Pseudomonas aeruginosa lectin is purified and nanoparticle‐conjugated in an attempt to inhibit biofilm formation. Thirteen (23.6%) P. aeruginosa isolates are obtained from chicken meat samples, of which 30.8% are biofilm producers and 69.2% are lectin producers. Lectin is purified 36.8‐fold to final specific activity of 506.9 U/mg. Four nanoparticle types are prepared via laser ablation: platinum (Pt), gold (Au), silica oxide (SiO2), and tin oxide (SnO2). The four types are characterised, and pulse feeding is used to conjugate the lectin and nanoparticles. Pt, Au, SiO2, and SnO2 nanoparticles inhibit biofilm formation, especially SiO2 nanoparticles, which have higher effectiveness when conjugated with purified lectin. SiO2‐conjugated lectin significantly (p < 0.05) inhibits biofilm formation more effectively than control and other nanoparticle‐conjugated lectins. Au‐, Pt nanoparticle‐, and SnO2‐conjugated lectins inhibit biofilm significantly compared with control (p < 0.05), and rhlR gene expression is decreased in the presence of SiO2‐conjugated lectin. Furthermore, lectin and Pt, Au, SiO2 and SnO2 nanoparticles separately, and their conjugated lectins, are effective biofilm inhibitors. Of these, SiO2‐conjugated lectin was most significant as an anti‐biofilm. Moreover, virulence factors regulon and RhlR were reduced by SiO2‐conjugated lectin, indicating that this conjugation may also decrease the virulence of P. aeruginosa. Viruses, bacteria, and mould can contaminate chicken meat during processing [70nm×100].2.9rhlR gene) expression was measured by real‐time RT–PCR after and before treatment with the lectin–SiO2 nanoparticles that exhibited the best antibacterial ability. SiO2 nanoparticles and lectin at 1.01 ng/ml of purified lectin were used. Late log phase culture of P. aeruginosa was used for RNA extraction by the RNeasy kit . The rpsL (16S rRNA gene‐above primer set) served as the normalising gene. The primers for rhlR expression were 5ʹ‐GACCAGGAGTTCGACCAGTT‐3ʹ and 5ʹ‐GGTAGGCGAAGACTTCCTTG‐3ʹ, and the probe was 5ʹ‐CCGACGACCGACGCCCGAC CT‐3ʹ. The Brilliant QRT–PCR Master Mix  kit was used for the gene expression experiments. The samples were run three times [Quorum‐sensing (QS) regulatory gene  as shown in Figure Thirteen (23.6%) rpsL gene amplicon of P. aeruginosa. Lane M: 100 bp DNA ladder, lanes 1 and 2 are positive results with 201 bp amplicon and negative control. The electrophoresis was run in 1.5% agarose gel, 7 V/cm for 90 min.The agarose gel electrophoresis of 3.2P. aeruginosa isolates showed haemagglutination activity. P. aeruginosa C9 showed a higher haemagglutination value and reached a higher titre (128 U/ml), as shown in Figure Haemagglutination activities in a glass slide and microtitre plate revealed that 9 69.2%) of 13 % of 13 P3.3P. aeruginosa isolate, and haemagglutination activity was raised from 128 to 256 U/ml, and specific activity was 62.43 U/mg. The extracted lectin subjected to the first step of purification included precipitation of lectin by ammonium sulphate at 60% saturation as reported in Table First, lectin was extracted from C9 3.43.4.12, and SnO2 sizes increased with increases in the spectra of the absorption.The variation of the absorbance (A) with the wavelength (nm) for the nanoparticle thin films is shown in Figure 3.4.2The X‐ray diffraction spectrum of the four nanoparticles is shown in Figure 2, and 100 oriented SiO2 were calculated using the following equation:The crystallite size of 200 oriented Au, 111 oriented Pt, 110 oriented SnO3.4.32, and SnO2 nanoparticles were 25, 17, 18, and 15 nm, respectively. Figure 2‐conjugated lectin.Figure 3.5P. aeruginosa isolates, only four (about 30.8%) produced different capabilities to form a biofilm. The anti‐biofilm activity of the purified lectin (1.01 ng/ml) was only about 70%. On the other hand, the anti‐biofilm activity of the nanoparticles separately, as shown in Figure P. aeruginosa that formed biofilm were also used. The presence of lectin with SiO2 and Au separately led to inhibition of biofilm formation  inhibits biofilm formation compared with results from control and other nanoparticle‐conjugated lectins . Considering these results, SiO2‐conjugated lectin was chosen for the gene expression experiments.SiO3.6rhlR gene was determined after and before treatment with lectin and SiO2 nanoparticles. The expression of the rhlR gene was decreased to 5.26‐fold (Table The expression of the ld Table .4P. aeruginosa isolates in the current study. The Pseudomonas species are one of the causes of food decomposition [Salmonella spp, Escherichia coli, Klebsiella spp, Pseudomonas spp, and others—are contaminants of chicken meat. The odours that are mostly associated with spoiled poultry, such as discharge odours, were produced by Pseudomonas spp [Pseudomonas is the predominant spoilage bacteria found in fresh broiler chicken.About a quarter of the samples of the chicken meat are contaminated with position , 27, 28.position  found thonas spp . Kanatt onas spp  also revP. aeruginosa isolates can produce biofilm as reported here and elsewhere [P. aeruginosa biofilm [2 and Au separately, leading to inhibition biofilm formation ability by the CRA plate method. In the polystyrene plate method, biofilm inhibition was observed with the four nanoparticles that were conjugated with lectin. This may be the first report of such an effect.lsewhere . Howeverlsewhere , 35, 36.lsewhere . Ag nano biofilm . In thisP. aeruginosa to form biofilms on medically main devices [P. aeruginosa. Biofilms and QS are affected by the adjacent environment. Nevertheless, numerous associations between biofilm formation and QS have been documented. Two QS systems of P. aeruginosa were discovered; they are the N‐acyl‐homoserine lactone–based synthases, Rhl and Las. The Rhl system is composed of the RhlR and N‐butyryl‐l‐homoserine lactone [rhlR gene of P. aeruginosa has a fundamental effect on QS and encodes for the transcriptional regulator RhlR [https://www.ncbi.nlm.nih.gov/pubmed/14600219. It was found that rhlR gene expression decreases in the presence of SiO2‐conjugated lectin. RhlR regulon regulates a set of virulence factor genes such as elastases, rhamnolipids, and pyocyanin [The QS systems contribute to the capability of  devices . QS play lactone . The rhltor RhlR , 42 httpyocyanin . Conjuga2, and SnO2 nanoparticles individually are biofilm inhibitors. Pt‐, Au‐, SiO2‐, and SnO2‐conjugated lectins are effective biofilm inhibitors, while SiO2‐conjugated lectin is the most significant as an anti‐biofilm. Moreover, SiO2‐conjugated lectin lowered the levels of virulence factors regulon and RhlR, and as a consequence, reduced the virulence of P. aeruginosa.In conclusion, lectin and Pt, Au, SiO


Paratylenchus nanus collected from the type locality in Four Mile Run, Fall Church, Virginia using COI, D2-D3 of 28 S rRNA and ITS rRNA gene sequences was provided. We molecularly also characterized, Paratylenchus specimens collected from grasses in Devils Lake, Ramsey County, North Dakota indicated as the type locality in the original description of P. nanus by Cobb (1923). These nematodes were identified as representatives of the species P. projectus. Populations of P. nanus belonging to the molecular types A and B, and previously designated by Van den Berg et al. (2014) should be now identified as P. nanus and P. projectus, respectively.In this study, molecular characterization of  Paratylenchus nanus was described by P. nanus , which were well separated using molecular criteria, but had very similar morphometrics. These authors also noticed intraspecific variation in shapes of lip region and tail. Thus, the designation of the true P. nanus species remained to be unresolved.P. nanus from the type locality, Four Mile Run, Fall Church, Virginia, USA designated by Paratylenchus species from the roots of grasses collected in Devils Lake, North Dakota, which was also indicated as the type locality in the original description of P. nanus by The objective of this work was to molecularly characterize Paratylenchus nematodes (nematode sample code = CD3326)Several sampling trips were conducted to Devils Lake, Ramsey County, North Dakota and Four Mile Run, Fall Church, Virginia to collect nematodes. Soil samples were arbitrarily collected from the grassland in Devils Lake, Ramsey County, North Dakota in 2015 and 2016 . Sampling was conducted from June to October each year. In each sampling spot, the top, dry soil (1–2 cm) was removed and the remaining soil was collected up to a depth of 30 cm using a soil probe (2.5 cm in diameter and 30 cm in depth). Each soil sample consisted of a composite of 10 to 15 soil cores and soil samples were placed in plastic bags. The soil with grasses was also placed in a glasshouse for several weeks and then sampled for nematodes . Several soil samples were also collected along Four Mile Run, Fall Church, Virginia in September 2020. Nematodes were extracted from soil using sieving and decanting as well as the sugar centrifugal-flotation method . One samParatylenchus female specimens extracted from grass soil samples were morphologically examined and photographed with an automatic Infinity 2 camera attached to a compound Olympus BX51 microscope equipped with a Nomarski interference contrast, and then these specimens were used for molecular study.Several COI gene  were aligned using ClustalX 1.83  with their corresponding published gene sequences  were applied to generate the D2–D3 of 28 S rRNA gene alignment. Sequence datasets were analyzed with Bayesian inference (BI) using MrBayes 3.1.2 as described by The new sequences for each gene  µm; W = 17.0 ± 0.9 (16.0–18.9) µm; a = 22.2 ± 1.1 (20.6–23.5); b = 3.7 ± 0.2 (3.4–3.9); c = 16.0 ± 2.3 (13.7–18.4); V = 83.0 ± 1.6 (80.9–85.0)%; stylet = 31.2 ± 0.8 (30.3–32.4) µm; pharynx = 101.2 ± 2.9 (96.0–104.3) µm; anterior end to median bulb = 56.9 ± 1.8 (53.8–60.0) µm; anterior end to excretory pore = 78.3 ± 3.2 (73.8–83.3) µm; tail = 23.1 ± 3.4 (20.2–26.9) µm.Several P. projectus. Females had short (330 µm) body and stylet 28–31 µm long.Two females and several juveniles extracted from samples collected from grasses in Devils Lake, Ramsey County, North Dakota were similar to those described by P. nanus is very similar to P. projectus and the characters most important in distinguishing these two species as indicated by P. projectus, head rounded with distinct annuli, not set off, in P. nanus P. nanus .P. nanus within selected Paratylenchus are given in P. nanus from Virginia were identical to that of P. nanus type A from California provided by P. projectus from North Dakota were identical to those previously identified as P. nanus type B from California (P. nanus from North Dakota (P. projectus.The alignment generated with modified parameters was 756 bp in length and contained 41 sequences. Phylogenetic relationships of lifornia  or as P.h Dakota  and South Dakota . All theP. nanus within selected Paratylenchus are given in P. nanus from Virginia was identical to that of P. nanus type A from California. New sequence of P. projectus from North Dakota was identical to those of previously identified as P. nanus type B from South Africa (P. nanus from North Dakota (P. projectus.The alignment generated with default parameters was 885 bp in length and contained 35 sequences. Phylogenetic relationships of h Africa  or as P.h Dakota  and South Dakota , and nowP. nanus within selected Paratylenchus are given in P nanus from Virginia were identical to those from P. nanus type A from Belgium provided by Paratylenchus projectus formed two clades: a and b in the phylogenetic trees. New sequence of P. projectus from North Dakota was identical to those of this species from this state by P. nanus type B from South Korea (P. projectus.The alignment generated with default parameters was 721 bp in length and contained 39 sequences. Phylogenetic relationships of th Korea  and now P. nanus. Specimens of Paratylenchus collected from this location belong to P. nanus molecular type A according to P. nanus. Paratylenchus specimens collected from the roots of grasses collected in Devils Lake, North Dakota, in the location also mentioned in Cobb’s description of P. nanus are considered here as representatives of P. projectus as they have been already identified by In this study, we consider Four Mile Run, Fall Church, Virginia, USA as the type locality for


Forsythia suspensa (Thunb.) Vahl (F. suspensa) is a traditional Chinese medical herb and only its fruit is currently used in clinical therapies. However, the discarded parts like leaves also contain a large number of active components. In this study, we used macroporous adsorption resin to enrich the effective components from F. suspensa leaves. The separated active compounds were then identified and quantified by ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC/Q-TOF MS) and high-performance liquid chromatography Active components with antibacterial properties extracted from F. suspensa leaves were confirmed in vitro and the corresponding mechanisms were explored. In sum, a stable and effective method for extracting antibacterial active components from F. suspensa leaves was established in this study, which proved the practicability of F. suspensa leaves as traditional Chinese medicine and is conducive to the more comprehensive utilization of the plant. Forsythia suspensa (Thunb.) Vahl (F. suspensa) has been widely used in clinical settings. However, the leaves of Forsythia suspensa receives little attention, in spite of their great medicinal value. Currently, F. suspensa leaves are mainly used for the treatment of pyrexia, inflammation, gonorrhea, carbuncle, and erysipelas. Modern pharmacological research has revealed that its pharmacological effects include anti-inflammatory, antioxidant, antibacterial, anti-cancer, anti-virus, anti-allergy, neuroprotective functions  was developed, and the antibacterial mechanism was discussed . The HPLC analyses were performed on Shimadzu 2010 CH HPLC system . The vertical automatic high-pressure steam sterilization pot was used for the bacteria experiment . Nitrogen blowing was carried out on a CM-12J type nitrogen blowing instrument . A scanning electron microscope (SEM) Hitachi S-3400N equipped with an X-radiation detector EDS (Voyager of noran instruments). Gold thin films and nanoparticles were prepared by an ion coater . UV-visible spectrometry and multi-function microplate reader were used . Reverse transcription polymerase chain reaction (RT-PCR) amplifications were performed on LineGene 9660 model FQD-96A .The UPLC/Q-TOF MS analyses were performed on an AB SCIEX Triple-TOFTM 5600AB-8 MARs were purchased from Suzhou Donghong Chemical Co., Ltd . HPLC-grade ethanol, methanol, and acetonitrile were purchased from Merck . Gentamicin sulfate injection was purchased from Henan Runhong Pharmaceutical Co., Ltd . 2.5% EM grade glutaraldehyde was purchased from AGAR Scientific . TRIzol™ reagent was purchased from Thermo Fisher Scientific . Water was purchased from Guangzhou Watsons Food & Beverage Co., Ltd  and phosphate-buffered saline (PBS) was purchased from Invitrogen Co. Carlsbad . Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich Company Ltd. . Forsythiaside A, forsythin, chlorogenic acid, and rutin standard  . Reverse transcription kit and Real-Time PCR kit were purchased from Takara Biotechnology Co., Ltd  and an alkaline phosphatase assay (AKP) Kit was purchased from MyBioSource, Inc . Mueller-Hinton (MH) agar medium, MH broth medium, and Lysogeny broth (LB) broth were prepared according to the manufacturer’s instructions and purchased from Qingdao Gaokeyuan Haibo Biotechnology Co., Ltd .Escherichia coli (E. coli) O157:H7 (CICC 10899) and Staphylococcus aureus (S. aureus) (CICC 21601) strains were obtained from the China Center of Industrial Culture Collection .The F. suspensa leaves were collected in May 2019 from Tunliu County, Shanxi Province, and identified as F. suspensa leaves by Jinhai Huo . Finally, the leaves were picked and dried in a ventilated place.The We modified a method of Sun partially and obtained a stable method for enriching phenethyl alcohol glycosides in leaves . A totalSample preparation for HPLC: The forsythiaside A standard was dissolved in methanol to prepare a sample with a concentration of 0.106 mg/ml. The A, B, C, D, and E elution were concentrated 5 times and filtered to obtain the test solution ). HPLC performance parameters were in the Supplementary Material.HPLC analysis: The chromatographic system is equipped with a quaternary gradient pump, online degasser, auto-sampler, column thermostat, and Shimadzu liquid chromatography workstation. The chromatographic separation was performed on an Agilent ZORBAX C18 column . Methanol-0.8% glacial acetic acid was used as the mobile phase, the flow rate was 0.8 ml/min. The detection wavelength was 280 nm, the injection volume was 10 μL, and the column temperature was 30 °C. The solvent gradient adopted was as follows: 0–6 min, 85%–74% B; 6–20 min, 74% B; 20–25 min, 74%–70% B; 25–30 min, 70%–63% B; 30–45 min, 63% B; 45–60 min, 63%–61% B.Sample preparation for UPLC/Q-TOF MS: Standards of forsythin, chlorogenic acid, and rutin were dissolved in methanol solution to prepare a sample with a concentration of 95, 304, 138 μg/ml. The C elution solution (FC) was diluted 5 times and filtered ).UPLC/Q-TOF MS analysis: The chromatographic separation was performed on a Waters ACQUITY UPLC BEH C18 column with 1.7 μm, 2.1 mm × 100 mm, and equipped a BEH C18 VanGuard Pre-Column with 1.7 μm, 5 mm × 2.1 mm, from the same supplier. The composition of the two mobile phases was 0.1% (v/v) formic acid in water (A) and acetonitrile (B): 0–5 min, 5%–25% B; 3–13 min, 25%–80% B; 13–23 min, 80%–100% B; 23–23.1 min, 100%–5% B; 23.1–25 min, 5% B. The separations were performed with a constant flow rate of 300 μL/min. The column oven and tray cooler temperatures were set to 30 and 4°C, respectively. 5 μL of samples were injected in a full loop injection mode. The electrospray MS detection was performed in positive and negative mode detection mode with the ion source voltage set to 5.5 kV/4.5 kV. Nitrogen was used as the sheath gas and helium as auxiliary gas with a flow rate of 10 and 0 arbitrary units, respectively. The mass spectrum scanning range is 80–1500 Da. The 80 V declustering potential, 35 eV/-35 eV collision energy, and 15 eV/-15 eV collision energy spread were used in mass detection. PeakView 2.0 was used to load published compounds into the MasterView function block for analysis.7 CFU/ml bacterial suspension was prepared by diluting an overnight culture in LB broth, and then spread evenly on the LB agar plate. Filter paper (6 mm diameter) was impregnated with 20 µL of FC and gentamicin sulfate injection (0.16 mg/ml) placed on inoculated agar, and incubated at 37 °C for 24 h. The antibacterial activity was evaluated by measuring the diameter of the inhibition zone against the tested bacteria.A standard agar disc diffusion method was used for the antibacterial assay. A standardized inoculum (100 µL) containing 107 CFU/ml (100 uL) were added to each well of a 96-well plate containing 100 uL of serially diluted FC in MH broth. After incubating at 37 C with shaking for 24 h, the MICs were obtained by measuring the optical density (OD) at 600 nm. Furthermore, the aliquots from wells displaying no growth were spread on nutrient agar plates and incubated at 37°C for 24 h. Compared with the initial bacterial inoculum, the lowest concentration of the sample solution in the well that reduced the growth of bacteria on the agar plate by 99.9% was expressed as MBC.The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of FC were determined by the method of two-fold broth micro-dilution . The str7 CFU/ml and incubate at 37°C with a 150 r/min shaking incubator for viable bacteria count. At 0, 20, 40, 60, 90, 120, 150, 210, and 300 min, 50 µL of bacterial suspension was applied to the surface of the MH nutrient agar which incubates in a 37°C incubator for 24 h and counts. 