To investigate the prevalence of overweight and obesity, as well as the association between body mass index (BMI) and disease activity in patients with axial spondyloarthritis (axSpA).2), overweight (BMI ≥ 25 to < 30 kg/m2), and obese (≥ 30 kg/m2).Norwegian axSpA patients from the European Map of Axial Spondyloarthritis (EMAS) survey were included in this analysis. Sociodemographic, anthropomorphic, and disease-related variables  were reported. Patients were categorized into under/normal weight (BMI < 25 kg/mp < 0.001. Obese patients had significantly higher disease activity (BASDAI mean (SD) 5.87 ± 1.78 vs 4.99 ± 2.08, p < 0.001), degree of spinal stiffness , and number of comorbidities  than under/normal weight patients. After adjusting for gender and age, obesity proved to be independently associated with disease activity.Of the 509 participants in the EMAS survey, 35% were categorized as under/normal weight, 39% overweight, and 26% obese. Compared to under/normal-weight patients, overweight patients had significantly higher degree of spinal stiffness (mean (SD) 7.91 ± 2.02 vs 7.48 (2.15) and number of comorbidities , both Obesity was associated with higher reported BASDAI score, and being overweight or obese was associated with a higher degree of spinal stiffness and number of comorbidities compared to under/normal weight respondents. The results highlight the serious impact of obesity on health status, and obesity should therefore be considered as a modifiable risk factor for disease activity within the disease management of axSpA.The online version of this article (10.1007/s11926-020-00917-4) contains supplementary material, which is available to authorized users. Axial spondyloarthritis (axSpA) is a chronic inflammatory rheumatic disease that primarily affects the axial skeleton . The axSRecent evidence from population-based studies points toward a combined effect of physical activity and body weight on mortality and health profile . IncreasThe aim of this study was to investigate the prevalence of overweight and obesity and to explore the association between body mass index (BMI) and disease activity, in Norwegian patients with axSpA included in the European Map of Axial Spondyloarthritis Survey ••.The design and survey development of EMAS was recently reported ••. In brThe patient questionnaire included information about sociodemographic variables , anthropomorphic (BMI), comorbidity, living habits , and disease-related variables described below. BMI was classified according to WHO Europe. BASDAI was used to assess patient-reported disease activity. BASDAI includes six questions (Q) addressing fatigue/tiredness (Q1), neck, back or hip pain (Q2), pain/swelling in joints other than the neck (Q3), back or hips, discomfort of any areas tender to touch or pressure (Q4), morning stiffness from time of awakening (Q5), and duration of morning stiffness from time of awakening (up to 120 min) (Q6). Mean values for each of the six questions (Q1–6) and total BASDAI score  were calculated from 0 (no activity) to 10 (maximum activity) .This index, developed specifically for EMAS, assessed the self-reported degree of stiffness experienced by patients in the cervical, dorsal, and lumbar areas of the spinal column. Possible responses range from least to most affected column and total scores are obtained by adding together the responses in each of the areas of the spine, resulting in a scale ranging from 3 (low degree of stiffness) to 12 (high degree of stiffness). This index showed acceptable internal reliability  ••.The respondents’ self-reported comorbidities, including sleep disorders, anxiety, depression, obesity/overweight, hypertension, hypercholesterolemia, fibromyalgia, severe infections requiring antibiotics, psoriatic arthritis, cardiac arrhythmia, spinal or other fractures, cataracts, gout, diabetes, Crohn’s disease, severe infections requiring hospital admission, atherosclerosis, genital lesions, liver disease, and episcleritis.Sample selection inclusion criteria were age ≥ 18 years, resident of Norway, self-reported diagnosis of axSpA , and visit a healthcare professional for axSpA in the 12 months prior to participation.χ2) was used to check independence between two categorical variables by means of a contingency table. The Mann-Whitney U test (2 groups) or Kruskal-Wallis H test for independent samples (> 2 groups) was used to test group differences. The BASDAI scores  for the different BMI categories were visualized in bar graphs. The association between BMI and BASDAI was tested with a Pearson correlation test. The level of statistical significance was set at p < 0.05.Sociodemographic and disease-specific variables are presented as means and standard deviations (SD). BMI was categorized into three groups . BASDAI was categorized into two groups: high (≥ 4) or low (< 4) disease activity. Chi-square test  did not smoke and 50.3% reported never or occasionally drink alcohol (Table n = 180) of the participants were under/normal weight (BMI < 25), 39.1% (n = 199) were overweight (BMI 25–20), and 26.5% (n = 130) were obese (BMI > 30), resulting in 65.6% of the respondents in this study categorized as overweight or obese , while the remaining reported being diagnosed with nr-axSpA 12.6%) or just axSpA without specifying the subtype (21%) (Table .6% or jup < 0.001) (Table p = 0.001).To study associations between weight and disease activity, we compared disease variables in groups of under/normal weight, overweight, and obese patients. Being overweight or obese was associated with a higher degree of spinal stiffness and number of comorbidities compared to normal weight respondents Table . The ave1) Table , Fig. 1.1) Table . The assThe results of this study indicate a higher burden of disease in overweight or obese patients compared to under/normal-weight patients with axSpA. Obese patients reported worse scores on all subscores as well as the total score of the patient-reported disease activity index (BASDAI). Furthermore, being overweight or obese was associated with a higher degree of spinal stiffness and number of comorbidities compared to normal weight respondents. There was also seen a significant association between BASDAI and BMI (higher BASDAI with higher BMI) when adjusted for gender and age.According to large population-based Norwegian studies, 60–70% of adults are either overweight or obese, and the minority is normal weight , 17. In The two main drivers of overweight and obesity are unhealthy diet and physical inactivity. The direct evidence of effects of diet as a disease modifier in rheumatic diseases is scarce, but a meta-analysis of six randomized controlled trials addressed the effects of weight management, showing that weight loss could prevent the onset of psoriasis and improve pre-existing psoriasis in obese individuals ••. FurthA healthy diet and physical activity are the most important factors in achieving a beneficial body composition. Physical inactivity leads to the accumulation of visceral fat mass and increased abdominal fat and is associated with impaired glucose and lipid metabolism as well as higher production of pro-inflammatory cytokines (adipokines), as for example, interleukin-6 (IL-6) and TNFα . FurtherOverweight or obesity may also represent a biomechanical factor that can be considered as a trigger of the inflammatory process that may influence the pathogenesis of SpA in terms of new bone formation. This hypothesis was investigated in an animal study, concluding that mechanical strain may drive both entheseal inflammation and new bone formation . In lineWeight control and physical fitness are well-documented pathways toward health and disease control , but PinWe acknowledge the data from the EMAS study has some limitations. The survey depended on self-reported data and did not attempt to confirm participant diagnosis nor to support participant responses with clinician-reported assessments. Accordingly, clinical data including BASDAI and spinal stiffness score may also suffer from response bias. Nevertheless, the sample characteristics were consistent with previous cohorts including patients with confirmed axSpA –35.The medical treatment options have revolutionized the field of rheumatology in the last decades, and many patients experience reduced disease burden as a result of effective biological medication , 31, 36.ESM 1(PDF 918 kb)


The results indicated that intercellular iron overload plays an essential role in TBHP-induced ferroptosis of human NPCs. Mechanistically, FPN dysregulation is responsible for intercellular iron overload under OS. The increase in nuclear translocation of metal-regulatory transcription factor 1 (MTF1) restored the function of FPN, abolished the intercellular iron overload, and protected cells against ferroptosis. Additionally, hinokitiol enhanced the nuclear translocation of MTF1 by suppressing the JNK pathway and ameliorated the progression of IVDD in vivo. Taken together, our results demonstrate that ferroptosis and FPN dysfunction are involved in the NPC depletion and the pathogenesis of IVDD under OS. To the best of our knowledge, this is the first study to demonstrate the protective role of FPN in ferroptosis of NPCs, suggesting its potential used as a novel therapeutic target against IVDD.Ferroptosis is a specialized form of regulated cell death that is charactered by iron-dependent lethal lipid peroxidation, a process associated with multiple diseases. However, its role in the pathogenesis of intervertebral disc degeneration (IVDD) is rarely investigated. This study is aimed at investigating the role of ferroptosis in oxidative stress- (OS-) induced nucleus pulposus cell (NPC) decline and the pathogenesis of IVDD and determine the underlying regulatory mechanisms. We used tert-butyl hydroperoxide (TBHP) to simulate OS conditions around human NPCs. Flow cytometry and transmission electron microscopy were used to identify ferroptosis, while iron assay kit, Perl's staining, and western blotting were performed to assay the intracellular iron levels. A ferroportin- (FPN-) lentivirus and FPN-siRNA were constructed and used to explore the relationship between FPN, intracellular iron homeostasis, and ferroptosis. Furthermore, hinokitiol, a bioactive compound known to specifically resist OS and restore FPN function, was evaluated for its therapeutic role in IVDD both  Intervertebral disc degeneration (IVDD) is a leading cause of