GERD‐related symptoms but who do not primarily intend to undergo treatment for RE. In addition, 8.9% patients had already undergone PPI therapy before surgery, which may have caused bias.Our study is the first to report the prevalence of RE in severely obese Japanese patients. We show that the prevalence of RE in severely obese Japanese patients was significantly higher than the average prevalence of RE in Japan. However, no significant difference was observed in BMI between the RE and non‐RE groups. Although our study has certain limitations, these findings can still provide valuable reference data in the epidemiology field. In the future, a well‐designed, large‐sample, multicenter study is required to confirm these findings.


Gentiana rigescens Franch, a traditional Chinese medicine. It prolonged the replicative and chronological lifespans of yeast. Autophagy, especially mitophagy, and antioxidative stress were examined to clarify the mechanism of action of this compound. The free green fluorescent protein (GFP) signal from the cleavage of GFP-Atg8 and the colocation signal of MitoTracker Red CMXRos and GFP were increased upon the treatment of GPS. The free GFP in the cytoplasm and free GFP and ubiquitin of mitochondria were significantly increased at the protein levels in the GPS-treated group. GPS increased the expression of an essential autophagy gene, ATG32 gene, but failed to extend the replicative and chronological lifespans of ATG32 yeast mutants. GPS increased the survival rate of yeast under oxidative stress condition; enhanced the activities of catalase, superoxide dismutase, and glutathione peroxidase; and decreased the levels of reactive oxygen species and malondialdehyde. The replicative lifespans of Δsod1, Δsod2, Δuth1, and Δskn7 were not affected by GPS. These results indicated that autophagy, especially mitophagy, and antioxidative stress are involved in the antiaging effect of GPS.Gentiopicroside (GPS), an antiaging secoiridoid glycoside, was isolated from  Aging is a time-dependent functional decline that is the primary risk factor for many diseases , 2, suchGentiana rigescens (G. rigescens) grows in the southwest region of China. The roots of this Chinese herb medicine have been used to treat inflammation, hepatitis, and functional dyspepsia [Sheng Nong's Herbal Classic, a classic book of traditional Chinese medicine on Materia Medica, described that G. rigescens has effects on cognition improvement and antiaging. In our previous study, 11 novel neuritogenic benzoate-type molecules (named gentisides A–K) were isolated from G. rigescens [G. rigescens on the molecular basis, which was mentioned in Sheng Nong's Herbal Classic.yspepsia . Sheng Nigescens , 7. The igescens . These rG. rigescens. Budding yeast Saccharomyces cerevisiae is a common aging model that has replicative and chronological lifespans. The K6001 strain, a yeast mutant strain derived from W303, has a characteristic that only mother cells can produce daughter cells and daughter cells do not continue to mitosis in glucose medium [In the present study, we investigated the antiaging molecular base of e medium , 9. The Caenorhabditis elegans (C. elegans), fruit fly, and mice, but the effects are abolished when the autophagy-related genes are knocked out or knocked down, suggesting that autophagy plays an important role in the life-prolonging effect of rapamycin [C. elegans, depending on the expression levels of the mitophagy genes pink-1, dct-1, and skn-1 and improves the muscle function in old mice with a high expression of mitophagy (Park2) transcripts [Autophagy is a highly conservative process used to degrade large structures, such as long-lived or damaged organelles and protein aggregates . Accordiapamycin , 15. Mitapamycin . Many stnscripts .SOD1, SOD2, SUN family protein Uth1 (UTH1), and SKN7 genes were confirmed to be involved in oxidative stress and played an important role in the antiaging effects [Oxidative stress is a disturbance in the balance between the production of free radicals and antioxidant defences, thereby causing cell aging. Free radicals, known as reactive oxygen species (ROS) and reactive nitrogen species , will be effects .