107 CFU/ml text bacterial suspension was mixed with the FC to make the final concentration of MIC, and cultured at 37°C, 150 r/min shakers, with sterile water as a blank group. The OD value of the solution at 600 nm was measured by a spectrophotometer every 2 h , and cultured in a 37 °C incubator for 24 h. The specimens were collected and fixed in 2.5% (v/v) glutaraldehyde for 8 h, and then continuously exposed to ethanol in a concentration range of 30–100% to dehydrate the samples. The ethanol was replaced by 100% tertiary butyl alcohol. The specimens were dried with CO2 and the dried cells were coated with gold in a sputter coater after dehydration.Effect on cell wall permeability: According to Juan Xie’s method, the AKP kit was used to detect cell wall permeability (5 CFU/ml) in 2 ml of LB broth was inoculated with the MIC level of FC and incubated at 37°C and 150 r/min in a shaking incubator. In addition, the sample was replaced with sterile water as a blank group. After mixing and culturing, 1 ml of the bacterial suspension was centrifuged at 3,500 r/min for 10 min. The absorbance of the supernatant was measured every 2 hs at 680 nm by a microplate reader.eability . Each 10Effect on membrane integrity: The FC at the MIC level was added to the tube with the bacterial inoculum. The cells were centrifuged at 3,500 r/min after 0, 60, 120, 180, 240, 300, and 360 min of treatment, and the absorbance of the obtained supernatant was determined at 260 nm by a microplate reader (e reader .Effect on membrane efflux pump gene expression: Total RNA samples were isolated from the bacterial cells using the TRIzol™ reagent (−ΔΔCt method using quantitative RT-PCR ( reagent . Reversee RT-PCR . ApproxiAfter the reaction, the RT-PCR amplification curves and splitting curves were observed and derived, and the Ct values of each group were output, and the reverse transcription polymerase chain reaction products were analyzed.All the assays were carried out in triplicate and all the data were recorded as mean ± SD.F. suspensa leaves was determined by HPLC (The content of forsythiaside A in  by HPLC . The con2 chromatogram and product ion spectrum of forsythiaside A is shown in The chromatographic conditions and mass spectrometry conditions were optimized for analysis and displayed the total ion chromatogram . 31 compin vitro antibacterial activity of FC against two bacteria was assessed based on the presence of inhibition zones (The on zones . The diaS. aureus and E. coli. The MIC of S. aureus and E. coli was 7.81 and 3.91 mg/ml, and the MBC was 7.81 and 15.63 mg/ml.The FC showed antibacterial activity against S. aureus and E. coli.By measuring the viability of the tested bacteria, their survival during the 300 min with FC treatment was inhibited. The growth of the two tested bacteria was inhibited at the MIC, and 50% inhibition of cell viability was detected within 20 min. After 210 min, 80% bacterial inhibition was observed, and all tested bacteria were completely inhibited by FC after 300 min . AccordiE. coli in the blank group had a regular rod shape, intact surface. The shape of most E. coli treated with FC became irregular, and shrinked to varying degrees. The shape of S. aureus in the blank group was a regular spherical shape, with complete cells and a smooth surface. After S. aureus was exposed to FC solution, its cell membrane appears pitted, shrunk, holes appear on the surface, which may even cause the cell to rupture and the contents to flow out. The changes of test bacteria may be caused by the destruction of the membrane and the loss of intracellular substances in E. coli and S. aureus caused by FC. (Through SEM, it was observed that d by FC. ., therefore, we conclude that FC can destroy the cell walls of S. aureus and E. coli. Proteins and nucleic acids are extremely important macromolecules for cells, providing cellular structure, function, and genetic information. According to the method of Paul, the absorbance of nucleic acids and proteins at 260 nm could be an indicator of membrane integrity (AKP is an intracellular enzyme. Normally, its activity can not be detected outside the cell. However, when the cell wall or membrane is destroyed, the permeability is increased and then AKP leaks out of the cell . Such pe, therefntegrity . With tintegrity . This rentegrity .E. coli to be resistant to many drugs. The expression of the norA gene can cause S. aureus to be resistant to many drugs. Therefore, the expression of the active efflux gene acrA/norA plays a crucial role in the drug resistance of E. coli/S. aureus. The dissociation curve of RT-PCR indicated that the melting temperature of the amplification product is stable, the amplification product is single, the primer design is correct, there are no unspecific products, and the test results are accurate. As shown in F. suspensa leaves in the next step.An in-depth study of specific drug resistance mechanisms was outside the scope of this study. However, a targeted study of some drug delivery pumps with clear pump functions can be used to screen new antibacterial drugs and resistance inhibitors . The expF. suspensa leaves, which had the advantages of being fast, efficient, economical, and recyclable. Compared with the traditional HPLC analysis, this research used UPLC/Q-TOF MS for the first time to identify 31 compounds in the extract of F. suspensa leaves, which deepened the understanding of their chemical constituents. At present, the research on F. suspensa leaves mainly focuses on the extraction, isolation, and structural identification of individual components, without the overall analysis and in-depth study of its pharmacological effects and the antibacterial mechanism has not been clearly explained. In this article, the antibacterial effect of leaves and its antibacterial mechanism have entered in-depth research, including the determination of the inhibition zone diameter, MIC and MBC, the growth curve, viable counts, external morphology changes, permeability/integrity of the cell wall/membrane, and changes in the expression of a cell membrane efflux pump gene. Taken together, our findings provide evidence that the extract of F. suspensa leaves exhibited an antibacterial effect that involves damaging the bacterial cell membrane and raising bacterial cell wall permeability abnormally. This is conducive to more comprehensive utilization of medicinal plant resources.In this paper, MARs were used to enrich the active components of


Polygonum multiflorum Thunb (PM) has been restricted or banned in many countries, due to its hepatotoxic adverse effects. Its toxicity research has become a hot topic. So far, the pharmacokinetic studies of PM, focusing on prototype compounds such as 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), emodin, and physcion, have been considered the main basis of pharmacodynamic material or of toxic effect. However, pharmacokinetic studies of its phase II metabolites have not yet been reported, mainly because the quantifications of such metabolites are difficult to do without the reference substance. In addition, pharmacokinetic studies on different pathological models treated with PM have also not been reported. On the other hand, toxic effects of PM have been reported in patients diagnosed with different liver pathologies. In the present work, a simultaneous quantitation method for eight prototypes components of PM and their five phase II metabolites has been performed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and used for the pharmacokinetic study of PM in two different liver pathological models in rats , and carbon tetrachloride (CCl4)). The results showed that the main blood-entering components of PM are TSG, emodin, physcion, emodin-8-O-β⁃D⁃glucoside (E-Glu), physcion-8-O-β⁃D⁃glucoside (P-Glu), aloe-emodin, gallic acid, resveratrol and catechin, among which TSG, emodin, and catechin were primary metabolized in phase II, while resveratrol was converted to all phase II metabolites, and the others were metabolized as drug prototypes. Meanwhile, their pharmacokinetic parameters in the different models also exhibited significant differences. For instance, the AUC (0-∞) values of the TSG prototype and its phase II metabolites were higher in the ANIT group, followed by CCl4 group and the normal group, while the AUC (0-∞) values of the emodin prototype and its phase II metabolites were higher in the CCl4 group. To further illustrate the reasons for the pharmacokinetic differences, bilirubin metabolizing enzymes and transporters in the liver were measured, and the correlations with the AUC of the main compounds were analyzed. TSG and aloe-emodin have significant negative correlations with UGT1A1, BSEP, OATP1A4, OCT1, NTCP, MRP2 and MDR1 (p < 0.01). These data suggest that when the expression of metabolic enzymes and transporters in the liver is inhibited, the exposure levels of some components of PM might be promoted in vivo.The clinical use of  Polygonum multiflorum Thunb (PM) is a traditional tonic Chinese medicine used to fortify the liver and kidneys, benefit the essence and blood, and darken the hair. Also, it has been widely used in clinical and health care products  and cholestatic/mixed liver injury (R < 5) according to the Roussel Uclaf Causality Assessment Method (RUCAM), with a high proportion of hepatocellular injury in clinical cases  . The metal cases . Bile dur injury . Herein,Polygonum multiflorum Thunb (PM) supplied by Shanghai Kangqiao TCM, Co., Ltd . The standards 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), emodin, aloe-emodin, physcion, emodin-8-O-β⁃D⁃glucoside (E-Glu), physcion-8-O-β⁃D⁃glucoside (P-Glu), gallic acid, catechin, resveratrol, and puerarin (≥98% purity) were all procured from the National Institute for the Control of Pharmaceutical and Biological Products . Sulfatase was obtained from Helix pomatia . The peptides LTIIPQDPILFSGSLR, GVALPETIEEAENLGR, AAATEDATPAALEK, TFQFPGDIESSK, LLLSGFQEELR, STALQLIQR, NTTGALTTR, EENLGITK, SVQPELK, and TYPVPFQR, as well as stable isotope-labeled internal standards (≥98% purity), were synthesized by Bankpeptide Biological Technology, Co., Ltd . The ProteoExtract native membrane protein extraction kit was purchased from Calbiochem . The BCA protein assay kit and in-solution trypsin digestion kit were obtained from Pierce Biotechnology . Formic acid was MS grade; ammonium bicarbonate (98% purity) and sodium deoxycholate (98% purity) were purchased from Sinopharm Chemical Reagent, Co., Ltd . Acetonitrile and methanol, all MS grade, were obtained from Merk . Acetonitrile and methanol, all MS grade, were obtained from Merk .Polygonum multiflorum Thunb (AE-PM) was prepared by immersing 90 g of PM in 540 ml of 75% ethanol  for 0.5 h. The sample was refluxed twice, each time for 1 h. The extraction solution was mixed and passed through a paper filter. Then, the filtrate was concentrated to 30 ml, and the final solution containing the crude drug presented a concentration of 3 g/ml. After that, we carried out a multicomponent assay, as described by Zhao MJ's .Male Sprague Dawley rats (200–220 g) were purchased from B&K laboratory Animal, Corp. Ltd , fed in the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine, and housed in an environmentally