low back pain , which aThe term ferroptosis, coined in 2012, describes a form of regulated cell death induced by the small molecule erastin, which inhibits the cystine-glutamate antiporter and causes the depletion of glutathione. Ferroptosis is characterized by iron-dependent excessive accumulation of lipid hydroperoxides, which is distinct from necrosis, apoptosis, and autophagy based on biochemical, morphological, and genetic criteria . FerroptFerroportin (FPN), a multitransmembrane protein, is the only known mammalian iron exporter that transports iron from the cytoplasm to the extracellular space . FPN is Chymacyparis taiwanensis. As a component of essential oils, hinokitiol has been shown to resist OS and restore FPN function [Metal-regulatory transcription factor 1 (MTF1) is responsible for regulating various metals at the cellular level, especially iron and zinc . When refunction , 23, inhfunction , 24, 25.In the current study, OS was induced in NPCs via exposure to tert-butyl hydroperoxide (TBHP). The results indicated that ferroptosis was involved in TBHP-induced human NPC death and IVDD. TBHP aggravated ferroptosis in NPCs by suppressing FPN expression via MTF1. Suppression of the JNK pathway using hinokitiol reversed the ferroptosis induced by the TBHP treatment. We explored the ferroptotic mechanism underlying OS-induced IVDD with the expectation of detecting potential treatment strategies for IVDD.n = 13), aged 11–24 years with a mean age of 15.8 years, who had undergone surgery for idiopathic scoliosis. Degenerative NP tissues were obtained from 6 males and 7 females (n = 13), aged 28–65 years with a mean age of 45.2 years, who underwent disc excision surgery for lumbar disc herniation. The degenerative grade of NP tissue samples was classified according to Pfirrmann grades using magnetic resonance images as previously described [All experimental were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (No. S214). Having obtained informed consent from all patients, normal NP tissues were acquired from 7 males and 6 females  for 6 h or 12 h. For the purpose of inhibiting ferroptosis, NPCs were treated with medium containing ferrostatin-1 5 μM , liproxstatin-1 5 μM , deferoxamine 100 μM , hinokitiol 8 μM , or SP600125 20 μM, a specific inhibitor of JNK , and harvested for subsequent analysis.Human NPCs were isolated as described previously . BrieflyCell viability was performed using the Cell Counting Kit-8 (CCK-8)  according to the manufacturer's instructions. The absorbance was measured at 450 nm using a spectrophotometer . NPC proliferation was detected via the incorporation of EdU (5-ethynyl-2′-deoxyuridine)  according to the manufacturer's instructions, and fluorescence images were acquired using a fluorescence microscope .μM C11-BODIPY 581/591, following which the preparation was incubated at 37°C for 30 min. Next, cells were washed thrice with PBS before being trypsinized and resuspended in fresh PBS. Fluorescence emission peaks were analyzed using a flow cytometer . The peak from ~590 nm to ~510 nm is proportional to lipid ROS generation.Lipid ROS levels were assayed using C11-BODIPY 581/591  according to the manufacturer's instructions. Briefly, cells were treated with the intended compounds and 10 Cells were fixed in 2.5% glutaraldehyde  for 1 h, followed by fixation in 2% osmium tetroxide for 3 h. After washing, cells were stained with 0.5% uranyl acetate for 12 h. Following dehydration and polymerization, samples were cut into 70–90 nm ultrathin sections with an ultramicrotome  and observed using a transmission electron microscope .g, 10 min) at 4°C. The supernatant was collected and 5 μl of iron reducer was added to each sample and incubated for 30 min at 37°C. Next, 100 μl of iron probe was added to each sample and incubated for 60 min at 37°C away from light. Absorbance was measured at 593 nm using a microplate reader (Thermo Fisher Scientific).Iron concentrations were measured using an iron assay kit  following the manufacturer's instructions. After being treated with various compounds, cells were collected, homogenized in 5x volume of iron assay buffer on ice, and centrifuged . Histological sections were deparaffinized and rehydrated. Next, the iron stain solution was produced by combining equal volumes of potassium ferrocyanide and hydrochloric acid solutions. Slides containing tissue sections were incubated in the solution for 3 min, washed with distilled water, stained using Abcam nuclear fast red solution for 5 min, and then washed with distilled water. Finally, the sections were dehydrated in 95% ethanol, followed by absolute ethanol. The final blue stain directly correlates with nonchelated iron in the human NP tissue.Total proteins, cytoplasmic proteins, and nuclear proteins were extracted from cultured cells using the corresponding kits  according to the manufacturer's instructions. The protein samples were incubated with the following primary antibodies: TFRC (1 : 500), FTL (1 : 500), DMT1 (1 : 1000), MTF1 (1 : 500), JNK (1 : 1000), beta-actin (1 : 5000), GAPDH (1 : 1000), and Histone 3 (1 : 1000) , FPN , and Phospho-JNK . This was followed by incubation with a horseradish peroxidase- (HRP-) conjugated secondary antibody . Beta-actin, GAPDH, and Histone 3 were used for normalization.TRIzol reagent (Invitrogen) was used to extract total RNA from cultured cells according to the manufacturer's instructions. The following primers were used: Homo FPN ; Homo TFRC ; Homo DMT1 ; and Homo GAPDH . GAPDH was used for normalization.Knockdown of FPN in human NPCs was achieved via transfection of FPN-siRNA (siFPN)  using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After verifying high silencing efficiency, the NPCs were used as the treatment group.Overexpression of FPN or MTF1 in human NPCs was conducted via transfection of lentiviral plasmid generating Lenti-FPN and Lenti-MTF1, respectively,  according to the manufacturer's instructions.n = 8 for each group), which were obtained from the Laboratory Animal Center of Huazhong University of Science and Technology . After the rats were weighed and anesthetized with 2% (w/v) pentobarbital (40 mg/kg), the experimental level (Co7/8) was located by digital palpation on the coccygeal vertebrae and confirmed by counting the vertebrae from the sacral region in a trial radiograph. The tail skin was sterilized, and the tail vertebral disc of Co7/8 was punctured though the tail skin, parallel to the end plates with a 27-gauge sterile needle from the lateral side of the tail and was held with a 4 mm depth for 30 s. After surgery, DFO, Fer-1, or hinokitiol was diluted with normal saline and injected intraperitoneally for 8 weeks at a dose of 200 mg/kg/d, 150 mg/kg/d, or 150 mg/kg/d, respectively. The degeneration group was administered an equal amount of normal saline daily until the rats were killed. All animals were allowed free unrestricted weight bearing and activity.All procedures involving animal were conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the ethical standards of the Animal Experimentation Committee of Huazhong University of Science and Technology. A simple annulus fibrosus puncture rat model was developed as previously described , 28. BriImmunofluorescence analysis was performed as previously described . First, μm sections. For histological analysis, the sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE) and safranin-O (SO). Immunohistochemistry was conducted as previously described [NP tissue was fixed in 10% formaldehyde for 24 h, embedded in paraffin, and sliced into 4 escribed . Sectiont-test or one-way ANOVA. Significance was defined as p < 0.05.Data are expressed as the mean ± SD of at least three independent experiments. Statistical analyses were performed using GraphPad Prism 7 software . Significance of difference was determined by unpaired Previous studies have demonstrated that peroxide-induced cell death is involved in ferroptosis  and thatFerroptosis is an iron-dependent regulated cell death, and cellular iron is required for ferroptotic ROS accumulation . To testConsidering that the divalent metal transporter 1 (DMT1), transferrin receptor (TFRC), and FPN are mainly responsible for regulating the intercellular iron levels, we measured the corresponding mRNA and protein expression levels using RT-qPCR and western blotting, respectively. The results showed that FPN, but not TFRC or DMT1, was significantly downregulated in human NPCs treated with TBHP Figures , suggestTo further determine the role of FPN in generating intercellular iron overload and ferroptosis in human NPCs, we knocked down FPN using siFPN and overexpressed FPN by transfecting a lentiviral plasmid (Lenti-FPN) in control NPCs and TBHP-induced NPCs. Western blotting results showed the knockdown of FPN was efficient, and it increased intercellular iron levels in both control NPCs and TBHP-induced NPCs, indicated in ferritin and LIP Figures . FPN knoNext, we aimed to identify the regulator responsible for the decrease of FPN during TBHP treatment. Based on a literature search, MTF1 could regulate the genetic expression of FPN at the transcriptional level , 33, 34.