Acta Pharmaceutica最新文献

{"title":"Combined shake-flask, chromatographic and <i>in silico</i> approaches for evaluating the physicochemical and ADME properties of aloin A and aloe-emodin.","authors":"Daniela Amidžić Klarić, Jelena Kovačić, Petra Bajt, Nikša Turk, Željko Krznarić, Emma Riordan, Ana Mornar","doi":"10.2478/acph-2025-0014","DOIUrl":"https://doi.org/10.2478/acph-2025-0014","url":null,"abstract":"<p><p><i>Aloe vera</i> has a long history of medicinal use due to its diverse biological activities, including antioxidant, anti-inflammatory and antimicrobial. This study investigates the physicochemical and ADME (absorption, distribution, metabolism, excretion) properties of two primary anthraquinones from <i>Aloe vera</i>, aloin A and aloe-emodin. The focus of this research was to evaluate the lipophilicity, solubility, and pharmacokinetic profiles of aloin A and aloe-emodin through a combination of computational predictions, the shake-flask method, and chromatographic techniques. The optimised shake-flask method was successfully employed to determine the log <i>P</i> values of phyto-chemicals. Aloin A was found to be more hydrophilic than aloe-emodin, likely due to the presence of an attached β-d-glucopyranosyl unit. All RP-TLC and RP-HPLC lipophilicity indices were higher for aloe-emodin compared to aloin A, aligning with their log <i>P</i> values (obtained through <i>in silico</i> and shake-flask methods). IAM (immobili sed artificial membrane)-HPLC results suggest that unlikely partitioning in the <i>n</i>-octanol/water system or C18 chains, partition into phospholipids involves not only hydrophobic intermolecular recognition forces but also electrostatic interactions. The presence of a sugar moiety (β-d-glucopyranosyl unit) at the C-10 position of aloin A considerably enhanced its affinity to phospholipids compared to its affinity to alkyl chains. HSA (human serum albumin)-HPLC and AGP (α1-acid glycoprotein)-HPLC data confirmed aloe-emodin's stronger affinity to plasma proteins. The integration of computational and experimental approaches provided a detailed understanding of aloin A and aloe-emodin physicochemical and ADME properties.</p>","PeriodicalId":7034,"journal":{"name":"Acta Pharmaceutica","volume":" ","pages":""},"PeriodicalIF":2.1,"publicationDate":"2025-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144214532","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of two different eco-friendly label-free platforms for analysis of selumetinib.","authors":"Sarah Alrubia, Wafa A Alshehri, Nourah Z Alzoman, Ibrahim A Darwish","doi":"10.2478/acph-2025-0013","DOIUrl":"https://doi.org/10.2478/acph-2025-0013","url":null,"abstract":"<p><p>Selumetinib (SEL) is a recently approved medication for paediatric patients who have neurofibromatosis type 1. It is the first approved therapy for this rare, debilitating, and disfiguring disease. Development of proper analytical platforms for SEL analysis in its marketed pharmaceutical formulation (Koselugo^® capsules) and blood plasma is highly warranted. Availability of such analytical tools would ensure SEL capsules' quality and effective therapy. This study introduces, for the first time, the development of two label-free and sensitive platforms for SEL quantification in capsules and human plasma. These platforms are microwave-assisted with an ultraviolet absorbance microplate reader (MW-UV) and reverse-phase high-performance liquid chromatography with a photodiode-array detector (HPLC-PDA). Both platforms employed the SEL native UV absorption as an analytical signal. The MW-UV measured the UV absorption in 96-well transparent plates at 255 nm. The HPLC-PDA involved chromatographic separation of SEL and tozasertib (TOZ), internal standard, on a C18 column both were detected at 255 nm. The optimum procedures of both platforms were established and validated following the ICH guidelines. The linearity ranges were 15-500 μg mL^-1 and 0.8-100 μg mL^-1, with limits of quantification of 15.3 and 3.5 μg mL^-1, for MW-UV and HPLC-PDA, resp. Both platforms displayed high precision with relative standard deviation values ≤ 1.8 %, and high accuracy with recovery ranging from 98.3 to 102.3 %. The platforms were successfully applied to quantify SEL in bulk form, Koselugo^® capsules, and were preliminarily applied to human plasma analysis. Eco-friendliness assessment confirmed the adherence of both platforms to green analytical approaches. MW-UV and HPLC-PDA are simple and fast, enabling high-throughput analysis, thus introducing valuable tools for routine use in quality control and clinical laboratories for SEL quantification.