β-L-galactopyranoside from Gastrodia elata, apple branches, Pedicellus melo, fruits of Momordica charantia L and G. elata Blume, under the guidance of the K6001 yeast bioassay system [G. rigescens using the same bioassay system. Here, the antiaging activity and the mechanism of action of GPS were reported.In our previous study, many antiaging substances were isolated from natural products, such as parishin, phloridzin, cucurbitacin B, cucurbitane glycosides, and a new compound named bis(4-hydroxybenzyl)ether mono-y system , 20–23. G. rigescens was purchased from Huqingyutang Chinese Pharmacy Hangzhou, Zhejiang Province. The identity of this plant was confirmed by Associate Professor Liurong Chen , and a voucher specimen (no. 20190620) was preserved in Zhejiang University, Institute of Materia Medica. Preparative high-performance liquid chromatography (HPLC) analysis was performed using a HPLC system equipped with Elite P-230 pumps . An Agilent Technologies 6224A Accurate-Mass time-of-flight liquid chromatography–mass spectrometry (TOF LC/MS) system was used to measure the mass spectra . A Bruker AV III-500 spectrometer was used to record the nuclear magnetic resonance (NMR) spectra . NMR chemical shifts in δ (ppm) were referenced to solvent peaks of δC 49.0 for CD3OD . Octadecyl-silane  and RP-18 plates (0.25 mm)  were used for column chromatography and thin-layer chromatography analysis, respectively [imen no. 0190620 wG. rigescens. Dried and powdered G. rigescens (100 g) was extracted in methanol  at room temperature for 24 h with shaking. The methanol extract was filtrated, and the filtrate was concentrated, suspended in water, and successively partitioned with n-hexane, ethyl acetate, and n-butanol to obtain the active n-butanol fraction (7.5 g). The n-butanol fraction was separated by ODS open column and eluted with MeOH/H2O  to give seven fractions. A part of the third fraction (30 mg) was purified through HPLC (Cosmosil 5C30-UG-5 (10 mm × 250 mm), flow rate: 3 mL/min, 30% aq. MeOH) to yield a pure active substance . The chemical structure of the active molecule was identified to be gentiopicroside (GPS) by comparing HR ESI-MS and 13C NMR data with those reported in literature [13C NMR : δ = 166.3 (C-11), 150.6 (C-3), 135.0 (C-8), 127.0 (C-5), 118.5 (C-10), 117.2 (C-6), 105.0 (C-4), 100.2 (C-1′), 98.5 (C-1), 78.4 (C-5′), 78.0 (C-3′), 74.6 (C-2′), 71.5 (C-4′), 70.9 (C-7), 62.8 (C-6′), and 46.6 (C-9); and high-resolution ESI-TOF-MS m/z 379.099, calcd for C16H20NaO9 (M+Na)+379.100. The chemical structure of GPS is shown in GPS was isolated from Δuth1, Δskn7, Δsod1, Δsod2, and Δatg32 yeast mutants with K6001 background, YOM36, Δatg32 of YOM36, YOM38-Atg8-GFP, and BY4741 yeast strains were used in the present study. K6001 yeast was provided by Professor Breitenbach , and other mutant yeast strains were constructed by Professor Matsuura . The replicative lifespan assay was performed in accordance with the protocol described in our previous study [μM. The well-known antiaging molecule, Res, at a concentration of 10 μM was used as positive control. The agar plates were then incubated at 28°C, and 40 microcolonies formed on agar plate were randomly selected and counted daughters produced by each mother cell under microscopy. The replicative lifespan assay methods of mutants with K6001 background  were the same as that of the K6001 strain. The chronological lifespan assay was conducted as described in our previous report [μM with the initial OD600 value of 0.01 and incubated in a shaking incubator at 180 rpm and 28°C. On the third day, 200 cells from each treat or untreated groups were spread on glucose agar plates and cultured in an incubator at 28°C for 2 days. The colony-forming units (CFUs) of each plate were counted, and the CFUs were denoted as 100% survival at day 3. On the fifth day, the steps of the third day were repeated, and the survival rate of each group was calculated (CFUs of each plate/CFUs of the same plate on the third day × 100%). The steps were repeated every two days until the survival rate decline to less than 10%. The chronological lifespan assay methods of YOM36 mutants (Δatg32 of YOM36) were the same as those of YOM36 strain.The K6001 yeast, us study . Brieflys report . YOM36 yμM or Res at 300 μM and cultured for 22 h in the dark. Subsequently, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) 20 μg/mL) for 10 min in the dark and then washed thrice with PBS. Yeast cells were observed and photographed with a two-photon confocal fluorescence microscope . The experimental procedure for mitophagy was similar to that of autophagy. The difference is that the cells were stained with 250 nM MitoTracker Red CMXRos  at 