μM hinokitiol was cytotoxic to human NPCs, whereas doses with <8 μM hinokitiol did not cause cytotoxicity (μM hinokitiol for the current study to determine its therapeutic role in protecting against ferroptosis of NPCs in vitro. Western blotting indicated that hinokitiol significantly suppressed activation of the JNK pathway by decreasing the phosphorylation level of JNK in a manner consistent with SP600125 (a specific inhibitor of JNK)  . We next of JNK) . Additio of JNK) . Further of JNK) . Finally of JNK) . Togethein vivo, we constructed a tail disc percutaneous needle puncture animal model of IVDD using Sprague–Dawley rats as previously described [in vivo. Administration of hinokitiol may ameliorate IVDD progression though increasing the nuclear translocation of MTF1 and restoring the function of FPN in vivo.To further investigate the relationship between ferroptosis and IVDD and the therapeutic efficacy of hinokitiol escribed , 28. Aftin vitro and in vivo.Lower back pain has a negative impact on quality of life, with IVDD being its major cause. Although much effort has been made to diagnose lower back pain disorders in advance , 36, theOS is a common pathological phenomenon involved in various diseases, including diabetes, nonalcoholic fatty liver disease, and Alzheimer's disease . The levin vivo. This confirms the involvement of ferroptosis in TBHP-induced decline of NPCs and IVDD pathogenesis.Ferroptosis, an iron-dependent regulated cell death, was first identified while investigating cell death induced by the small molecule erastin, which inhibits the function of cystine-glutamate antiporter. Initial studies indicated that ferroptosis was biochemically, morphologically, and genetically distinct from necrosis, apoptosis, and autophagy . HoweverThe sensitivity to ferroptosis is tightly linked to various biological processes, including lipid metabolism, iron metabolism, amino acid and glutathione metabolism, and the biosynthesis of GPX4, p53, coenzyme Q10, ferroptosis-suppressor-protein 1 (FSP1), and NADPH , 46–48. FPN plays a critical role in iron homeostasis, and alterations in FPN may result in either iron overload or deficiency. The regulation of FPN expression is complex, with important layers of control at the transcriptional level via the regulation of levels and splice variants of mRNA, at the posttranscriptional level via an iron-regulatory element in the 5′-untranslated region of FPN mRNA and at the posttranslational level via direct interaction between FPN and hepcidin, a peptide hormone. These different regulatory mechanisms could influence FPN activity at its various sites of expression . Previouin vivo [in vivo. Our study has some limitations. Although our study found that TBHP attenuated ferroptosis in human NPCs by reducing FPN via MTF1, further investigation is warranted to elucidate the mechanisms underlying TBHP and MTF1 interaction. Furthermore, only a ferroptosis-associated decrease in human NPCs subjected to OS was investigated in our study. Additional investigations are warranted to confirm the universality of ferroptosis in IVDD via the simulation of other stress factors, such as inflammation, hypoxia, and compression.Hinokitiol, a natural tropolone derivative, exhibits multiple biological activities such as antibacterial, antifungal, antiviral, anti-inflammatory, and anticancer. This compound has been used to treat decubitus ulcers, pulmonary tuberculosis, and lung gangrene in clinical practice , 51. Hinin vivo . Inhibitin vivo . The curTo the best of our knowledge, the current study is the first to demonstrate the protective role of FPN in ferroptosis of human NPCs and the pathogenesis of IVDD. Furthermore, we revealed that TBHP downregulated FPN expression by reducing the nuclear translocation of MTF1, which causes intracellular iron overload and ferroptosis in human NPCs. Moreover, hinokitiol alleviated TBHP-induced OS and ferroptosis of NPCs by restoring the FPN function. Therefore, we inferred that repairing FPN function and inhibiting ferroptosis in human NPCs may be potentially effective therapeutic targets against OS-associated IVDD.


Nanobiotechnology has grown rapidly and become an integral part of modern disease diagnosis and treatment. Biosynthesized silver nanoparticles (AgNPs) are a class of eco-friendly, cost-effective and biocompatible agents that have attracted attention for their possible biomedical and bioengineering applications. Like many other inorganic and organic nanoparticles, such as AuNPs, iron oxide and quantum dots, AgNPs have also been widely studied as components of advanced anticancer agents in order to better manage cancer in the clinic. AgNPs are typically produced by the action of reducing reagents on silver ions. In addition to numerous laboratory-based methods for reduction of silver ions, living organisms and natural products can be effective and superior source for synthesis of AgNPs precursors. Currently, plants, bacteria and fungi can afford biogenic AgNPs precursors with diverse geometries and surface properties. In this review, we summarized the recent progress and achievements in biogenic AgNPs synthesis and their potential uses as anticancer agents. In receical use .Particles with at least one dimension smaller than 100 nm are defined as NPs. Their physical  and chemical  properties arise from their surface geometry, which determines the area/volume ratio. When NPs diameter decreases for a spherical particle, the surface area increases proportionately to the square of diameter and there is a resulting increase of surface activity compared to bulk materials with larger dimensions. In some cases, a decrease in size combined with an increase in the area can improve material biocompatibility . AlthougMetallic NPs such as silver, gold and platinum NPs have been widely tested in humans. For medicinal applications, NPs synthesis must be biocompatible and either non-toxic or low-toxicity protocols should be used. The most common method which has been used to produce AgNPs is chemical synthesis, recruiting reagents whose function is to reduce the silver ions and stabilize the nanoparticles. However, these reagents are toxic and may have potential health hazards . In addiAgNPs can cause cytotoxicity of the cancer cells by altering their morphology and reducing the viability along with oxidative stress in different cancer cells ,22. In aUnique physicochemical properties of AgNPs provides them the ease of entrance into the cells and gives them the ability to efficiently interact with biomolecules inside the cells or on the cell surfaces . Some ofXanthomonas oryzae, spherical as well as triangular and rod-shaped particles with an average size of 14.86 nm were obtained depending on the experimental conditions [Pseudomonas stutzeri AG259 resulted in triangular and hexagonal particles with an average size of 200 nm [Fusarium acuminatum, spherical AgNPs in the range of 5–40 nm (average size 13 nm) were reported [Trichoderma viride, spherical and sporadically rod-like particles in the range of 10–40 nm at 27 °C. Decreasing the temperature to 10 °C increased the particle size to 80–100 nm [Nelumbo nucifera can be used as an example. Synthesis using the leaf extract of this plant resulted in AgNPs with spherical, triangular, truncated triangular and decahedral morphologies. Sizes of the particles ranged between 25 and 80 mm averaging 45 mm [Camellia sinensis (green tea) extracts [While picking a drug material for the treatment of certain disease types including cancer, hemocompatibility of that material should be kept in mind . Kwon etnditions , whereasf 200 nm . In the reported . On the 0–100 nm . Studies0–100 nm . To undeng 45 mm . Vilchisextracts .AgNPs efficiently interact with light through a phenomenon known as surface plasmon resonance (SPR) more efficiently than any known organic or inorganic chromophores. This strong interaction is a result of both the restriction of a large density of conducting electrons to smaller dimensions compared to the mean free path and the unique frequency dependence of the dielectric function in metallic silver. The combination of these two factors gives rise to SPR related properties. The size and shape of NPs and the dielectric function of the surrounding medium determines the frequency and resonance strength . The ligOriganum vulgare [One of the important electrical properties of AgNPs in a colloidal suspension is zeta potential which is influenced by the size of the NPs. AgNPs possess a large absolute value of zeta potential corresponding to a high surface electrical charge which can result in a strong repulsive force among particles to resist agglomeration . Agglome vulgare .Both “top-down” and “bottom-up” approaches can be adopted for NPs synthesis, including AgNPs . A top-dThe downside of most AgNPs synthesis protocols involving chemical and physical methods include associated high cost and the use of toxic and potentially hazardous materials which may pose environmental and biologic threats . NPs pro+) by several bacteria species, such as Streptomyces sp. LK3 [Bacillus licheniformis [3−) in silver nitrate (AgNO3) are reduced to nitrite (NO2−) by accepting two protons then releasing two electrons and water. Electrons released in this reaction are transferred to the Ag+ ion to form elemental silver (Ag0) [Biosynthesis of NPs may occur extracellularly or intracellularly. A widely utilized extracellular mechanism for AgNPs synthesis via bacteria employs NADH-dependent nitrate reductase-mediated reduction of silver ion (Ag sp. LK3  and Baciniformis . During er (Ag0) . This meLactobacillus A09, where Ag+ reduction occurred on the bacterial cell surface [Lactobacillus A09, contain numerous anionic surface groups. These groups can provide silver ion biosorption sites [+ on Lactobacillus A09, suggesting competition between proton and silver ion binding to negatively charged sites [+ adsorption sites on the cell surface. The two electron released while forming the aldehyde group from an alcohol can reduce Ag+ ions to elemental Ag0 [However, enzymes are not always necessary for bacterial synthesis of AgNPs. For example, non-enzymatic intracellular synthesis was reported in  surface . Gram-poon sites . The pH ed sites . Potentintal Ag0 . The steVerticillium model system: Ag+ ions interact electrostatically with negatively charged carboxylate groups in the mycelial cell wall and are trapped at the cell surface. Subsequently, trapped ions are reduced by specific fungal enzymes [Aspergillus flavus, a 32-kDa reductase protein secreted by the fungal isolate, reduces Ag+ ions [Due to their inherent ability to efficiently secrete protein, fungi can produce substantially larger amounts of AgNPs than bacteria . However enzymes . Enzymes enzymes . For exaAg+ ions . A schemPlant extracts have been used for AgNPs synthesis and can be superior to bacterial and fungal synthetic systems due to their availability, low toxicity and safety. A wide range of extracted phytochemicals can rapidly reduce silver ions. Reduction can be rapid compared to microbial fermentation , as this+ ions [+ ions by specific functional groups. For example, Punica granatum peel extract contains the flavonoids- kaempferol and naringin along with their glycosides. All of these compounds have hydroxyl (–OH) groups that cause the reduction of the Ag+ ions, resulting in the formation of AgNPs [Numerous phytochemicals have been identified that are capable of producing AgNPs. As examples, flavonoids, terpenoids, terpenes, flavones, phenolics, polysaccharides, saponins, tannins and alkaloids have all been used for AgNPs synthesis ,65. Phyt+ ions ,66. Due of AgNPs . A typic+ ions reduction and only certain phytochemicals