controlled animal room at a temperature of 22–24°C and a relative humidity of 60–65%. The animals were maintained on a 12:12 h light–dark cycle (lights on at 7:00 am) with regulated temperature and humidity. During the entirety of the experimental process, the rats were fed with certified standard rat chow and tap water To explore the pharmacokinetic properties of PM following intragastric administration in rats, a rapid and sensitive method involving the use of UHPLC-MS/MS  was developed and validated for the simultaneous quantification of nine active components in rat plasma. The quantification was conducted in ESI negative ionization mode, and mass spectrometry conditions were set up as follows: capillary voltage of 3500 V; gas flow at 12 L/min; nebulizer of 40 psi; the gas temperature of 350°C; and delta EMV (−) of 400. A 10 µl extraction sample was injected into the column  and eluted at 0.4 ml/min with a gradient elution of water (with 0.1% v/v formic acid) (A) and acetonitrile (B) . Multiple reaction monitoring parameters and chemical structures of nine chemical components in PM  are shown in A selective sample preparation method was applied to eliminate endogenous proteins’ interference and optimize extraction recovery. Among several chemical reagent methanol and acetonitrile, and acetonitrile combined with methanol and formic acid, were used. Moreover, the amounts of extraction solvent were all tested before. The application of triple methanol resulted in the highest sensitivity level and convenience, especially minimizing endogenous interference and enhancing extraction recovery. Therefore, we selected it as the optimal solvent for sample preparation.The sulfatase chosen for this experiment contains β-glucuronidase activity, which could hydrolyze both glucuronide and sulfate conjugation metablites of components in PM into a prototype drug. According to the product information, the optimal working temperature of the sulfatase is 37°C, and the pH value if 5. According our previous results , the maxA 50 µl aliquot of blood plasma sample was placed in a centrifuge tube with 50 µl of puerarin  solution , followed by 150 µl methanol, which was mixed for 5 min. Then, the mixture was centrifuged for 10 min at 15,000 rpm (4°C), the supernatant was transferred to a clean centrifuge tube and dried with nitrogen gas at room temperature. The residue was resuspended in 100 µl of methanol, then centrifuged at 15,000 rpm , and 10 µl of the supernatant was analyzed by UHPLC-MS/MS.Additional plasma samples (50 µl) were enzymatically hydrolyzed with 50 µl of enzyme solution  for phase II metabolite quantification, in accordance with our previous study . After bThe UPLC-MS/MS method for determining the nine ingredients in rat blood plasma was validated according to the current US FDA Bioanalytical Method Validation Guidance  . The folThe purpose of specificity analysis is to eliminate the interference of endogenous substances on the determination. Specificity was determined by comparing chromatograms of blank rat blood plasma obtained from six individual subjects with chromatograms of blood plasma samples obtained after AE-PM administration at a dose of 50 g/kg.2) least squares linear regression analysis. The LLOQ was determined at the lowest concentrations at the signal-to-noize ratio (S/N) ≥ 10.Calibration curves were constructed using the peak area ratios of the analytes to puerarin and by applying a weighted (1/xPrecision [expressed as the relative standard deviation (RSD)] and accuracy [expressed as the relative error (RE)] were calculated for three QC points . Six replicates of each QC point were analyzed to determine the interday accuracy and precision. This process was repeated three times over three consecutive days to determine the intraday accuracy and precision.Recovery was evaluated in six replicates at three different QC concentrations . The percentage recovery was determined by comparing the concentrations of the pre-extraction spiked QC samples prepared in a blank matrix  with the peak area of the post-extraction spiked QC samples prepared in an extracted blank matrix . Matrix effects were investigated on six independent sources of blank rat blood plasma by calculating the ratio of the peak area in the presence of matrix to the peak area in the absence of matrix at three different QC concentrations .The stability of standard analytes in rat plasma was evaluated under several conditions (time and temperature) by analyzing six replicates of the QC samples at three concentrations . Stability was investigated in terms of short and long-term stability, freeze and thaw stability, and post-preparative stability by using the developed method. Short-term stability was evaluated by storing QC samples at room temperature (25°C) for 24 h. Long-term stability was assessed after 60 days by a storage at −80°C. Freeze and thaw stability were determined after three freeze–thaw cycles at -80°C. In addition, post-preparative stability during storage in an auto sampler at 4°C for 24 h was investigated.n = 12 for each group: 1) normal group  ANIT group  CCl4 group (group 3). Group 1 served as non-treated controls, whereas group 2 was treated with 4% ANIT at a dose of 50 mg/kg (diluted in olive oil) to induce cholestatic liver injury. Additionally, group 3 represented the CCl4-induced hepatocytes injury model, which was treated with pure CCl4  on day 0, 50% CCl4 (diluted in olive oil)  on day 3, and 20% CCl4 (diluted in olive oil)  on day 6. After 24 h, five rats selection for serum and liver collection from each of the three groups. Blood samples were collected in a coagulation tube, and were centrifuged at 4°C for 15 min , The resultant serum was used for the ALT, AST, TBIL, DBIL, TBA and ALP assays. Liver samples were dissected and stored at −80°C for further analysis, whereas the central part of the right large lobe of the liver was used for histological examination.The rats were randomly distributed into three groups . This dosage was chosen based on previous studies to liver injury in normal rats after long-term administration. Blood was collected in heparinized tubes at 5, 15, 30, 45, 60, 120, 240, 360, 480, 720, 1,440, 2,880, and 4,320 min after administration. The blood samples were centrifuged at 5,000 rpm (4°C) for 7 min, and the supernatant plasma was harvested.4 model groups.The detection method for UGT1A1 and nine other transporter proteins in the liver was comprehensively investigated in our study (35). We analyzed the different expression levels of bilirubin metabolizing enzyme and transporters in the liver of rats from the healthy animals, ANIT, and CCl.An Agilent 1290 Infinity series UHPLC system coupled to an Agilent 6460 series MS/MS system  was applied to quantitate the signature peptides in ESI positive ionization mode. The mass spectrometry conditions were set up as follows: capillary voltage of 2000 V; gas flow at 8 L/min; nebulizer at 30 psi; gas temperature, 300°C; delta EMV (+) of 400. A 5 µl digest sample was injected into the column  and eluted at 0.4 ml/min with a gradient elution of water (with 0.05% v/v formic acid) (A) and acetonitrile (B) . The sequence of characteristic peptides that corresponded to UGT1A1 and the other nine transporter proteins were based on our previous experiments and listed in Total membrane protein was isolated (in triplicate) from liver tissue samples according to the Native Membrane Protein Extraction Kit protocol. Subsequently, protein concentrations were determined by the BCA Protein Assay Kit. 10 µl of 5 mg/ml (or lower concentration) of a hepatocyte membrane protein incubated with 20 µl of dithiothreitol (100 mM) and 50 µl of ammonium bicarbonate buffer . After incubation at 95°C for 5 min, 20 µl of iodoacetamide (20 mM) was added to the mixture, followed by incubation at 37°C for 20 min in the dark. To concentrate the samples, ice-cold methanol (0.5 ml), chloroform (0.2 ml), and water (0.2 ml) were added. After centrifugation at 4°C for 5 min at 16,000 g, the supernatant was discarded and the pellet was washed once with ice-cold methanol (0.25 ml) and resuspended with 40 µl of reconstitution solution  and 5 mM ammonium bicarbonate buffer). Finally, the protein sample was digested with 10 µl of trypsin. The protein-to-trypsin ratio was 25:1 (w/w). After incubation at 37°C for 24 h, the digestion reaction was quenched by 60 µl of IS cocktail (prepared in 15% acetonitrile in water). The samples were centrifuged at 5,000 g for 5 min at 4°C, and 5 µl of the supernatant was injected in the UHPLC-MS/MS system.p < 0.05 and highly significant when the p-value was <0.01 or p < 0.001. Spearman rank correlation coefficient, calculated by bivariate correlation analysis was used to evaluate the correlation between AUC values and expression levels of metabolic enzyme and transporters. All statistical analyses were performed with the SPSS Statistics system (SPSS vision 21.0). Pharmacokinetic data analyses were performed using DAS 2.1.1 software .All data were presented as mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) was used to compare the data between three groups. The differences were considered to be statistically significant when 4 groups, and the livers also showed different pathological changes compared to the control group. In order to confirm the feasibility of our two pathological models, we examined the serum biochemical parameters and liver sections. The biochemistry serum results of ALT, AST, ALP, TBIL, DBIL, and TBA were presented in 4 dose efficiently increased the serum levels of ALT and AST to 4.45 and 2.68-folds (p < 0.001) respectively, compared with the control group, indicating that severe hepatocyte damage occurred after CCl4 administration. TBIL, DBIL, TBA and ALP were significantly increased in rats with cholestatic liver injury. As shown in p < 0.001) respectively, suggesting a severe cholestatic liver injury occurred after continuous administration of ANIT compared with the control group.The serum biochemical parameters such as TBIL, DBIL, ALT, and AST, were significantly increased in ANIT and CCl4 group showed disturbed hepatocyte arrangement, large number of hepatocytes with fatty degeneration in the form of vacuoles, few inflammatory infiltration and necrosis compared with the healthy control group. Two models of pathological liver injury were successfully established considering the results of serum biochemical and histopathological examination.Liver sections from the healthy control group and the pathological model groups stained with hematoxylin and eosin were examined by microscopy to provide visual evidence. As shown in The method selectivity was evaluated by comparing chromatograms of six extracted blank plasma samples of different sources with those of spiked plasma samples containing nine compounds and puerarin. 