</p>","PeriodicalId":7034,"journal":{"name":"Acta Pharmaceutica","volume":" ","pages":""},"PeriodicalIF":2.1,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144214598","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phytochemical composition, antioxidant, antiglycation, and antihyperlipidemic activity of flowering parts from five plant species before and after <i>in vitro</i> digestion.","authors":"Valerija Vujčić Bok, Domagoj Bosiljevac, Ivana Šola, Ana Vukres, Gordana Rusak, Željan Maleš","doi":"10.2478/acph-2025-0012","DOIUrl":"https://doi.org/10.2478/acph-2025-0012","url":null,"abstract":"<p><p>This study evaluates the antihyperlipidemic (pancreatic lipase inhibition assay), antiglycation (inhibition of bovine serum albumin glycation, BSA glycation), and antioxidant activity (ABTS, DPPH and FRAP assays) of ethanolic extracts from flowering parts of five widely distributed plant species in Croatia - <i>Crocus heuffelianus</i> Herb. (tepals), <i>Nicotiana tabacum</i> L. (petals), <i>Malva sylvestris</i> L. (petals), <i>Calendula officinalis</i> L. and <i>Helianthus annuus</i> L. (both sterile ligulate flowers). An <i>in vitro</i>-simulated system of human digestion was employed to assess the bioaccessibility of the selected phenolics and the stability of the extracts' antioxidant, hypolipidemic, and antiglycation potential following each digestion phase. The concentrations of l-ascorbic acid, individual flavonoids, and phenolic acids were determined using RP-HPLC analysis. Principal component analysis revealed significant differences in the content of bioactive compounds and their biological activity among the investigated plant species. All original extracts exhibited high antioxidant capacity (> 70 %) in at least one assay, with <i>N. tabacum</i> and <i>H. annuus</i> demonstrating the strongest antioxidant capacity throughout digestion. <i>H. annuus</i> contained the highest levels of total identified phenolic acids, total identified phenols, and total identified compounds, while <i>N. tabacum</i> and <i>C. heuffelianus</i> exhibited the highest total flavonoid content. Among individual compounds, protocatechuic acid, quercetin, and ferulic acid significantly contributed to antioxidant activity. <i>N. tabacum</i> had the strongest antihyperlipidemic potential in the original extracts, as well as in the most digestion phases. Strong BSA glycation inhibition (70-100 %) was observed in all plant extracts across various digestion phases, with the exception of <i>C. heuffelianus</i>, which exhibited moderate inhibitory effects. These findings suggest that the analyzed flower-derived plant materials, some of which are often considered agricultural waste, can serve as sustainable and valuable resources of bioactive compounds for functional food, dietary supplements, and pharmaceutical applications.</p>","PeriodicalId":7034,"journal":{"name":"Acta Pharmaceutica","volume":" ","pages":""},"PeriodicalIF":2.1,"publicationDate":"2025-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144214599","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Design, synthesis, and evaluation of a novel fluorescent probe for competitive fluorescence polarization assay to screen galectin-8 inhibitors.","authors":"Edvin Purić, Marina Marinović, Zala Kojek, Barbro Kahl-Knutson, Hakon Leffler, Ulf J Nilsson, Marko Anderluh, Janez Mravljak","doi":"10.2478/acph-2025-0010","DOIUrl":"https://doi.org/10.2478/acph-2025-0010","url":null,"abstract":"<p><p>In the present work, we describe the design, synthesis, and evaluation of a galectin-8- binding fluorescent probe designed for a competitive fluorescence polarization (FP) assay for screening new galectin-8N inhibitors. The probe was characterized for its photophysical properties and its binding affinity for galectin-8N was determined by using FP. We evaluated the probe in a competitive FP assay with three known galectin-8N inhibitors and demonstrated its suitability for high-throughput screening.