are involved in this reduction. For example, phytochemical screening revealed the presence of phenols, glycosides, flavonoids, reducing sugars, resins, tannins, terpenoids, steroids, alkaloids and saponins in Anagallis tenella leaf extract  spectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD) and energy dispersive spectroscopy (EDS) are commonly used for AgNP characterization ,76.UV-Vis light spectroscopy is used to determine AgNPs formation by measuring the optical absorbance spectra. The wavelength of the light absorbed is heavily affected by the size and aspect ratio of these NPs. Variations in sizes cause each solution to have a different color. The vibrant colors occur because the conduction electrons on the surface of each nanoparticle vibrate when a particular wavelength of light excites them (“surface plasmon resonance” phenomenon as discussed earlier). These vibrations result in extremely bright colors which can be tweaked by altering the particle size and shape. In case of spherical NPs, the larger they are, the “bluer” they appear. The color change results from the interaction of the individual metal atoms with each other .DLS is a technique that can be utilized to determine particle size and particle size distribution . Since tSEM and TEM can be utilized to characterize AgNPs shape and morphology . It has −1 is attributed to O–H stretching, whereas the peaks at 2974 and 2887 cm−1 correspond to C–H stretching vibrations of methyl, methylene or methoxy groups. The other noticeable peaks positioned at 1656 and 420 cm−1 are attributed to the C=O stretching in carbonyl group, while the peak at 1049 cm−1 is attributed to the C=O stretching of the alcoholic group [FTIR spectroscopy has been used to characterize AgNPs surface chemistry, i.e., the functional groups present on NPs surfaces can be identified with the help of this technique . The suric group .T. baccata was 27% when the temperature of the sample was raised from 0–521 °C. Another weight loss of 8.3% followed when the temperature reached 678 °C. The final result was a total weight loss of about 36.89% after increasing the temperature up to 700 °C [The types of bio-organic compounds or functional groups on the AgNPs surfaces can be further confirmed by TGA analysis. TGA identifies the organic compounds on the NPs surfaces by determining the thermal stability of the compounds. TGA detects the characteristic weight losses at certain ranges of temperatures which is attributes to specific compounds on the AgNPs surfaces. For example, Kajani et al. reported that the weight loss of AgNPs synthesized using ethanolic extract of o 700 °C .D’ is the particle diameter size, ‘K’ is a constant, ‘λ’ is the wavelength of X-ray source, ‘β’ is the full width half maximum (FWHM) and ‘Phase identification and crystal structure determination of NPs can be determined by X-ray diffraction of monochromatic collimated X-rays (XRD) by NPs . With thon angle .Elemental analysis of NPs can be performed by EDS . In the Optimal procedures for anti-cancer AgNPs preparation are broadly being examined. It is worth mentioning that we have noted some biosynthesized AgNPs from bacterial and fungal sources in the 50 values of these extracts against Michigan Cancer Foundation (MCF)-7 cells ranged from 3.04–250 μg/mL (50) of AgNPs was dependent upon the dose of the extract given [Literature searches have uncovered studies of 23 plant extracts which were used for AgNPs preparation. Many of these AgNPs were either toxic to the breast cancer cell line MCF-7 or inhibited its growth. The sizes of NPs reported in these studies ranged from 5–80 nm; their shapes also varied . The IC50 μg/mL . Moreovect given ,103,104.50 values were dose-dependent [50 of ≤4.25 μg/mL [50 values varied depending on the preparation method of the AgNPs and also depended upon the plant extracts used [50 values ranging from 5.5–100 μg/mL [Biosynthesized AgNPs have also been found to inhibit the brain cancer cell line HNGC2 . These sependent . Biosynt25 μg/mL . In contcts used ,111,112.00 μg/mL ,115,116.50 values ranging from 78.58–83.57 μg/mL [50 of 21.05 μg/mL [50 values ranged from 10.02 to more than 30 μg/mL, the load of detection was 31.25 ng/mL, there was 13.86% viability at 25 μL, and some of the other results demonstrated a dose dependent relationship [50 value was greater than 30 μg/mL. Biosynthesized, spherical AgNPs with sizes less than 40 nm blocked the Hek-293 kidney cancer cell line in a dose-dependent manner [50 values of 3.42 μg/mL and 12.5 μg/mL, and an AgNPs concentration of 500 nM [50 value of 5.33 μg/mL and in a dose-dependent manner, respectively [Cuboidal and spherical bio-AgNPs with sizes ranging from 59–94 nm, prepared from different plant extracts, inhibited the A431 cell line, an epidermoid carcinoma, with IC57 μg/mL . Inhibit05 μg/mL . A hepattionship ,119,120.t manner ,98. The f 500 nM ,121,122.ectively ,123. The50 and LD50 values as mentioned below [50 was 21.05 μg/mL [50 value of 7.6 ± 0.8 μg/mL, following a dose-dependent relationship [50 was 0.6 μg/mL [50 value of 7.5 μg/mL and in a dose-dependent manner [50 was 38.9 μg/mL [50 values ranging from 6.85–87.33 μg/mL [According to a literature review, 13 bio-extracts have been successfully used as reducing agents to prepare AgNPs that can block the activities of the A549 lung cancer cell line ,130,131.ed below ,130,131.05 μg/mL . The B16tionship ,133. The.6 μg/mL . The 15–t manner ,127. Inh.9 μg/mL . The 9–933 μg/mL ,123,135.The cytotoxicity of AgNPs depends upon their size and shape. For example, it was reported that AgNPs with diameters of 100–160 nm, lengths of 1.5–25 μm, and spherical shapes (30 nm) showed potential cytotoxic effects on human lung epithelial A549 cells . The mosBlood vessels play a vital role in supplying nutrients and clearing waste products at the tissue level; the formation of new blood vessels from existing vessels is termed angiogenesis. Wound healing and granulation tissue formation are some potential results of angiogenesis . AccordiThe cell cycle incorporates a complex series of signaling pathways, by which a cell grows replicates its DNA and divides. This phenomenon plays a vital role in cancer progression. However, due to the genetic mutations that occur in cancer, this regulatory process malfunctions, which leads to uncontrolled cell proliferation. DNA synthesis (S), Gap2/mitosis (G2/M), Gap1 (G0/G1) and subG1 are all vital checkpoints for cell cycle arrest  in FigurThe toxicity of AgNPs has been mentioned in different studies; Bharadwaj Punita reported that AgNPs could be highly toxic to mammalian cells, e.g., brain cells, liver cells and stem cells . BesidesStudies have revealed that the mitochondrial activation and reactive oxygen species (ROS) overproduction are the key factors of AgNPs in antimicrobial and anticancer defense. Interestingly, the ROS production and the subsequent damages resulting from oxidative stress are AgNPs size-dependent, smaller NPs cause better ROS overproduction . AgNPs cPlasmodium falciparum) and its related vector  [AgNPs have been investigated intensively regarding their promising anticancer effects exhibited in different human cancer cell lines, such as endothelial cells, IMR-90 lung fibroblasts, U251 glioblastoma cells and MDA-MB-231 breast cancer cells ,155,156.osquito) .+ and other soluble complexes like AgCl(aq) and AgCl2− can be absorbed by the gastrointestinal tract into the systemic circulation, where Ag+ can bind to proteins with thiol groups such as serum albumin and small thiol molecules like glutathione (GSH). These complexes generate H+ and GS-Ag which then produce Ag-GSH complexes that are distributed throughout the body. However, biliary pathway is the major elimination route for AgNPs [Bioavailability of AgNPs is low in oral, dermal and inhalational exposures, however, it depends on the particle size, dose, surface coating and soluble fraction. For examples, Park and his team revealed that in a single oral exposure in rats, the bioavailability of citrate-coated 7.9 nm AgNPs was 1.2% and 4.2% in the 1 and 10 mg/kg, respectively . After oor AgNPs .Despite all the recent advancements in cancer treatment, cancer remains one of the most common causes of death around the globe. It is already known to us that the conventional treatment strategies often have many side effects of their own. Therefore, scientists are looking to design novel strategies for the diagnosis and treatment of cancer. Recently, green synthesis of AgNPs has gained much attention in the pharmaceutical field. The use of green chemistry is non-toxic, cheap and environmentally friendly, although there are some disadvantages of biologic methods as listed in Box 1These methods are inexpensive, eco-friendly and non-toxic.No complex setup is required to conduct the synthesis process.It is not necessary to use stabilizing agents to prevent agglomeration of the NPs.Since these processes are carried out in ambient conditions, they are not energy intensive.These methods offer finer tuned control of the size and shape of the NPs compared to chemical and physical methods.Advantages of biologic methods:Synthesis by biologic methods is not as fast as synthesis by chemical methods.Because of the presence of numerous biomolecules present in the biologic sources, it is difficult to pinpoint the exact biomolecules responsible for the synthesis of the NPs.If the biologic synthesis is conducted on a large scale, an ecological imbalance may kick in from overuse of different biologic species.Microbe toxins may be brought together by biologic synthesis.Disadvantages of biologic methods:Business experts have extrapolated that the global market of nanotechnology has a potentially bright future. Commercially produced AgNPs are being manufactured at a significantly large scale and the application of NPs as therapeutic agents is increasing. AgNPs are promising drug leads based on the successful results of previous research studies as well as their cost. However, there are some basic hindrances to using the AgNPs as therapeutic agents in terms of toxicity. To overcome these hindrances—and for use in preclinical trials on humans or any other living bodies—the AgNPs should be biocompatible, non-toxic and free from side effects. Finally, usage of biogenic AgNPs as cancer nanomedicine will require elaborate datasets that uncover potential toxicity and pharmacological issues such as the side effects.