2) was greater than 0.9811. The linear regression equations and correlation coefficients (R2) of the analytes are listed in The calibration curves of the nine compounds were constructed respectively by plotting the peak-area ratio of each analyte to puerarin (y) vs each analyte concentration (ng/ml) (x) in spiked blank rat plasma. The method was linear over the concentration range of 7.64–29,650.00 ng/ml for TSG, 7.67–38,375.00 ng/ml for emodin, 5.92–5,800.00 ng/ml for aloe-emodin, 6.06–13,100.00 ng/ml for physcion, 6.01–12,700.00 ng/ml for E-Glu, 6.42–2,468.00 ng/ml for P-Glu, 6.66–2,560.00 ng/ml for gallic acid, 9.28–9,500.00 ng/ml for catechin, and 8.39–17,200.00 ng/ml for resveratrol. A signal noise ratio (S/N) ≥ 10 at the LLOQ was observed for all the analytes. In addition, it was observed that the LLOQ was7.64 ng/ml for TSG, 7.67 ng/ml for emodin, 5.92 ng/ml for aloe-emodin, 6.06 ng/ml for physcion, 6.01 ng/ml for E-Glu, 6.42 ng/ml for P-Glu, 6.66 ng/ml for gallic acid, 9.28 ng/ml for catechin, and 8.39 ng/ml for resveratrol. And the square of correlation coefficients  and five phase II metabolites  could be detected by UPLC-MS/MS method. Resveratrol has been fully converted into a phase II metabolite and could not be detected directly. The mean plasma concentration-time profiles of TSG, emodin, physcion, aloe-emodin, callic acid, catechin, E-Glu, and P-Glu in normal, cholestatic and hepatocytes damaged rats are shown in 4 model. The AUC (0-∞) values of emodin, physcion and E-Glu increased by 75.69, 29.24, and 655.90% respectively in CCl4 model group, and the Cmax values increased by 57.02, 173.72, and 620.15% respectively. While in the ANIT model, the AUC (0-∞) values of emodin, physcion, and E-Glu decreased by 55.98, 49.77 and 8.25%, respectively, the Cmax values decreased by 85.18, 29.48, and 22.98%, respectively.Compared with the normal group, the AUC (0-∞) and Cmax values of TSG in the ANIT model increased by 2,548.17% and 560.12%, respectively, and 802.14 and 236.59% respectively in the CClin vivo is to combine these radicals with endogenous α-D-glucuronide to produce β - glucuronide. PM mainly contains stilbene glycosides, anthraquinones, tannins and other components, as shown in in vivo. The metabolic pathway of these compounds are related to the metabolic pathway in bilirubin, which may affect the key enzymes and transporters of bilirubin metabolism , while its metabolism in ANIT group was significantly inhibited. The trends of AUC(0-∞) and Cmax of TSG in different models were consistent with the prototype, and emodin exhibited a similar pattern. The AUC(0-∞) and Cmax values of II phase metabolite of TSG were the highest in the ANIT model, and increased by 2,943.73% and 1917.18% respectively compared with the normal group; the AUC(0-∞) and Cmax values of emodin II phase metabolites were the highest in the CCl4 group, and increased by 522.45 and 250.27%, respectively, when compared with the normal group.The pharmacokinetic curves of phase II metabolites are illustrated in p < 0.05), respectively, when compared with the control group. Although the mechanism of CCl4-induced liver injury was significantly different from that of ANIT, it also showed a more obvious inhibitory effect on the expression levels of liver metabolic enzymes and transporters. Compared with the control group, the expression levels of UGT1A1, BSEP, OATP1A4, OCT1, NTCP and MDR1 were reduced by 31.18, 26.92, 46.11, 22.30, 15.75, and 34.15%, respectively (p < 0.05).The expression levels of metabolic enzyme and transporters in the rat livers were used to evaluate the liver status of rats in different pathological models. The quantitative results of the metabolic enzyme UGT1A1 and the nine transporters  are listed in in vivo. The critical enzymes and transporters located on the hepatocyte membrane are involved in the uptake, transformation and excretion of endogenous or exogenous substances by the liver. To further investigate the reasons for the differences in the pharmacokinetics of PM in different liver pathological models, we made a correlation analysis between the AUC values of the active ingredients in PM and the expression levels of metabolic enzymes and transporters in the liver. The results are shown in The area under the curve (AUC) is an important indicator to evaluate the degree of drug absorption, reflecting the exposure characteristics of the drug ρ = −0.832, ρ = −0.875, ρ = −0.914), BSEP , OCT1  and MDR1  (p < 0.01) were significantly correlated with the exposure characteristics of TSG prototype and its phase II metabolites and aloe-emodin in vivo, respectively. In addition, the expression levels of NTCP, MRP2 and OATP1A1 were found in relation to the AUC values of TSG prototype  and its phase II metabolites , aloe-emodin  (p < 0.01), emodin , physcion , P-Glu  and gallic acid  (p < 0.05), respectively. And the expression levels of OATP1A2 and MATE1 significantly correlated with the AUC values of phase II metabolites of resveratrol , emodin , physcion , P-Glu , gallic acid  (p < 0.01) and E-Glu  (p < 0.05). Besides, the expression levels of OATP1A4 were significantly correlated with the AUC values of aloe-emodin (ρ = −0.702), the phase II metabolites of emodin (ρ = −0.834) (p < 0.01), the prototypes of TSG (ρ = −0.573) and catechin (ρ = 0.560) (p < 0.05) and their phase II metabolites  (p < 0.01).The expression levels of UGT1A1  values of phase II metabolite of resveratrol in control, ANIT, and CCl4 groups were 18,593.48, 10,375.62, and 91,942.21 μg/L, respectively. The conversion rates of phase II metabolites of TSG and emodin among the three groups did not present significant differences. However, overall, the exposure levels of phase II metabolites of TSG and emodin were higher in both pathological models. Catechins and resveratrol were more inclined to undergo phase II metabolism in the CCl4 model. However, the expression level of UGT1A1 involved in phase II metabolism was significantly inhibited in ANIT and CCl4 models. It is possible that the drug might be more susceptible to phase II metabolism due to the compensatory increase of UGT1A1 in both pathological models.The pharmacokinetic characteristics of the nine components of AE-PM were studied by using UPLC-MS/MS. The sulfatase hydrolysis technique was used for the indirect determination of phase II metabolites of TSG, emodin, resveratrol and catechins. Physcion also undergoes glucuronidation and sulfation, consistent with the results of the Zhang J.’ study , but the4 models was 120 min, to the other components in the different groups were less than 60 min. Aloe-emodin, emodin prototype and its II phase metabolite presented Tmax values smaller than 30 min, indicating that these components absorbed rapidly. The AUC(0-∞) and Cmax values of the TSG prototype and its phase II metabolites were higher in the ANIT model, followed by CCl4 group, and the normal group. These two components had higher exposure and slower elimination in both pathological states. The AUC(0-∞) and Cmax values of emodin, physcion, phase II metabolites of emodin, and resveratrol were higher in the CCl4 model, followed by the normal group and the ANIT group. The liver cell damage caused by CCl4 would promote their absorption into the blood and inhibit its elimination, while cholestasis could hinder the absorption. Aloe-emodin had higher exposure and slower elimination in ANIT group. Catechins and P-Glu could be rapidly absorbed and eliminated in the CCl4 model, but cholestasis could inhibit their elimination. The absorption and elimination of gallic acid could be inhibited in the case of cholestasis. The phase II metabolite of catechin could be absorbed and eliminated rapidly in the normal group but inhibited in ANIT and CCl4. The exposure levels of physcion, emodin, E-Glu, phase II metabolites of emodin and catechin were higher in CCl4-induced pathological state. In contrast TSG, aloe-emodin, gallic acid and the phase II metabolite of TSG were higher in the cholestasis state.The Tmax value of phase II metabolite of TSG in CCl4 groups after administration of AE-PM for 72 h, compared with those when AE-PM was not administered. Our results might suggest that PM could upregulate the expression levels of bilirubin metabolizing enzymes and transporters in injured liver and downregulate them in the normal liver. Finally, the mechanism of hepatic metabolizing enzymes and transporters that affect the metabolism of the active ingredients in PM needs further investigation.There was a correlation between the exposure characteristics of the active ingredients in PM and the expression levels of liver metabolizing enzymes and transporters, with TSG and aloe-emodin being the most pronounced. The AUC values of TSG and aloe-emodin showed a significant negative correlation with the expression levels of UGT1A1, BSEP, OATP1A4, OCT1, NTCP, MRP2, and MDR1. This result suggests that the metabolisms of TSG and aloe-emodin by the liver were weakened when metabolic enzymes and transporters were inhibited, resulting in the elevated occurrence of TSG and aloe-emodin in the blood. The AUC values of emodin, physcion, and resveratrol were positively correlated with the expression levels of OATP1A2, NTCP, and MRP2, suggesting that the excretions of emodin, physcion and resveratrol were promoted when the expression levels of OATP1A2, NTCP, and MRP2 were reduced. Besides, the expression levels of UGT1A1 decreased in normal rats, while the expression levels of UGT1A1, MATE1, BSEP, and OCT1 increased in the ANIT and CClA more comprehensive description of the pharmacokinetics of PM was investigated. The phase II metabolites TSG, emodin, resveratrol and catechin were preliminarily analyzed. In addition, a priliminary correlation analysis of the expression levels of hepatic metabolizing enzymes and transporters with the pharmacokinetic parameters of PM was done in this work. At the same time, the specific mechanisms of the interaction between these two variables that affect each other still needs further experimental investigation.