</p>","PeriodicalId":7034,"journal":{"name":"Acta Pharmaceutica","volume":" ","pages":""},"PeriodicalIF":2.1,"publicationDate":"2025-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143951887","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimization of 6-(trifluoromethyl)pyrimidine derivatives as TLR8 antagonists.","authors":"Nika Strašek Benedik, Valerij Talagayev, Troy Matziol, Ana Dolšak, Izidor Sosič, Günther Weindl, Gerhard Wolber, Matej Sova","doi":"10.2478/acph-2025-0011","DOIUrl":"https://doi.org/10.2478/acph-2025-0011","url":null,"abstract":"<p><p>Toll-like receptors (TLRs) are essential for the innate immune system as they recognize pathogen-associated molecular patterns and trigger immune responses. Overactivation of TLR8 by endogenous nucleic acids is associated with the development of autoimmune diseases and promotes inflammatory responses. This study presents the design, synthesis, and evaluation of a series of TLR8 antagonists based on the optimization of previously reported 6-(trifluoromethyl)pyrimidin-2-amines, with targeted modifications to further explore structure-activity relationships (SAR) and increase potency. A two-step synthesis involving nucleophilic aromatic substitution and Suzuki coupling was used to prepare two series of new compounds. The biological evaluation revealed that compounds <b>14</b> and <b>26</b> exhibited promising TLR8 antagonistic activity with <i>IC</i> ₅₀ values of 6.5 and 8.7 μmol L^-1, respectively. Compound <b>14</b> showed reduced cell viability at higher concentrations, while compound <b>26</b> showed no cytotoxic effects, making it a promising candidate for further investigation.</p>","PeriodicalId":7034,"journal":{"name":"Acta Pharmaceutica","volume":" ","pages":""},"PeriodicalIF":2.1,"publicationDate":"2025-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143959648","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"FOXD2-AS1 is modulated by METTL3 with the assistance of YTHDF1 to affect proliferation and apoptosis in esophageal cancer.","authors":"Zijin Wang, Xing Chen Liu, Zhen Gya Gao, Wo Da Shi, Wen Cai Wang","doi":"10.2478/acph-2025-0009","DOIUrl":"https://doi.org/10.2478/acph-2025-0009","url":null,"abstract":"<p><p>This study aims to investigate the regulatory mechanisms of METTL3, YTHDF1, and the long non-coding RNA FOXD2-AS1 in the proliferation and apoptosis of esophageal cancer, with the goal of providing a basis for molecular diagnosis and targeted therapies. Gene expression was evaluated using qRT-PCR (METTL3/14) and Western blot analysis. The Cell Counting Kit-8 (CCK-8) assay, flow cytometry, and Transwell assay were employed to assess cell proliferation and apoptosis. The EpiQuik m6A RNA Methylation Quantification Kit was utilized to quantify total m6A levels. The interaction between YTHDF1, FOXD2-AS1, and METTL3 was confirmed using RNA Binding Protein Immunoprecipitation (RIP), Co-Immunoprecipitation (CO-IP), and RNA pull-down assays. Methylated RNA Immuno preci pitation (MeRIP) was employed to assess the m6A modification levels of FOXD2-AS1. Tissue samples from animal models were analyzed via Hematoxylin-eosin staining (HE) staining and immunohisto-chemistry to assess METTL3 expression. The expression of <i>METTL3</i> was up-regulated in esophageal cancer tissues and cells. Flow cytometry and CCK-8 detection showed that silencing <i>METTL3</i> could inhibit the proliferation of esophageal cancer cells but accelerate their apoptosis. MeRIP-qPCR and Prediction of m6A-modified sites indicated that METTL3 regulated the m6A modification of FOXD2-AS1. <i>In vitro</i> and <i>in vivo</i> experiments showed that YTHDF1 binds to METTL3 and regulates the m6A modification of FOXD2-AS1 to affect esophageal cancer. Our results indicate that METTL3 regulates FOXD2-AS1 in an m6A-dependent manner through its interaction with YTHDF1, thereby influencing EC proliferation and apoptosis. This suggests a potential therapeutic target for the treatment of esophageal cancer.