Arabian horses, regardless of the gender. Gender, regardless of the breed, had no significant impact on the antioxidants' activity and concentration. Total SOD and MnSOD activities were significantly higher in Hucul's mares when compared to Hucul's stallions. Concentrations of TAC and TOS were significantly lower in Arabian stallions than in Arabian mares. Commercially handled horses expressed a higher level of oxidative stress markers in comparison to breeds living in seminatural conditions. We conclude that antioxidants are important biomarkers of animal health, whether they are under maintenance care or performing physical exercise.Hucul and Arabian horses differ in the physiological constitution and exposition to environmental conditions. Oxidative stress plays a pathogenic role in many diseases and enables further injuries. The objective of this study was to compare the levels of enzymatic and nonenzymatic oxidative stress markers in Hucul horses living in seminatural conditions and in commercially handled Arabian horses. We tested the serum samples for total superoxide dismutase , Cu-Zn-superoxide dismutase (CuZnSOD), and Mn-dependent superoxide dismutase (MnSOD) activity; for lipofuscin (LPS), ceruloplasmin (CER) and malondialdehyde (MDA) concentration; and for total antioxidant capacity (TAC) and total oxidant status (TOS). Total SOD ( Hucul and Arabian horses differ in the physiological constitution, conformation traits, exposition to environmental conditions, and nutritional requirements . Huculs Domestic animals are routinely exposed to a variety of anthropogenic stressors. Stress can be defined as physiological response elicited when a threat to homeostasis is perceived , 8. OxidThe study included 20 sexually mature, healthy Hucul horses  and 20 sexually mature, healthy Arabian horses . The Hucul horses aged between 5 and 10 years (7.5 ± 2.5), originated from a herd kept on extensive pasture (Poland) throughout the year and had no contact with other horses. Arabian horses, aged between 4 and 11 years (7.35 ± 3.65), originated from 20 different commercial horse riding clubs in Poland and were kept on the pasture. All horses were untrained and not transported for at least one month before collecting the samples. Female horses were not pregnant or in lactation. Both Arabian and Hucul horses were pure-breed mating. No history of diseases such as respiratory symptoms, injuries, digestive tract symptoms, signs from the central nervous system, or pregnancy complications were previously reported. Therefore, none of the horses showed any clinical signs of tested viral or microbiological infections at the time of sampling. All horses were therefore considered healthy based on history results of physical examination and haematological and biochemical analyses (data not shown). In this study, the blood samples were collected during routine veterinary care .Horse blood specimens submitted for routine veterinary towards equine viral arteritis (EVA) were used to assess oxidant stress markers. Therefore, the additional approval of the Local Ethics Committee was not required. A written consent statement was obtained from the owners of animals included in the study.Vena jugularis to the vacuum tubes with EDTA. The samples were centrifuged at 4000 rpm for 10 min at 4°C. Then, the serum was collected and stored at –80°C until analysis.The blood samples were collected between 7:00 and 8:00 am during routine veterinary care. 10 ml of blood was collected from Antioxidant systems in the Hucul and Arabian horse's serum samples were analysed by determining the enzymatic activity of total superoxide dismutase , Cu-Zn-superoxide dismutase (CuZnSOD), and Mn-dependent superoxide dismutase (MnSOD); lipofuscin (LPS) and ceruloplasmin (CER) concentration; the total antioxidant capacity; (TAC) and total oxidant status (TOS). Lipid peroxidation rate was determined by measuring the malondialdehyde (MDA) concentration.2O was used to calculate the total SOD activity. SOD activity is presented as nitrite units (NU) per ml serum. One NU exhibits 50% inhibition of the formation of a nitrite ion under the method's condition [The activity of SOD isoenzymes was measured using the Oyanagui method  with KCNondition .v/v), shaken and centrifuged. The intensity of fluorescence in a dissolved solid was determined using a PerkinElmer spectrophotometer LS45 . The value is expressed in relative lipid extract fluorescence (RF), where 100 RF corresponds to the fluorescence of 0.1 μg/ml quinidine sulfate in 0.1 N sulfuric acid. The inter- and intra-assay coefficients of variations (CV) were 2.8% and 9.7%, respectively.LPS concentration was determined according to Tsuchida et al. . The serCER concentration in serum was assessed according to Richterich  using reTAC of serum was measured using a commercial kit . 2,2′-Azino-di-(3-ethylbenzothiazoline sulfonate) (ABTS) was incubated with a peroxidase (methmyoglobin) and hydrogen peroxide to produce the radical action ABTS+, which has a relatively stable blue-green colour that can be measured at 600 nm (PerkinElmer spectrophotometer LS45 for this study). The suppression of the colour was compared to the standard for TAC measurement assays (Trolox). The assay results are expressed as a Trolox equivalent (mmol/l). The inter- and intra-assay coefficients of variations (CV) were 1.1% and 3.8%, respectively.2+ to Fe3+ in an acidic medium. Fe3+ form a colourful complex with xylene orange ranging up to a blue-purple colouration. Absorption can be read at 560 nm wavelength. The measurements were done on VICTOR-X3 PerkinElmer reader. The concentration was calculated from the calibration curve using H2O2 as the standard. Values are expressed in μmol/l as described by Erel [The method uses the oxidation of Fe by Erel .Malondialdehyde (MDA) concentration in serum was determined using the reaction with thiobarbituric acid. The produced 1,1,3,3- tetraethoxypropane concentration can be read using the spectrophotometric method, employing 515 nm for excitation and 552 nm for emission using a PerkinElmer spectrophotometer LS45. MDA concentration was calculated from the standard curve prepared for 1,1,3,3-tetraethoxypropane .2O and adding 0.9 ml of solution A (2 g l−1 potassium sodium tartrate (KNaC4H4O6·4H2O) and 100 g l−1 sodium carbonate (Na2CO3) in 0.5 M NaOH) before incubation for 10 min at 50°C. In the next step, the samples were cooled down to room temperature, and 1 ml of solution B (0.2 g l−1 KNaC4H4O6·4H2O and 0.1 g l−1 copper sulfate pentahydrate (CuSO4·5H2O) in 0.1 M NaOH) was added and left for 10 min. In the final step, 3 ml of solution C ) was added before incubation for 10 min at 50°C. A standard curve was made of bovine serum albumin  and absorbance was read at 650 nm.Protein concentration was assessed according to the modified Lowry method , 27. Thep value < 0.05. All tests were two tailed. Interval data were expressed as mean value ± standard deviation for normal distribution or as median/lower–upper quartile range for data with skewed or nonnormal distribution. Distribution of variables, including normality, was evaluated by the Shapiro-Wilk test and the quantile-quantile plot; homogeneity of variances was assessed by Levene's test. The two-way parametric ANOVA with post hoc contrast analysis, nonparametric Kruskal-Wallis test, or Mann–Whitney U test were used for data comparisons. The idea of the two-way ANOVA analysis is to compare two main factors and its interaction. In our case, the p value for race deals with the comparison between Hucul and Arabian horse independently to sex. Then, the second main factor deals with the comparison between gender  independently to race. And finally, we get the interaction between those two main factors. When the two-way analysis of variance proved that one of the main analysed factors is statistically significant and when also, but not necessarily, the interaction between two main factors occurs, then contrast analysis may and should be conducted. It means that we may compare subclasses of the first factor between groups defined by the first factor, and also we may compare subclasses of the second factor between groups defined by the first factor (interaction). Those are contrast comparisons. They are defined as a priori in opposite to post hoc tests which compare all groups with each other . If at least one of the main factors was statistically significant, appropriate contrast analysis was performed and has been presented in Statistical analysis was performed using STATISTICA 12.5 PL . Statistical significance was set at a The results of antioxidant enzymatic activity and nonenzymatic antioxidants concentrations in the blood serum of Hucul and Arabian horses are presented in 2p < 0.001, Figures p < 0.05, p < 0.001, respectively, Figures p < 0.097, In the presented study, we observed breed-dependent differences in the antioxidant enzymatic activity and nonenzymatic antioxidants concentration. Total SOD, MnSOD, and CuZnSOD activities were significantly higher , TOS (p < 0.05), and MDA (p < 0.01) were significantly lower in stallions than in mares , accumulation of oxidative stress products (LPS and MDA), and total oxidant status (TOS), than Hucul horses.2·− in mitochondria. A product of MnSOD-catalyzed reactions is hydrogen peroxide (H2O2), thus increased MnSOD activity results in H2O2 accumulation [In this work, we observed that total SOD, MnSOD, and CuZnSOD activities were significantly higher in blood samples obtained from Huculs when compared to Arabian horses. The superoxide dismutase family (SODs) is the first line of defence against reactive oxygen species (ROS) . It is kmulation . The incLipofuscin is an undegradable material, composed of oxidatively modified protein and lipid residues. Its accumulation is proportional to changes observed in mitochondria and depends on individual's age and ROS formation intensity . MDA is Here, we observed that Arabian mares showed a higher level of oxidative stress, measured by increased MDA levels, higher mobilisation of cellular antioxidant agents (TAC), and elevated TOS, in comparison with Arabian stallions. That may suggest that Arabian mares, due to gender and constitution, are more sensitive, so their physiological answer to the environmental stressors is stronger than in Arabian stallions. It may also result from the stress connected to handling, environmental exposition, and nutritional requirements. On the contrary, the reaction to the oxidative stress in Hucul mares and Hucul stallions is at a comparable level except for the higher total SOD and MnSOD antioxidant activities observed for Hucul mares. This subtle mobilisation of SOD enzymes could result from gender differences in sensibility to the general stress conditions.The intergroup comparison shows that Huculs' antioxidant systems are more efficient than Arabian ones. Huculs are known for their excellent health, including disease resistance, tolerance for environmental conditions, and longevity . The anaHorse breeds chosen for this study differ in the morphological constitution, traits, and disposition. They also differ in terms of physiological and biochemical variables. Both breeds developed under severe environmental conditions, characterized by a deficiency of food and heavy workloads. Thus, both breeds share numerous common features, such as courage, strength, and resistance to difficult habitat conditions; good health; and balanced character. Antioxidant stress markers are important biomarkers of animal conditions, in this case, referring to the adaptation of the physiological processes under homeostatic conditions. Based on the results obtained in this study, we can conclude that the physiological level of oxidative/antioxidative defence measured with profiles of antioxidant stress markers was higher in commercially handled Arabian horses than in semi-isolated Hucul horses.