Context: Hyaluronic acid (HA) plays critical roles in the structural skeleton, joint lubrication, renal function and cell signaling. We previously showed that partially N-butyrylated, low molecular weight, hyaluronic acid (BHA) exhibited an anti-inflammatory effect in cultured human macrophage, where inflammation was induced either by a TL-4 agonist or the low molecular weight HA itself, in dose-dependent fashion.Objectives: To investigate the anti-inflammatory, antioxidative, and antihyperuricemic effects of BHA using animal models of acute gouty arthritis and hyperuricemia.Materials and methods: The anti-inflammatory effect of articular BHA (10 and 50 μg) injections was evaluated by measuring joint swelling and the serum levels of inflammatory cytokines in a model of acute gouty arthritis induced by intra-articular injection of monosodium urate crystals in Wistar rats (n = 10/group), in comparison to the control group with saline injection. Antioxidative and antihyperuricemic activities were investigated using intraperitoneal injections of oteracil potassium and yeast extract hyperuricemic Balb/C mice, which were treated with intraperitoneal injection of BHA at day 6–8 in the model.Results: In the gouty arthritis rat model, BHA at a higher dosage (50 μg) demonstrated a strong anti-inflammatory effect by reducing the degree of articular swelling and the serum levels of IL-1β, IL-8, IFN-γ, and MCP-1 by 5.56%, 6.55%, 15.58% and 33.18%. In the hyperuricemic mouse model, lower dosage BHA (10 μg) was sufficient to provide antioxidative activities by significantly decreasing the ROS levels in both serum and liver by 14.87% and 8.04%, while improving liver SOD by 12.77%. Intraperitoneal injection of BHA suppressed uric acid production through reducing liver XO activity by 19.78% and decreased the serum uric acid level in hyperuricemic mice by 30.41%.Conclusions: This study demonstrated for the first time that BHA exhibits anti-inflammatory, antioxidative and antihyperuricemic effects in vivo, suggesting a potential therapeutic application of BHA in gouty arthritis and hyperuricemia. Gout is one of the most prevalent metabolic disorders and is accompanied by a high level of uric acid in the blood and monosodium urate (MSU) crystal precipitation in joints and tissues. Joint swelling and pain of sudden onset are the major symptoms of acute gout  and interleukins-1β (IL-1β), which will then cause an influx of inflammatory cells and strengthen the inflammatory reaction  and URAT1 inhibitors  inhibit UA production  and d-glucuronic acid (GlcA)  is a linear polysaccharide composed of simple repeating disaccharide units of d (GlcA)  is the md (GlcA) . The intd (GlcA) . HMW HA d (GlcA) . HA is ad (GlcA) .N-butyrylated HA (BHA) with reduced molecular weight significantly inhibited the production of proinflammatory cytokines, such as the TNF-α, IL-1, IL-6 and IL-8 in the lipopolysaccharide (LPS) stimulated human cultured macrophages, while other N-acyl substitutions were less effective. In this publication we studied the pro-inflammatory effects of the semisynthetic polymers over a wide range of concentrations. We demonstrated induction of proinflammatory cytokine production by the low molecular weight HA. This HA polymer was initially deacetylated and then fully reacetylated, to an identical extent as the BHA, so that it could be precisely compared to BHA. In addition, we showed that increasing concentrations of the TL4 agonist, lipopolysaccharide from E. coli, in the presence of a fixed amount of BHA resulted in much lower of proinflammatory cytokine production from the macrophages than in its absence.We had previously shown  and yeast extract powder. The underlying mechanisms related to oxidative stress and inflammation were also investigated.The BHA was synthesized by two step reaction as previously described  spectrometer The degree of deacetylation can be calculated according to the equation: Deacetylation (%) = (1.0−(Y/1.5)100)  pressure = 25 psi, heater gas (N2) pressure = 50 psi, Curtain gas pressure = 25 psi, DP = −100 V and CE = −35 eV. Samples at concentrations of 10 μg mL−1 were injected into the mass spectrometer via a syringe pump to scan for specific fragments corresponding to HA and its derivatives as shown in MS analyses of the samples were performed using a Triple-TOF 5600 mass spectrometer  equipped with an electrospray ionization source operated in the negative scanning mode. MS parameters, which were optimized by a 10 μg mLn = 50, 8 weeks: 160–200 g) were purchased from Liaoning Changsheng Biotechnology Company, Jilin, China (SCXK (Liao)-2015-0001). These rats were housed in plastic cages and maintained on a 12 h light/dark cycle (lights on 7:00–19:00 h) under standard laboratory conditions of 55% relative humidity and at 23 °C ± 1 °C. They were given standard chow  and tap water ad libitum. The protocols of the animal experiments were approved by the Animal Ethics Committee of Jilin University (Reference No. 201605).Male Wistar rats  as shown in −1 MSU solution was intra-articular injected at the right ankles of all rats except the NC group which injected with saline solution. In the 10BHA and 50BHA groups, intra-articular injections of 10 or 50 μg BHA along with MSU solution. About 1 μg or 5 μg BHA was dissolved into 10 mL of 30 mg mL−1 MSU solution, and 100 μL of 30 mg mL−1 MSU solution contains 10 or 50 μg BHA were administered according to the stated BHA doses. On the 8th day, 1 h after the administration of the final COL solution, blood was sampled from the caudal veins of the rats. Immediately after sampling the right ankle joints of the rats of all of the groups of the rats were collected and fixed in 4% paraformaldehyde. Serum was separated and stored at −80 °C until analysis.An experimental model of MSU-induced gouty arthritis established as described in the literature /C0 × 100. In particular, Ct represents the circumference at time (t) (in h) and C0 represented the circumference at 0 h.The right ankle circumference of all groups of the rats at the certain time  after the intra-articular injection step was measured with a line method . The histopathological changes of the joint synovium were assessed for the degree of inflammatory cell infiltrate, by an experienced histopathologist.Supporting Information Table S1.The serum levels of interleukin-1α (IL-1α), IL-1β, interleukin-6 (IL-6), interleukin-8 (IL-8), IL-10, interleukin-16 (IL-16), C-X-C motif chemokine 10 (CXCL10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), interferon gamma (IFN-γ), TNF-α, tumor necrosis factor-β (TNF-β), nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), 6-keto-prostaglandin F-1α (6-Keto-PGF1α) and prostaglandin E2 (PGE2) in rats were determined by the ELISA method using related Elisa Kits  according to the manufacturer’s instructions. The absorbance recorded in different concentrations that used to establish the calibration curves were shown in the n = 50, 8 weeks: 18–22 g) were purchased from Yisi Experimental Animal Technology Company, Jilin, China (SCXK (Ji)-2016-0003). These mice were housed in plastic cages and maintained on a 12 h light/dark cycle (lights on 7:00–19:00 h) under standard laboratory conditions of 55% relative humidity and 23 °C ± 1 °C. They were given standard feed and tap water ad libitum. The protocols of the animal experiments were approved by the Animal Ethics Committee of Jilin University (Reference No. 201605).Male Balb/C mice (n = 10) as shown in −1 yeast extract powder (YEP) by gavage for 8 days. From day 6 to 8, 300 mg kg−1 of OXO was intraperitoneally injected to all the mice except the control group, which injected with saline solution. In MC group, 20 mg kg−1 AL was orally administrated after 1 h of the OXO injection. In the 10BHA and 50BHA groups, intraperitoneal injections of 10 or 50 μg BHA along with OXO were conducted according to the required BHA doses from day 6 to 8 . At 1 h after the final drug administration, blood was sampled from the caudal veins of the mice, serum was separated, and their livers were quickly collected  according to the manufacturer’s instructions. Tissue homogenate samples were prepared with a 0.9% sterile saline solution.The levels of reactive oxygen species , malondialdehyde (MDA) glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), creatinine (Cr) and urea nitrogen in the serum and livers of mice were determined using ELISA Kits  according to the manufacturer’s instructions.Blood was sampled from six healthy volunteers and six patients suffering with acute gout attack enrolled in the First Hospital of Jilin University, Changchun, China. The clinical trial protocol was approved by the Ethics Committee of Jilin University (Reference NO. 2015-015). We adhered to the Guidelines on Humane Treatment to Lab Animals (published in 2009). Inflammatory factors of human plasma were assayed using a Proteome Profiler Human Cytokine Array Kit , according to manufacturer’s instructions.post hoc Dunn’s multiple comparisons test by SPSS 19.0 Software . p values < 0.05 were considered to be statistically significant.All results collected were expressed as mean ± S.D. One-way analysis of variance (ANOVA) was performed to evaluate statistical significance of the data, and this was followed by δ2.50 , δ4.40–3.83 , δ5.09–5.08 , δ4.94–4.93 . DHA: δ2.50 , δ4.45–3.84 , δ5.10 , δ4.95 , δ5.31 , δ5.18 . BHA: δ2.50 , δ4.40–3.83 , δ5.10–5.09 , δ4.94–4.93 , δ5.13 , δ5.10–5.09 , δ2.74 , δ2.09–2.08 , δ1.42 . As shown in 2CH2CH3 proton signals indicating that a reacylation reaction had occurred and butylation-to-acylation ratio was calculated to be 25.4%.The NMR data are as follows, HA: The molecular weights of HA and its derivatives were estimated by agarose gel electrophoresis, as shown in m/z value was very similar to the predicted m/z value, as shown in m/z 396.1160), as well as singly  and doubly (m/z 387.1089) charged tetrasaccharides of GlcNAc and GlcA were observed in the mass spectra of the HA sample. The presence of additional singly charged disaccharide of GlcN and GlcA (m/z 354.1053) as observed via TOF-MS spectra showed that the sample of DHA was composed of partially deacetylated HA. The additional singly charged disaccharide of GlcNBu and GlcA (m/z 424.1462) observed in the TOF-MS spectra of BHA revealed that the sample contained partially butylated HA. In these specific fragments, singly charged disaccharides of GlcNAc and GlcA  were the most abundant species in all of the samples, and thus we used this signal as the target peak. The relative intensities of the other relevant peaks were calculated based on the target peak as shown in Following injection into the Q-TOF MS system via a syringe pump, the sample solutions were scanned in the TOF-MS mode. The observed p < 0.01, F = 66.51 to 203.12) compared with control rats  in comparison with MC group, but the effects did not reach statistical significance  at 4, 24 and 48 h. BHA injected along with MSU at the doses of 10 and 50 μg suppressed