</p>","PeriodicalId":7034,"journal":{"name":"Acta Pharmaceutica","volume":"75 1","pages":"69-86"},"PeriodicalIF":2.1,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143957214","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The impact of blood lipids and statins on renal function and mortality in patients with diabetic nephropathy: A meta-analysis.","authors":"Dongqin Tian, Qian Chen, Lingli Zeng, Yan Hao","doi":"10.2478/acph-2025-0002","DOIUrl":"10.2478/acph-2025-0002","url":null,"abstract":"<p><p>The aim of this study is to explore the impact of blood lipids and statins on renal function and all-cause mortality in patients with diabetic nephropathy (DN). PubMed, Embase, Web of Science, and Cochrane Library were systematically searched until April 9, 2024, for relevant studies of blood lipids and statins on renal function and all-cause mortality in patients with DN. After the selection, total cholesterol levels (TC), total triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), estimated glomerular filtration rate (eGFR), urinary albumin excretion (UAE), serum creati-nine (SCR), end-stage renal disease (ESRD), and all-cause mortality indexes were extracted for finally meta-analysis. In total, 25 papers containing 21,411 patients with DN were finally included in this study. Levels of TC and LDL-C, which are continuous variables, were higher in DN patients who developed ESRD [TC/weighted mean difference (WMD) = 0.517, 95 % confidence interval (CI): (0.223, 0.812), <i>p =</i> 0.001; LDL-C/WMD = 0.449, 95%CI: (0.200, 0.698), <i>p</i> < 0.001]. In addition, this study also observed that statins may reduce UAE levels [WMD = -46.814, 95% CI: (-71.767, -21.861), <i>p</i> < 0.001]. Finally, the survey indicated that statins may be associated with an ESRD reduction [HR = 0.884, 95% CI: (0.784, 0.998), <i>p</i> = 0.045]. Blood lipids, particularly TC and LDL-C, may slow the progression of DN to ESRD. Besides, statins may protect the kidneys by lowering the excretion of UAE levels and reducing the risk of ESRD. Based on the above outcomes, the findings of this study provided robust evidence-based medical support for the future prevention, surveillance, and management of DN.</p>","PeriodicalId":7034,"journal":{"name":"Acta Pharmaceutica","volume":" ","pages":"1-22"},"PeriodicalIF":2.1,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142833403","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cloning, expression, and purification of recombinant AKR1D1 for therapeutic applications.","authors":"Kristina Shutevska, Aleksandra Kapedanovska Nestorovska","doi":"10.2478/acph-2025-0003","DOIUrl":"10.2478/acph-2025-0003","url":null,"abstract":"<p><p>AKR1D1, a key enzyme in the aldo-keto reductase superfamily, plays a dual role in both steroid metabolism and bile acid synthesis by catalyzing the NADPH-dependent reduction of carbon-carbon double bonds, specifically converting 3-ketosteroid hormones into 5β-steroids. Positioned at the critical intersection of steroid hormone and bile acid metabolism, AKR1D1 has the potential to profoundly influence metabolic homeostasis and drug metabolism. Despite its importance, the enzyme's therapeutic implications and role in drug metabolism remain underexplored. This study presents an optimized methodology for the cloning, expression, and purification of AKR1D1 using an <i>Escherichia coli</i> expression system. We identified optimal conditions for ligation and precise DNA sequencing, emphasizing the need for lower DNA concentrations and higher purity. Protein expression was evaluated in <i>E. coli</i> strains BL21 and Rosetta, with the highest yields achieved under extended incubation at 25 °C with controlled IPTG concentrations. Using freshly transformed cells was essential for maintaining consistent protein expression. The enzyme's activity was confirmed using a spectrofluorometric assay, demonstrating efficient reduction of testosterone to 5β-DHT. This optimized methodology facilitates the production of AKR1D1 with high specific activity, establishing a valuable platform for future research. It enables a deeper investigation into AKR1D1's contributions to drug metabolism and its therapeutic potential.