Six vegetables, i.e., Sesbania grandiflora (turi) flower, Amaranthus hybridus L. (spinach), Carica papaya (papaya) leaves, Cosmos caudatus L. (kenikir) leaves, Vigna unguiculata ssp. Sesquipedalis (yard-long beans), and Vigna radiata (mung-bean) sprouts were cooked by boiling or steaming for 1 min or blanching for 20 s. Peanut (Arachis hypogaea), the raw material for peanut sauce, was fried in either fresh palm oil or repeatedly used palm oil. Our results revealed that the highest antioxidant capacity  was observed following boiling for 1 min in case of spinach (41.94 ± 9.8%), papaya (59.04 ± 5.35%), kenikir (54.93% ± 6.32%), and yard-long beans (70.21 ± 8.91%); steaming for 1 min in case of turi flower (60.25 ± 3.63%); and blanching for 20 s in case of mung-bean sprouts (49.27 ± 3.69%). Peanut sauce prepared by frying peanuts in fresh or repeatedly used palm oil reduces the natural antioxidant and lipid peroxidation inhibition properties. However, seasoning the peanut sauce with fresh garlic and lime leaves can restore the lost antioxidant properties. Our study provides the first and clear evidence of the optimal cooking method for Pecel vegetables and sheds light on the wisdom behind the existing traditional cooking method.Vegetables are essential in our diet to maintain health, partly due to their antioxidant properties. A well-known Javanese salad called “Pecel” is prepared by boiling the vegetables and dressed with seasoned peanut sauce. Cooking can reduce or improve the antioxidant properties of foods; therefore, the purpose of this study was to evaluate the effects of brief water boiling (1 min), steaming (1 min), and water blanching (20 s) of the Javanese Pecel vegetables, with or without the peanut sauce. We assessed the  Vegetables are well-known key components of the human diet and are beneficial because of the presence of diverse phytonutrients, such as polyphenols, flavonoids, and carotenoids, which have strong antioxidant properties –3. TheseSesbania grandiflora (turi) flower, Carica papaya (papaya) leaves, Cosmos caudatus L. (kenikir) leaves, Amaranthus hybridus L. (spinach), Vigna unguiculata ssp. Sesquipedalis (yard-long beans), and Vigna radiata (mung-bean) sprouts. Boiling each type of vegetable alternately using the same aluminium kitchen pot is the general practice, because of its simplicity and swiftness in preparation. From the food safety point of view, this practice inactivates microbial residues after vegetable washing. The dish can be consumed with or without steamed rice and protein-rich foods, such as cooked “tempe” (fermented soybean) or “tahu” (tofu), chicken egg, vegetable crackers, or a combination of a few of them. As one of the Javanese people, we (the author) have been consuming Pecel as a part of our daily diet since childhood.Pecel is prepared by boiling the vegetables briefly and dressing them with a seasoned peanut sauce. The vegetables most commonly used in Pecel consist of The practice of cooking Pecel vegetables by boiling may enhance their antioxidant properties depending on the cooking method and duration , 9–13. Tin vitro.It is important to understand the effect of cooking methods on the antioxidant activity of vegetables and the dressing sauce. Therefore, this study was carried out to evaluate the effect of different cooking methods  on the antioxidant capacity of Pecel vegetables. Furthermore, the antioxidant and lipid inhibition capacities of cooked and mixed Pecel vegetables before and after dressing with seasoned peanut sauce were evaluated S. grandiflora (turi), C. papaya (papaya) leaves, C. caudatus L. leaves, A. hybridus L. (spinach) leaves, V. unguiculata ssp. Sesquipedalis (yard-long beans), and Vigna radiata (mung-bean) sprouts, Citrus hystrix DC. (lime) leaves, Arachis hypogaea (peanut), Allium sativum (garlic), palm sugar, and salt were obtained from the local market. 2.2-diphenyl-1-picrylhydrazyl (DPPH), absolute (96%) ethanol, chicken egg yolk, phosphate buffer (pH 7.4), ferrous sulphate (FeSO4), trichloroacetic acid (TCA), and thiobarbituric acid (TBA) were obtained from Sigma-Aldrich.Fresh vegetables, i.e., The vegetables were cleaned under running water and drained. Edible parts/portions were collected, were weighed (EPW), sliced, and mashed using a blender, for analyses. Fresh peanuts were also mashed to analyse their antioxidant activity.All cooking was done using household kitchen utensils, i.e., aluminium pot and steamer with 25 cm diameter and 15 cm depth. Each type of vegetable was cooked by boiling for 1 min, steaming for 1 min, or water blanching for 20 s. The time duration was based on a preliminary observation and survey from some of the Pecel outlets (street vendors and restaurants). Our observation was that Pecel vegetables were cooked for a maximum of 2 min and the mung-bean sprouts for a minimum of 20 s. It is also based on a study that found cooking of vegetables for less than five minutes maintains a flavonol content of more than 80% . Based oApproximately 100 g of each type of Pecel vegetable  was added to boiling water  in a covered aluminium kitchen pot  and occasionally stirred to ensure even cooking of the vegetables. The vegetables were completely submerged in the boiling water. After 1 min, the vegetables were removed, drained, cooled to room temperature, and mashed using a blender for analyses.Approximately 100 g of each type of vegetable  was placed on a perforated tray in an aluminium kitchen-steamer  that contained boiling water . The boiling water was below the perforated tray. During the steaming, the lid was closed, and the vegetables were occasionally stirred to ensure even cooking of the vegetables. After 1 min, the vegetables were removed, cooled to room temperature, and mashed using a blender for analyses.About 100 g of each type of vegetable  was added to boiling water  in an uncovered aluminium kitchen pot. All vegetables were submerged in the boiling water. During the blanching process, the vegetables were stirred to ensure cooking. After 20 s, the vegetables were removed, cooled to room temperature, and mashed using a blender for analyses.A total of 250 g of fresh peanuts were added to 500 mL of palm oil in an aluminium frying wok at 100–120°C and fried until golden brown. The fried peanuts were removed, drained, and cooled to room temperature. The peanuts were deep-fried using either fresh palm oil, hereinafter referred to as “first oil (FO)-fried peanuts” or with palm oil that was already used five times for frying, hereinafter referred to as “repeatedly used oil (RO)-fried peanuts.”The ingredients for peanut sauce were fried peanut, water, garlic, lime leaf, and palm sugar . AlthougFrom a total of 100 g of each of the six cooked vegetables, 25 g of each of the vegetable was weighed, mixed in a ratio of 1 : 1 , and mashed using a blender for analyses. In the normal consumption practice, cooked vegetables are consumed without being mashed.The six cooked vegetables  were masWe considered two treatments: (1) combined vegetables mixed with FO peanut sauce, hereinafter referred to as “Pecel FO,” or (2) combined vegetables mixed with RO peanut sauce, hereinafter referred to as “Pecel RO.” The mixture was homogenised using a kitchen blender for analyses. In practice, cooked Pecel vegetables and its peanut sauce are mixed just before consumption, similar to a western salad.