the ankle swelling of each rat at 4, 12 and 48 h  in comparison with the MC group. The treatment with 10 µg of BHA provided the best suppression of swelling at 12 h, and the swelling ratio was reduced by 11.0% . Additionally, treatment with 50 μg of BHA provided the best swelling suppression at 48 h, with a swelling ratio reduction of 9.5% .As shown in rol rats . TreatmeHistopathologically, the injection of MSU crystals caused pronounced inflammatory cell infiltration in the synovium compared to the normal controls, as shown in Supporting Information Table S1. In comparison to the control group, the contents of MCP-1 and 6-Keto-PGF1α were significantly increased , IL-10 had decreased dramatically , while IFN-γ and NF-κB were not significantly improved  in the serum of rats that had received MSU injections. Compared to rats injected with MSU, oral administration with 0.3 mg kg−1 COL only decreased the level of MCP-1 by 9.93%  in the serum. Intra-articular treatment with 10 μg BHA significantly decreased the level of IL-8 and MCP-1 by 7.13% and 7.76% , respectively, and increased IL-10 by 9.49% , indicating that the effect of BHA is more obvious in such lower dosage. Lastly, treatment with 50 μg BHA significantly decreased the level of IL-1β, IL-8, MCP-1 and IFN-γ by 5.56%, 6.55%, 15.58% and 33.18% , respectively.As inflammatory factors are an important basis for gout attack  establishing the presence of hyperuracemia . Oral administration with 20 mg kg−1 AL suppressed the serum UA level by 46.38% .A reduction in elevated levels of uric acid needs to be established to demonstrate effective antihyperuricemic agents  and the levels of SOD, GSH-Px and CAT in the liver  exhibited no significant changes , while that of SOD in the serum decreased by 7.81% . Treatment with AL suppressed the levels of ROS in the serum by 9.48% and 17.86% in the liver, respectively. This treatment reduced the levels of MDA in the serum and liver by 6.66% and 11.43% . The administration of 10 μg BHA strongly decreased the ROS levels in both the serum and liver by 14.87% and 8.04% , respectively. Meanwhile, the administration of 50 µg intra-peritoneal BHA dose also significantly decreased the ROS levels in the serum and liver by 14.63% and 9.59% , respectively. Both 10 and 50 μg BHA treatments significantly improved liver SOD by 12.77% and 13.69% . Both the serum and liver levels of Cr and urea nitrogen have been accessed in this hyperuricemic mouse model . Treatment with AL and BHA significantly lowered the levels of urea nitrogen by 25.25%, 11.65% and 12.81%  in the liver and the Cr by 11.52%, 8.21% and 12.02%  in the serum. The administration of AL and the 10 μg BHA dose dramatically lowered the levels of Cr by 15.28% and 8.14% , respectively in the liver.XO catalyzes the production of ROS during the synthesis of UA, which poses an elevated risk to renal function  could be related to the feedback regulation.HMW HA was shown to impede phagocytosis of uric acid crystals A Brandt . More geA Brandt . HoweverA Brandt , while MA Brandt . In addiA Brandt . The devA Brandt , indicatA Brandt . Thus, tA Brandt . TherefoA Brandt . In addiin vivo, the activity of XO in liver homogenate was not affected in vitro. Because AL acts as an inhibitor of XO, which not affects the expression level of XO, this result could be due to the fact that in the in vitro assays, the activity of XO in liver homogenate was determined by adding exogenous XO substrates, but without the addition of AL. In the meantime, 10 μg BHA showed a better XO inhibitory effect in vitro and exhibited similar serum uric acid-lowering effects with 50 μg BHA as well as with AL. This result suggests that BHA may lower uric acid with a different mechanism, such as down-regulate the expression of XO in liver, and lower dosage exhibited a better effect. However, its mechanisms of action of uric acid-lowering remain unrevealed.Hyperuricemia is considered as a necessary condition for the development of gout, and XO that plays a key catalytic role in UA production  and the promotion of the anti-inflammatory cytokine (IL-10), which in turn reduces the level of inflammation and inhibits the XO activity by regulating oxidative stress. At the same time, BHA also protects the renal function of the hyperuricemic mice. In conclusion, BHA is a novel compound, derived from a naturally occurring carbohydrate polymer, which has translational potential for the clinical treatment of gout due to its bimodal pharmacological activity.Click here for additional data file.


Echinococcus granulosus (E. granulosus) larvae in liver and lungs of both humans and animals. Surgical intervention is the mainstay for CE treatment, using scolicidal agents that inactivate live protoscolices. This study evaluated the scolicidal effects of Silybum marianum ethanolic extract and its combination with albendazole in vitro for the first time. Moreover, in a literature review, we investigated the effects of a wide range of Iranian medicinal plants on protoscolices of E. granulosus.Cystic echinococcosis (CE), is a parasitic zoonosis caused by S. marianum ethanolic extract was prepared and high-performance liquid chromatography (HPLC) analysis was used to establish the proportions of its component compounds in the extract. Cytotoxicity was evaluated in mouse macrophage cells (J774A.1 cell line) using MTT method. Next, the scolicidal activity of the extract alone and combined with albendazole was tested as triplicate at various concentrations incubated for 5, 10, 20, 30, and 60 min. Finally, protoscolex viability was determined using 0.1% eosin as a vital stain. PCR–RFLP and DNA sequencing techniques were used to characterize the genotype of E. granulosus.S. marianum ethanolic extract contained mostly silydianin (14.41%), isosilybin A (10.50%), and silychristin (10.46%). The greatest scolicidal effects were obtained with the combination of S. marianum with albendazole (79%), S. marianum ethanolic extract alone (77%) and albendazole (69%), at a concentration of 500 μg/ml for 60 min, respectively (P < 0.05). Molecular analysis showed that all the cysts used were G1 genotype.HPLC analysis showed that S. marianum ethanolic extract is a potential scolicide in vitro; however, further investigations are required to determine its efficacy in vivo.The data suggest that  Echinococcus granulosus (E. granulosus), is a genetically diverse metazoan parasite causing cystic echinococcosis (CE) or hydatid disease [E. granulosus consists of canids (definitive hosts) and herbivores/omnivores species (intermediate hosts) [E. granulosus eggs from soil, water and vegetables contaminated with infected canid feces [The zoonotic tapeworm,  disease , 2. Huma disease , 4. The e hosts) , 6, and id feces , 8. In hid feces , 10; as Currently, there are three treatment approaches for CE, surgical removal, PAIR , and chemotherapy using benzimidazole compounds –14. GlobSilybum marianum (Asteraceae), is a medically important plant species native to North and South America, Africa, Australia and the Middle East [S. marianum contain a powerful substance called silymarin, which has long been used to support liver health [S. marianum have also been demonstrated against prostate, skin and breast tumors, cirrhosis and kidney disease [S. marianum seeds [Medicinal plants are considered as huge treasuries for a wide spectrum of valuable therapeutic compounds , 22. Sildle East , 24. Ther health . Historir health , 25. The disease , 27. Addum seeds –31.S. marianum seeds are lacking; hence, the current in vitro investigation was designed to evaluate the scolicidal efficacy of S. marianum ethanolic extract alone or combined with albendazole. Also, in a literature review, we investigated the effects of a wide range of Iranian medicinal plants on protoscolices (PSCs) of E. granulosus.Experimental studies of the scolicidal activity of ethanolic extract of Livers and lungs of sheep infected with hydatid cysts were collected from the industrial slaughterhouse, Urmia, Northwestern Iran, and transferred, on ice, to the Parasitology Department, Faculty of Medical Sciences, Tarbiat Modares University at Tehran. Hydatid cyst fluid and PSCs were collected in sterile 50-mL tubes, then centrifuged at 2000 rpm for 5 min, as described by Smyth and Barret . The supThe genomic DNA of 50 μl of viable PSCs was extracted using a commercial DNA extraction kit,  according to the manufacturer’s instructions. Purified DNA samples were kept at -20 °C until further use. In the next step, PCR assay was set up to amplify a 462-bp segment of the ribosomal DNA internal transcribed spacer 1 (ITS1) gene and restriction fragment length polymorphism (RFLP) method was used for parasite genotyping . FinallyS. marianum plants were collected from Tehran suburbs, followed by species authentication (herbarium number 514) by the Botany Division of the Iranian Biological Resource Center, Karaj, Iran. Plant seeds were ground to 0.4 mm diameter using a blender [In the present investigation,  blender . Next, 1HPLC is normally used to identify compounds present in plant extracts and compare their components. For this purpose, we used an Agilent 1290 Infinity LC System (USA). Extracts re-dissolved in ethanol and applied to a standard 250 mm × 4.6 mm HPLC column at a flow rate of 1 mL/min with the detector set at 288 nm for analysis of the chromatogram.S. marianum in equal proportions were 500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90, and 1.95 μg/ml.Albendazole  was dissolved in DMSO and used as a reference drug at concentrations of 500, 250, 125, 62.5, 31.25, 15.62, 7.81, 3.90, and 1.95 μg/ml, respectively. Combined concentrations for albendazole and ethanolic extract of 5 macrophages were seeded into each well of 96-well culture plates and incubated for 24 h. Then, S. marianum concentrations were added to macrophages in different concentrations and the plates were incubated for 1 day at 37 °C. After addition of 20 μl of MTT solution (5 mg/ml), the plates were incubated at 37 °C for an additional 3–5 h, and the supernatant portion was discarded from each well. Finally, 100 μl DMSO was added to the wells, and 15 min later, optical absorption was read using an ELISA reader  at 570 nm. The cell survival was measured as follows:Mouse macrophage cell line (J774A.1), bought from Pasteur Institute of Iran, was cultured in cell culture flasks containing DMEM supplemented with 10% FBS at 37 °C, in a 5% carbon dioxide atmosphere [Survival rate = (AT-AB)/(AC-AB) × 100; where AB is the optical absorption of the blank well, AC is the optical absorption of the control well, and AT is the optical absorption of the treated well.S. marianum extract and its combination with albendazole, were added to the respective wells, mixed gently and incubated for 5, 10, 20, 30, and 60 min at 37 °C. All experiments were done in triplicate. Occasionally, the number and viability of PSCs were determined using 0.1% eosin during incubation periods. The control for the study was