37°C in the dark for 1 h before staining with DAPI. The percentage of cells with green fluorescence and the colocation of red and green fluorescence were calculated, and the data obtained were analysed on software.The experiment was performed following a previously published literature . BrieflyμM or Res at 300 μM and cultured for 22 h in the dark. Subsequently, yeast cells of different groups were collected and sonicated for 5 min. The cell lysates were centrifuged at 12,000 g for 15 min, and the supernatant was obtained for western blot analysis. The mitochondria were isolated from yeasts to obtain the protein, as described in references [μg protein was separated with SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies followed by secondary antibodies. The primary antibodies used are as follows: anti-GFP , anti-β-actin , antiubiquitin , and anti-VDAC1/Porin (Abcam Trading (Shanghai) Company Ltd., Shanghai, China) antibodies. The secondary antibodies used are as follows: horseradish peroxidase-linked antirabbit and antimouse IgGs . Antigens were visualised using an ECL Western Blot Kit  and digitised with ImageJ software.YOM38 yeast cells containing pR316-GFP-Atg8 plasmid were cultured in liquid glucose medium in a shaking incubator at 180 rpm and 28°C under dark conditions. After 24 h, the cells were collected and washed with SD medium and divided into several different groups with OD600 value of 0.1. The cells were then treated with GPS at concentrations of 0, 1, and 3 ferences , 26. BriμM in glucose medium for 12 h at 28°C with shaking at 180 rpm. Total RNA was extracted using a hot phenol method. A reverse transcription method was utilised to synthesise cDNA using a HiFi-MMLV cDNA Kit  and 5 μg of RNA. Quantitative RT-PCR was performed by using CFX96 Touch  and SYBR Premix EX Taq , as described in our previous study [ATG32, sense 5′-ACC GTC TCA TCC CTT TAA AC-3′ and antisense 5′-CTT CCT CAA AAG CCT CAT CT-3′ and for TUB1, sense 5′-CCA AGG GCT ATT TAC GTG GA-3′ and antisense 5′-GGT GTA ATG GCC TCT TGC AT-3′. The relative gene expression data were analysed using the 2ΔΔCt− method. The levels of ATG32 mRNA were normalised to those of TUB1.The wild-type BY4741 were incubated with GPS at concentrations of 0 and 1 us study . The theμM or Res as positive control at 10 μM for 24 h. Subsequently, the yeast cells from each group were diluted to 1.5 OD600 value. Approximately 5 μL of yeast culture from each group was dropped on glucose agar plates containing H2O2 at a dose of 10.5 mM and incubated at 28°C for 3 days. The growth of yeast was observed and photographed.BY4741 yeast cells in glucose medium with initial OD600 value of 0.1 were treated with GPS at doses of 0, 1, and 3 μM or 10 μM Res at 28°C for 24 h. Then, 200 yeast cells from each treated group were painted on glucose agar plates with or without 5 mM of H2O2. After 2 days, the growth of yeast was observed, and the number of microcolonies in each plate was counted to evaluate the antioxidative activity. The survival rate was calculated as the ratio of the number of microcolonies with H2O2 at 5 mM divided by the number of microcolonies in the absence of H2O2.To quantify the antioxidative activity of GPS, BY4741 yeast cells were cultured with GPS at concentrations of 0, 1 and 3 μM or Res at 10 μM and cultured in a glucose medium for 24 h. Subsequently, the yeast cells from different groups were collected and sonicated for 5 min. The cell lysates were centrifuged at 12,000 g for 15 min, and the supernatant was obtained to test the activity of enzymes. The activities of CAT, GPx, and SOD were determined with CAT and GPx assay kits  and SOD assay kits  in accordance with the manufacturer's instructions. All experimental procedures were conducted in strict accordance with the protocol instructions provided by the manufacturers.BY4741 yeast cells were cultured in liquid glucose medium to reach the logarithmic growth phase. The cells were divided into five groups with OD600 value of 0.1 and treated with GPS at 0, 1, 3, and 10 μM or Res at 10 μM and cultured for 24 h. Subsequently, DCFH-DA  was added to the culture to obtain a final concentration of 40 μM. The cells were then incubated in a shaker under the dark condition at 28°C for 1 h. The cells were then collected and washed with PBS thrice. The DCF fluorescence intensity of 1 × 107 cells were detected with a fluorescence plate reader using 488 nm as excitation and 525 nm as