</p>","PeriodicalId":7034,"journal":{"name":"Acta Pharmaceutica","volume":" ","pages":"147-157"},"PeriodicalIF":2.1,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142833396","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comparative efficacy and safety of vedolizumab and antitumor necrosis factor alfa in patients with inflammatory bowel diseases: A meta‑analysis.","authors":"Yafang Li, Jin Ding, Chong Lu, Yiping Hong, Qunying Wang","doi":"10.2478/acph-2025-0004","DOIUrl":"10.2478/acph-2025-0004","url":null,"abstract":"<p><p>This meta-analysis directly compares the efficacy and safety of vedolizumab and tumor necrosis factor-α (TNF-α) inhibitors for patients with inflammatory bowel disease (IBD), contrary to the previous one which provided an indirect comparison. In this meta-analysis, only the studies that directly compared two treatments (vedolizumab and TNF-α inhibitors) to each other (head-to-head approach) were considered. A comprehensive literature search was conducted using the following databases: PubMed, Embase, the Cochrane Library, and Web of Science. The pooled estimates of efficacies and safety were calculated as relative risk (RR) and 95 % confidence interval (CI). The presence of bias in the published material was evaluated using Begg's test. Sensitivity analysis was used to evaluate the pooled results' robustness. In total, 32 eligible studies were finally included. Results showed that the efficacy of vedolizumab was superior to TNF-α inhibitors in clinical remission [1.26, 95 % CI: 1.15-1.39]. Moreover, the vedolizumab group showed a reduced incidence of severe adverse events (RR = 0.63, 95 % CI: 0.42-0.94) compared to TNF-α inhibitors. Our results revealed superior efficacy and safety of vedolizumab compared to TNF-α inhibitors, which provided direct evidence for the use of vedolizumab in IBD treatment. Future studies are needed to confirm our findings.</p>","PeriodicalId":7034,"journal":{"name":"Acta Pharmaceutica","volume":" ","pages":"23-40"},"PeriodicalIF":2.1,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143603302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Acute toxicity and hepatoprotective effect of <i>Arum maculatum</i> on rat liver cirrhosis induced with thioacetamide.","authors":"Riyadh Zainadin Mawlood, Kamaran Abdoulrahman","doi":"10.2478/acph-2025-0007","DOIUrl":"https://doi.org/10.2478/acph-2025-0007","url":null,"abstract":"<p><p><i>Arum maculatum</i> is a medicinal plant that has been employed in traditional medicine for treating liver diseases. The objective of the current study was to evaluate the hepatoprotective impacts of ethanolic extract of the <i>A. maculatum</i> leaves on cirrhosis induced by thioacetamide (TAA) in Sprague--Dawley rats. The rats were treated for two months with thioacetamide (TAA) administered intraperitoneally thrice weekly. Histopathological examination revealed severe liver damage in the thioacetamide control group, while the silymarin treatments (<i>p</i> < 0.05). Furthermore, <i>A. maculatum</i> treatment led to the normalization of pro-inflammatory cytokines TNF-α and IL-6, and increased expression of the anti-inflammatory cytokine IL-10 (<i>p</i> < 0.05). Thus, <i>A. maculatum</i> leaves might have a hepatoprotective role in rat liver cirrhosis induced by TAA, along with antioxidant and anti-inflammatory effects.</p>","PeriodicalId":7034,"journal":{"name":"Acta Pharmaceutica","volume":"75 1","pages":"87-102"},"PeriodicalIF":2.1,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143959809","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}