μM DPPH in ethanol . We obtained the maximum absorbance for the DPPH solution at 517 nm. Antioxidant activity was determined by adding 0.1 g of each sample to 5 mL of 60 μM DPPH. The reaction mixture was homogenised using a vortex and incubated for 1 h at room temperature in the dark. The absorbance of the mixture was recorded against a blank (ethanol) at 517 nm using a UV-Vis spectrophotometer (Spectroquant®Pharo300). This absorbance is designated as sample absorbance (Asample), and each sample was analysed in triplicate. Antioxidant activity was expressed as percent inhibition of DPPH radicals using the following equation:Acontrol is the absorbance of the 60 μM DPPH solution and Asample is the absorbance of the sample.DPPH is a stable free radical that has a deep violet colour in methanol or ethanol and has maximum absorbance at 517-520 nm. When an antioxidant neutralises DPPH, the solution becomes colourless or pale yellow and absorbance is reduced. As antioxidant activity increases, the absorbance of the DPPH solution decreases. The antioxidant activity of samples  was evaluated according to the method described by Rezaie et al.  and opti4 to induce lipid peroxidation. The mixture was shaken vigorously and incubated at room temperature for 1 h. Next, 0.5 mL of 15% TCA and 1 mL of thiobarbituric acid (TBA) were added to the mixture and incubated in a boiling water bath for 10 min. The tube containing the reaction mixture was cooled to room temperature and centrifuged at 3500 g for 10 min to precipitate the proteins. The supernatant was sampled to measure the formation of TBA reactive substances by recording the absorbance at 532 nm using a UV/Vis spectrophotometer (Spectroquant®Pharo300). A mixture of phosphate buffer, egg yolk, and FeSO4 without the sample was used as the control. The analysis was carried out in triplicate. The percentage of lipid peroxidation inhibition was calculated using the following formula:A0 is the absorbance of the control and AS is the absorbance of the sample from the reaction mixture containing egg yolk, phosphate buffer (pH 7.4), FeSO4, 15% TCA, and TBA.Lipid peroxidation inhibition of egg yolk was performed according to the method described by Rahman et al. in 2015 . Brieflyn = 3). The data were tested for normality using the Shapiro–Wilk test (n < 50). Normally distributed data were further tested using a one-way ANOVA and an independent t-test. The difference between the means was further analysed using the smallest significance difference at 95% confidence (p < 0.05). The statistical analyses were performed using SPSS 15.All data are presented as the mean ± SD (p > 0.05) for turi flower, spinach, papaya, and kenikir leaves. However, it does not affect the moisture content of yard-long beans and mung-bean sprouts.The water content of the cooked Pecel vegetables is shown in p < 0.05)  .p < 0.05). Blanching was done for a brief time, i.e., 20 s, during which some of the Pecel vegetables  were not cooked yet, except the bean sprouts. This uncooked condition of the vegetables, after blanching, reduced their antioxidant activity compared to that of the control. However, after blanching, the antioxidant activity of the bean sprout that had been cooked well increased (to 49.27%) significantly (p < 0.05), compared to that of the control (24.37%) (The antioxidant activity of each Pecel vegetable is shown in (24.37%) .p < 0.05), while that of yard-long bean (70.21%) was the same as that of the control. Long yard bean has the highest antioxidant activity, as indicated by an almost instant change in the colour of DPPH solution to pale yellow, as soon as the yard-long bean sample was added to the DPPH solution. However, the antioxidant activity of the bean sprout was reduced to 10.18% after boiling when compared to that of the respective control (24.37%).Boiling the Pecel vegetables for 1 min affected the degree of cooking and colour, indicating a change in texture and their phytochemical content. As shown in p < 0.05) after steaming, from 33.20% when raw to 60.25% after one-minute steaming. The highest antioxidant activity was achieved after boiling for 1 min in case of spinach (43.15%), papaya leaves (59.04%), kenikir leaves (54.93%), and yard-long beans (70.21%). However, the highest antioxidant activity for turi flowers was achieved with steaming for 1 min (60.25%) and for mung-bean sprouts with blanching for 20 s (49.27%).Water steaming of Pecel vegetables for 1 min affects the degree of cooking, and their colour change indicates a textural and chemical change within the vegetable matrix. The antioxidant activity of turi flowers increased significantly (p > 0.05) was not significantly different, although the numerical values of steamed turi flowers were higher.Moist heating increases the phenol content of vegetables. Short heat exposure softens the vegetable tissues, opens the seed coat, and facilitates the extraction of phenolic compounds and carotenoids. It could also be attributed to the possible breakdown of complex polyphenolic compounds, such as tannins to simple polyphenols, during heat processing , 21. HowOverall, it is evident from the results that brief boiling can retain the highest antioxidant activity in case of green leafy vegetables, yard-long beans, and turi flowers. Such brief cooking has been in use for decades, because of its ease and speed of preparation. Moreover, brief boiling can reduce or eliminate microbial residues from prior vegetable washing. The only vegetable that needs to be cooked separately is mung-bean sprouts; its highest antioxidant was achieved with brief blanching. Blanching is similar to boiling, but is performed for a shorter time (20 s compared to 60 s). The practice of boiling mung-bean sprouts together with other vegetables depends on the duration for which they are boiled. The sprouts can be placed on top of other vegetables in a boiling pot, removed, and drained after 20 s. Our results confirmed that cooking of Pecel vegetables increases their antioxidant activity, possibly because of the increased availability of antioxidant compounds , 23. Forp < 0.05). Compared to fresh peanuts, FO peanuts had reduced antioxidant activity (9.8% reduction). This activity was further reduced in RO peanuts (16.92% reduction). The reduction in antioxidant activity could be because of the partial loss of antioxidant compounds in the heated cooking oil [The results show that fresh raw peanuts have significant antioxidant activity . Antioxiking oil .The reduction in antioxidant activity of FO peanut or RO peanuts was compensated after they were processed into peanut sauce, with their antioxidant activity increasing significantly . It is possible that the increase in the antioxidant activity of peanut sauce is likely due to the added seasonings, that is, raw garlic and lime leaves. Raw garlic and fresh lime leaves have strong antioxidant activity , 29.Both the fresh seasonings used can scavenge free radicals in fried peanuts and, therefore, improve the antioxidant activity of peanut sauce. Our results suggested that to reduce or neutralise radicals from RO peanut, use of fresh garlic (not only one clove) in peanut sauce preparation is recommended.p < 0.05).Lipid peroxidation diminishes the nutritional quality of vegetables and form toxic compounds. Antioxidants are substances that can retard the oxidation of easily oxidisable biomolecules such as lipids and proteins. Lipid peroxidation inhibition is the percent inhibition of lipid peroxidation by antioxidant compounds. The higher the lipid peroxidation inhibition, the higher is the amount of antioxidant components in the material. Inhibition of egg yolk peroxidation by the combined cooked vegetables was low 23.81%) . Low inh.81% . LoThe inhibition of egg yolk peroxidation decreased significantly after mixing cooked vegetable with FO peanut sauce (19.58%) or RO peanut sauce (6.23%) . The redhttps://nilaigizi.com/) as presented in We estimated the nutritional value of Pecel salad in our study  based on the proximate composition available from Indonesian Food Composition Data (Following the guide of “My-plate” (“isi piringku”) from the Ministry of Health, Republic of Indonesia, regarding portions of a healthy diet, one-third of the plate must be vegetables . The standard average requirement for teenagers and adults per day is 400–600 g mixed ready-to-eat vegetables. One portion of Pecel salad (cooked vegetable) is approximately equal to 300–500 g  of vegetables. As the moisture content of raw and cooked Pecel vegetables is similar , one porThe results from this study demonstrated that to achieve the highest antioxidant activity, the Pecel vegetables, green leafy vegetables, flowery vegetables, and mung-bean sprouts, should be boiled briefly for 1 min, water-steamed for 1 min, or water-blanched for 20 seconds. Seasoned peanut sauce for Pecel should be prepared by frying peanuts in fresh palm oil and seasoned with fresh garlic and lime leaves, as has been the decade-old practice. Seasoned peanut sauce using raw peanuts fried in repeatedly used frying oil significantly reduced the antioxidant activity and the inhibition of lipid peroxidation. The results of this study can be used to make recommendations on the cooking methods to preserve the health benefits of Pecel vegetables.