emission wavelengths.ROS and MDA assays were performed by following the same methods as reported in literature . BrieflyμL PBS, and ultrasonicated on ice for 5 min. Subsequently, these cells were centrifuged at 4°C for 10 min at 12,000 g to obtain the supernatant, and the MDA level was measured with a MDA assay kit  in accordance with the manufacturer's instruction.To detect the MDA level in yeast, BY4741 yeast cells were cultured as described in the ROS assay for 24 h. These cells were then washed with PBS thrice, suspended in 250 t-tests with Bonferroni's correction on GraphPad Prism 5 (GraphPad Software Inc.). Survival analysis was used for chronological lifespan assay. A p value of less than 0.05 was considered statistically significant.Experimental data were expressed as mean ± SEM value of two or three independent experiments. Significant differences among the groups in all experiments were analysed through one-way ANOVA, followed by two-tailed multiple μM was evaluated using the K6001 replicative lifespan assay , respectively. The chronological lifespan assay of YOM36 yeast was performed to evaluate the antiaging activity of GPS. GPS significantly increased the survival rate of yeast at concentrations of 1 and 3 μM  (Yeast as an aging model is simple and amenable to genetic and molecular manipulations . It is wan assay  and S1.  < 0.01) . These rμM (p < 0.001 and p < 0.05), respectively. The positive control Res at 300 μM increased the percentage from 21.4 ± 1.6% to 31.7 ± 0.5% (p < 0.001). To confirm the effect of GPS on the regulation of autophagy, western blot analysis was performed to test the generation of free GFP that is released into the vacuole during the autophagy flux. The results of western blot analysis are displayed in μM (p < 0.001). These results implied that GPS can increase the level of autophagy in yeast.Autophagy, particularly mitophagy, is a degenerative process that degrades cellular components, such as the mitochondria, for recycling into amino acids and other metabolites. Autophagy and aging is closely related , 28. The2O2 and superoxide anion (O2·−) in eukaryotic cells [μM (p < 0.001). GPS increased the Atg8-GFP and free GFP protein levels in the mitochondrial fraction of yeast (p < 0.001). The ubiquitin-proteasome system is a major intracellular pathway for the degradation of proteins that govern important life processes of the cell [Mitochondria are the sites of oxidative phosphorylation and ROS production, such as Hic cells . Hence, ells [μM  (p < 0.0the cell . TherefoATG32 gene expression in yeast (ATG32 was increased by GPS at concentrations of 1 and 3 μM (p < 0.01 and p < 0.05), respectively. We examined the effect of GPS on the replicative lifespan of the atg32 mutant with K6001 background and chronological lifespan of the atg32 mutant with YOM36 background. The replicative lifespan of the atg32 mutant with K6001 background is shown in p < 0.001) and 9.95 ± 0.59 (p < 0.001) after treatment of Res at 10 μM and GPS at 1 μM, respectively. The replicative lifespan of the atg32 mutant of K6001 was 7.18 ± 0.53 generations in the control, and the generations were 7.45 ± 0.49 and 6.30 ± 0.37 after treatment with Res at 10 μM and GPS at 1 μM, respectively, suggesting that Res and GPS cannot extend the replicative lifespan of the atg32 mutant of K6001. These results implied that ATG32 was involved in the effect of extending the replicative lifespan of GPS. The chronological lifespan assay was performed using YOM36 wild type and atg32 mutant with YOM36 background yeast. The absence of ATG32 gene shortened the chronological lifespan of yeast , 66.4% ± 1.1 in the GPS at 1 μM treated group (p < 0.01), 66.0% ± 3.2 in the GPS at 3 μM group (p < 0.01) and 58.1% ± 0.9 in the treatment of GPS at 10 μM (p < 0.05), respectively. These results indicated that GPS has antioxidative stress and plays an important role in the antiaging activity of GPS in yeast.Oxidative stress is a major risk factor of aging and age-related diseases, and strong oxidative stress results in the damage of DNA, lipid peroxidation, and dysfunction of protein. Qualitative and quantitative experiments were performed to evaluate the antioxidative activity of GPS, as shown in Figures μM and GPS at 1, 3, and 10 μM to confirm the antioxidative effect of GPS. The enzyme activity (units/mg) of CAT significantly increased from 58.3 ± 3.4 to 72.2 ± 2.8, 72.0 ± 2.0, 68.0 ± 3.4, and 66.7 ± 3.7 , respectively , respectively. The enzyme activity (units/mg) of CuZn-SOD significantly increased from 46.1 ± 4.8 to 56.4 ± 1.0, 59.1 ± 0.7, 58.9 ± 1.2, and 44.8 ± 2.1  significantly increased from 102.4 ± 1.1 to 119.0 ± 3.6, 124.0 ± 5.4, 112.7 ± 4.4, and 107.7 ± 7.2 , respectively , respectively. The level of MDA decreased from 0.34 ± 0.02 to 0.23 ± 0.01, 0.27 ± 0.01, 0.27 ± 0.01, and 0.29 ± 0.01 , respectively. These results suggested that GPS can significantly reduce the ROS level and production of MDA.Increased production of ROS can cause mitochondrial deterioration and global cellular damage, thereby supporting the role of ROS in aging . MDA is SOD1, SOD2, UTH1, and SKN7 are antioxidative or oxidative-related genes. Replicative lifespan of K6001 strain and sod1, sod2, uth1, and skn7 mutants with K6001 background were performed to evaluate the effects of GPS on these genes. The results are demonstrated in Figures p < 0.01) and 9.88 ± 0.60 (p < 0.01) after the treatment of positive control (Res at 10 μM) and GPS at 1 μM, respectively  and 9.95 ± 0.59 (p < 0.01) after treatment of Res at 10 μM and GPS at 1 μM, respectively . The avectively , suggestectively , suggest Figures . The repectively , suggestectively , suggestG. rigescens Franch is a traditional Chinese medicine produced in Yunnan and Guizhou Provinces, China. Our research group has long been engaged in the chemical and biological studies on active natural products and their derivatives from G. rigescens for drug discovery against Alzheimer's disease and antiaging. In our previous studies, many novel molecules were isolated from G. rigescens and hundreds of their derivatives were synthesised. We discovered the lead compound, tetradecyl 2,3-dihydroxybenzoate through a structure–activity relationship study. This compound demonstrated significant neuritogenic effects in cell culture and animal models [G. rigescens, as described in Sheng Nong's Herbal Classic. However, the active molecules on antiaging effect described in Sheng Nong's Herbal Classic are still under discovery. In the present study, GPS, which is a major component in G. rigescens, was isolated as the antiaging molecule under the guidance of the yeast K6001 replicative lifespan bioassay system. A previous literature reported that GPS has antioxidant, hepatoprotect, anti-inflammation, analgesic, and antibacterial effects [l models . These r effects . Howeveratg32 mutant yeast with K6001 or YOM36 background, and RT-PCR. The increase in free GFP and ubiquitin in the mitochondria and gene expression of ATG32 and no changes in the replicative lifespan of the atg32 mutant with K6001 background after treatment of GPS (Figures β-L-galactopyranoside from the Gastrodia elata [Subsequently, two types of classical antiaging mechanisms, namely, autophagy and antioxidative stress, were investigated to understand the antiaging mechanism of GPS. Atg8 is an important component for autophagic machinery, participates in the entire process of autophagy, and is a biomarker of autophagy in yeast . In our  Figures  clarifiesod1, sod2, uth1, and skn7 yeast mutants with a K6001 background to provide many evidence for supporting the conclusion. The replicative lifespan assay results of yeast mutants in SOD1, SOD2, UTH1, and SKN7 were required in the antiaging effect of GPS.We investigated the effects of antioxidative stress on the antiaging effect of GPS. The increase in the survival rate under oxidative stress; enhancement of CAT, SOD, and GPx activities; and the decrease in ROS and MDA levels on yeast  suggesteIn the present study, we found that SD and SC mediums had different effects on the chronological lifespan of YOM36 yeast. The survival rate of yeast in the SC medium was better than that in the SD medium (Figures G. rigescens, a traditional Chinese medicine, has a significant antiaging activity on yeast. GPS prolonged the replicative and chronological lifespans through the regulation of mitophagy and antioxidative stress. GPS is an extremely high-content molecule in G. rigescens and is a representative component of the plant. This condition indicates that large quantities of the compound can be readily obtained for subsequent chemical and biological studies. This study provides an important foundation for future studies on the mechanism of GPS and evaluation of the antiaging effect of GPS in different antiaging animal models for novel drug development.The findings revealed that GPS isolated from