To investigate the relationship between fragmented QRS (fQRS) quantified by a new method and myocardial fibrosis (MF) and the diagnostic value of quantitative fQRS (Q-fQRS) to detect MF in hypertrophic obstructive cardiomyopathy (HOCM) patients based on histological validation.We performed a retrospective study that included 69 patients with HOCM who underwent ventricular septal surgery. Nine individuals who died from accidents were studied as a control reference for the histological parameters. Septal myocardium samples were subjected to Masson’s trichrome staining to quantify the collagen volume fraction (CVF). An fQRS pattern was defined as the presence of additional R waves or RSR’, evidenced by notched R or S wave on electrocardiography (ECG). The Q-fQRS was quantified as the total amount of deflections in the QRS complex in all 12 routine ECG leads together. Cardiac magnetic resonance imaging was conducted, and late gadolinium enhancement (LGE) was measured at 2, 4, 6 and 8 standard deviations (SDs).P < 0.001; P = 0.040). Q-fQRS was correlated with CVF , and Q-fQRS showed the best correlation with LGE measured at 8 SD . Multivariate regression analyses revealed that Q-fQRS was independently associated with the extent of CVF in HOCM patients after adjusting for age, sex, body surface area and the extent of LGE at 6 SD (P < 0.001). When the patients were divided into subgroups with normal CVF or high CVF according to the CVF in controls, Q-fQRS and LGE at 6SD showed similar diagnostic value in detecting patients with high CVF, with sensitivities of 66.7% vs 68.6%, specificities of 76.7% vs 72.4%, and accuracies of 71% vs 70.3%.Of the 69 patients, fQRS was documented in 38 (55.1%) patients, the mean number of leads with fQRS was 3.7 ± 1.6, and the mean Q-fQRS was 17 ± 7.2. Compared with HOCM patients without fQRS, HOCM patients with fQRS had a higher CVF and more LGE at 6 SD (HOCM patients with fQRS showed more extensive MF. Q-fQRS was an independent predictor for MF and had a good diagnostic value, with a sensitivity of 66.7% and specificity of 76.7%, in identifying patients with higher fibrotic burden. Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disease that is characterized as myocardial fibrosis (MF) . HCM patLate gadolinium enhancement (LGE) is a standard approach for the assessment of MF because collagen deposition and excessive retention of gadolinium lead to enhancement on CMR images . HoweverSixty-nine consecutive HOCM patients with symptomatic left ventricular (LV) outflow tract obstruction who underwent surgical myectomy at Fuwai hospital,Beijing between 2015 and 2020 were retrospectively studied. All patients underwent a detailed cardiovascular evaluation, including medical history, clinical examination, 12-lead ECG, and CMR.HCM was diagnosed by the criteria mentioned previously ..LV outfControl myocardium from the LV septal wall was collected at autopsy of 9 individuals  who died from accidents, showing no signs of macroscopic or microscopic cardiac lesions.A standard 12-lead ECG  was recorded in the supine position during quiet respiration before the surgeries. The fQRS was defined as previously described ,as folloTransthoracic echocardiography was performed by an experienced cardio-sonographer using the Phillips iE33 Color Doppler Ultrasound System . M-mode, twodimensional, and pulsed and continuous-wave Doppler studies were utilized in the standard evaluation according to the guidelines of the American Society of Echocardiography . All patCMR imaging was performed using a 1.5-T speed clinical scanner  before ventricular septal myectomy. All images were acquired with an electrocardiographically gated breath-hold technique.To evaluate functional parameters, electrocardiographic gating cine images were then acquired using a segmented, balanced, steady-state-free precession sequence. The images were acquired during multiple short breath holds (8–15 s). After scout images, cine imaging was performed in four chamber, three-chamber and two-chamber long- and short axis views with the following protocol: 6-mm-thick sections with a 2-mm gap between sections, repetition time, 2.7 ms; eho time, 1.2 ms; 70° flip angle; temporal resolution, 40 ms; field of view, 360 × 315 mm2; matrix, 192 × 162 pixels; pixel size, 1.9 × 1.3;slice thickness, 6 mm.All CMR images were analyzed using standard ventricular analysis software . For all patients, wall thickness at the septal, posterior and LV end-diastolic dimensions were all determined in the short-axis view (at the midpapillary level). Epicardial and endocardial borders of the LV myocardium were manually traced during the whole cardiac phase on each cine short-axis image to obtain LV and RV end-diastolic and end-systolic volumes, ejection fractions, and myocardial mass. Myocardial mass was calculated by multiplying the volume of the myocardium calculated at end-diastole by the specific gravity of the myocardium (1.05 g/ml). The end-diastolic volume index, end-systolic volume index, and mass index were indexed to body surface area. Late Gadolinium Enhanced (LGE) images were obtained 10–15 min after injection of 0.2 mmol/Kg gadolinium-DPTA.The extent of scarred myocardium was determined automatically by computer counting of all hyperenhanced pixels in the myocardium on each of the shortaxis images. Hyperenhanced pixels resembling LGE were defined as those with image intensities of 2SD, 4SD, 6SD, and 8SD above the mean of image intensities in a remote myocardial region in the same image. A percentage of LGE was then generated by the ratio of total LGE mass to total LV mass.We applied extended septal myectomy evolving from the classic Morrow procedure. The hypertrophic ventricular septal leading to systolic anterior motion of the anterior mitral valve and LVOT obstruction was resected. The resection range in the long-axis direction started from approximately 4 mm below the aortic ring to the apex of the left ventricle beyond the bases of the papillary muscles. In the shortaxis direction, the myectomy started rightward to the nadir of the right aortic cusp and to the left and terminated near the mitral anterior commissure. Part of the LV anterior free wall detached to the ventricular septal causing LVOT narrowing may also need to be resected. Furthermore, the anomalous chordal attachments between the mitral valve leaflflets or papillary muscle and the ventricular septal were also excised. Additional surgery was performed based on expert consensus among the experienced cardiac surgeons. If intraoperative transoesophageal echocardiography detected a postoperative LVOT gradient > 30 mmHg or more-than-moderate mitral valve regurgitation after weaning from cardiopulmonary bypass, reoperation was required.The septal myocardium samples were immediately fixed in 10% buffered formalin and embedded in paraffin. The samples were sectioned and stained with Masson’s trichrome staining for evaluating MF. Four images of every section were acquired with a projection microscope  or median and interquartile range according to their distribution. Categorical variables were presented as percentage frequency. Differences between groups were compared with the use of the Student t test (for parametric variables) and Mann–Whitney test (for nonparametric variables) for continuous variables. Categorical variables were compared with chi-square tests or Fisher’s exact chi-square tests. The Spearman’s correlation coefficient method was utilized to test for association between continuous variables. Patients were categorized in 2 subgroups according to values of CVF within  and above (subgroup with high CVF) the upper limit of normality . Receiver operating characteristic (ROC) analysis was plotted and the accuracy of the ROC curve was determined by measuring the area underneath it (AUC). Sensitivity, specifificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy for the presence of fQRS were defined as previously reported . MultivaSixty-nine HOCM patients who underwent ventricular septal surgery were included, and LGE was assessed in 64 patients. Among the HOCM patients, the mean age was 46.1 ± 14.4 years; 43 (62.3%) were male. fQRS was documented in 38 (55.1%) patients. A representative ECG with fQRS is presented in Fig. P > 0.05). The extent of LGE at 6SD was higher in patients with fQRS than those without fQRS . The baseline characteristics are listed in Table The LV mass index, septal thickness, LV end-diastole volume index, LV ejection fraction were all comparable between HOCM patients with and without fQRS . The mean CVF was higher in patients with HOCM than in controls , and 39 patients exhibited high CVF values [subgroup with high CVF 10.48% (7.16–13.33%)]. In the ROC analysis for the identification of patients with high CVF, the AUC, sensitivity and specificity of Q-fQRS, the number of leads showing fQRS and LGE at 6SD were 0.732 vs 0.715 vs 0.74, 66.7% vs 59% vs 68.6, and 76.7% vs 80% vs 72.4%, respectively. See Table .22%], anIn this study,we demonstrated the following points: 1. HOCM patients with fQRS exhibited more extensive MF than those with normal QRS wave morphology; 2. Q-fQRS showed good correlation with the fibrotic state of the myocardium and was an independent predictor for MF in HOCM patients; and 3. compared with LGE at 6SD, Q-fQRS showed a similar sensitivity and specificity in identifying patients with severe MF and could be used as an effective and cost-efficient predictor to evaluate fibrotic burden.MF is one of the main pathological characteristics in HCM patients . MF is aTwelve-lead ECG is a simple and cost-efficient tool. Patients with HCM frequently exhibit a wide spectrum of ECG abnormalities, especially in HCM patients with LV outflow track obstruction . fQRS waHowever, fQRS is not limited to patients with cardiac diseases. fQRS is also present in almost 19.7% of subjects without a known cardiac disease and was not associated with increased mortality in these subjects. Although fQRS morphologies could be early markers of subclinical cardiac disease , it cannOne limitation of this study was that this was a retrospective study, however, we prospectively enrolled HOCM patients from 2015, and all patients were diagnosed and managed by protocol. Second,because the controls were coming from autopsies of 9 individuals who died from accidents, we had no access to their medical history. Third, sample size was small and restricted to HOCM, which needed to be validated in a larger HCM patient cohort. Fourth, our study limited to single samples of the septum obtained at surgical myectomy, so sampling in different regions might be essential to detect distribution of MF in HOCM patients. Fifth, the lack of long-term longitudinal follow up data prevented us from analyzing the prognostic effect in HOCM patients with high CVF and the influence of collagen accumulation during the development of HOCM.In conclusion, HOCM patients with the presence of fQRS showed greater histological MF. Q-fQRS was an independent predictor to detect MF and had a good diagnostic value of 66.7% sensitivity and 76.7% specificity to identify patients with higher fibrotic burden.