non-treated parasites in DMEM. The PSCs fatality rate (PFR) was estimated using the following formula [For this purpose, 100/mL PSCs were added to DMEM containing 200 U/mL of penicillin, 200 mg/mL of streptomycin, and 0.5 mg/mL of amphotericin B in 96-well culture plates . Subsequ formula :\documenP-value of less than 0.05 was considered significant.ANOVA and t tests were used to determine the differences between tests and control groups; statistical analysis was performed using SPSS v. 17 . A E. granulosus”, “Herbal medicine AND E. granulosus”, “In vitro activity of plants AND Echinococcosis”, “Natural products AND Scolicidal”, and “Natural scolicidal”. Articles without full-text accessibility and those with confusing/unclear data and irrelevant papers were excluded.For the literature review, national and international databases were searched using the following keywords: “Scolicidal agents”, “Medicinal plants AND E. granulosus cysts were of G1 genotype , isosilybin A (10.50%), and silychristin (10.46%) at high level, while isosilybin B (3.04%) had the lowest relative concentration. All silymarin compounds are members of the flavonoid family.As shown in Table S. marianum extract)  in macrophage viability was seen at the highest dose . It is noteworthy that most of the effect on PSCs was seen within in the first 5 min and showed a linear dose relationship. Beyond that time, there was approximately 30% additional effect over the subsequent 55 min for all concentrations.The results clearly showed that maximum effect was nearly 77%% with 500 μg/mL of ethanolic extract of min Fig. . MoreoveThe highest PFR was 69% at a concentration of 500 μg/ml after 60 min. The scolicidal effects of albendazole concentrations were significant in comparison with the controls at different exposure times (p < 0.05)   [E. granulosus G1 strain is a major genotype in sheep, there have been reports of other genotypes such as G3, G6, and G7, in sheep [E. granulosus genotypes. Therefore, it is suggested that PSCs of different genotypes (G1 to G10) be studied to gain a better understanding of the resistance or susceptibility of these genotypes.In this study, we defined the (G1-G10) , 56. Our(G1-G10) . AlthougS. marianum and its combination with albendazole produced similar scolicidal activity compared to albendazole alone. Plants may induce or inhibit the activity of known anthelmintic agents due to their chemical content [S. marianum may be a co-factor to enhance albendazole anthelmintic activity. Traditionally, S. marianum is an effective herbal product for a wide range of infectious and non-infectious diseases, with silymarin seen as the highly beneficial substance in its seeds [S. marianum in each country; silychristin (isolated from fruit), silybin A (isolated from stems), and silybin B (isolated from seeds) have the highest concentrations in Russia [S. marianum extract has also shown significant antimicrobial effects against fungi  [Staphylococcus saprophyticus, Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae) [In general, finding a suitable alternative drug has become an important challenge. Over the past 4 decades, various herbal extracts and chemical compounds have been investigated in vitro and in vivo as potential treatments , 57–60.  content . Increas content , 62. Thets seeds , 64. Thits seeds –67. As sn Russia , Iraq [6n Russia , and Hunn Russia , respectn Russia , 72. Then Russia , 74. Basophytes)  and pathumoniae) .E. granulosus. Although it seems that the in vitro studies of Iranian medicinal plants on PSCs are extensive, it is possible that useful data were missed within the ‘grey’ literature. Leaves were the most frequently plant part used (31% of studies). The scolicidal effects of medicinal plants range from 17.4% to 100% and the S. marianum extract used in the present study is comparable in activity to other Iranian medicinal plants investigated.Iran has a great diversity in terms of medicinal plants. In our literature review, 25 studies were eligible for inclusion. As shown in Table S. marianum is a potential alternative scolicide during PAIR treatment of cysts, and to reduce the risk from hydatid cyst fluid PSCs leakage during surgery and the subsequent complications. In the future, more research is needed to evaluate the in vivo effects of this group of compounds in a clinical setting in animal models and ultimately in humans.In summary, an ethanolic extract of


The main aim of this study was to the determine relationship between Body Mass Index (BMI) and percentage body fat (BF%) in Singaporean adults, derive a prediction model to estimate BF%, and to report population BF%. The secondary aim was to determine the prevalence of overweight and obesity based on BF% threshold and the new risk categories for obesity in Singaporean population.This was a population-based study of 542 community-dwelling Singaporeans . Anthropometry and body composition were assessed. Relationship between BMI and BF% were analysed using multiple regression models. Prevalence of overweight and obesity were estimated using WHO and Singapore Ministry of Health (MOH) Clinical Practice Guidelines for BMI classification, and BF% cut-off points of 25 and 35% for men and women respectively.2) was 12.9% ; and 26.6%  according to the MOH classification (BMI ≥27.5 kg/m2). However, using the BF% cut-off (> 25% for men and > 35% for women) resulted in very high prevalence of obesity of 82.0% .We derived a prediction model to estimate BF% based on BMI, age and sex. The current cohort of Singaporeans when compared to Caucasians in the US and Europe as well as a Singapore cohort from 20 years age have higher BF% when matched for BMI, age, and sex. The overall population-adjusted prevalence of obesity according to WHO International classification (BMI ≥30 kg/mThere is a large discrepancy between BF% and BMI measured obesity in Singaporean adults. The results confirmed that Singaporean adults have higher BF% at lower BMI compared to US and Europe white counterparts; and that BF% in our population has increased over two decades.The online version contains supplementary material available at 10.1186/s12889-021-11070-7. In 201630 kg/m2 . Despite30 kg/m2 , 8.2 [Therefore, it is crucial to determine obesity or threshold of body fat that is associated with increased adverse health risk. Two commonly used methodology used to determine accumulation of body fat are waist circumference and BMI. Waist circumference (WC), measured at midpoint of the last palpable rib and top of iliac crest , has goo2 . Therefo2 , 15.2 as points for public health actions [2 for overweight, and 27·5 kg/m2 for obesity [In 2004, a WHO expert consultation was established to address the appropriate BMI classification for Asian populations . After a actions . Under i obesity , as Sing obesity . Therefo2 in Caucasians [Various methods have been developed to measure BF%, including densitometry, dilution technique and dual energy X-ray absorptiometry (DXA). While WHO has a clear BMI cut-off for defining obesity, there is no clear consensus on the threshold for BF% for overweight and obesity. Previous studies have suggested that BF% greater than 25% for men and 35% for women is the threshold for diagnosing obesity, which were derived from corresponding BMI of 30 kg/mucasians –19. In aucasians . Howeverucasians .2) at 5.9%, Indians at 14.0%, and Malays at 20.7% [2 and 27.5 kg/m2for overweight and obesity, the prevalence of obesity based on BMI 23 kg/m2 and 27.5 kg/m2should also be studied. The primary aim of this study was to the determine relationship between BMI and BF% in the multi-ethnic  population of Singapore, derive a prediction model to estimate BF%, and to report population BF%. The secondary aim was to determine the prevalence of overweight and obesity based on BF% threshold and the new risk categories for obesity in our population.Singapore is a multiracial and multicultural country, consisting of 74.4% Chinese, 13.4% Malays, 9.0% Indians, and 3.2% of various other races . In 2013at 20.7% . In a prat 20.7% . Relatioat 20.7% . Howeverat 20.7% . With that 20.7% , the relParticipants were recruited among community-dwelling adults ≥21 years) from a large north-eastern residential town of Yishun in Singapore, with residential population of 220,320 (49·4% men), with 12·2% older adults (≥65 years) [ years frRandom sampling methodology was employed to obtain a representative sample of approximately 300 male and 300 female participants, filling quotas of 20–40 participants in each sex- and age-group . Conventionally, the sample size of 30 or greater per age-group is sufficient for normative measures . BetweenBody weight to the nearest  0.1kg and height to the nearest 0.1 cm were measured using a digital balance and stadiometer . Waist and hip circumferences were measured using a non-elastic, flexible measuring tape around the navel and widest part of the hips respectively. These measurements were conducted by trained researchers at the research center. All participants underwent a DXA scan of the whole body . The DXA scan was conducted by experienced radiographers. Body composition information - lean mass, fat mass, and bone mineral content, were obtained from the scan.2, and BMI ≥30·0 kg/m2, respectively. Singapore MOH Clinical Practice Guidelines defined overweight as BMI 23·0–27·4 kg/m2 and obesity as BMI ≥27·5 kg/m2. The BF% cut-off points for obesity were set at 25% for men, and 35% for women [Classification of overweight and obesity by BMI were derived using WHO international criteria , and Sinor women , 27. Waior women .All statistical analyses were performed using SPSS Statistics version 22·0 . Relationship between BMI and BF% was analysed using forward-backwward stepwise linear regression models. BF% was considered the dependent variable; 1/BMI and age were independent variable. Data was analysed separately by sex. In exploratory analysis, the relationship between BMI and BF% was not linear, hence 1/BMI variable was used to linearise the data and to avoid the need for logarithmic conversion or the inclusion of power , 29. Potp < 0·005). BMI was not significantly different between men and women (p = 0·071).A total of 542 participants 43·1% men) aged 21 years and above were recruited for the study. Due to incomplete data from five participants, data from the remaining 537 participants  were analysed. The ethnic distribution was similar to that of Singapore’s population [% men agen = 438) was analyzed, as the ample sizes for Malay, Indian, and other races were too small. The relationship between BMI and BF% was curvilinear , and multiple R = 0.75 and SEE = 3.45% body fat for women (p < 0.05). Based on the estimated parameters of these equations, BF% values corresponding with BMI for men and women were derived (Table 2 and above .where multiple nd above .Table 3The prevalence of overweight and obesity are presented in Table In this study, we established the relationship between BMI and BF% in Singapore Chinese adults. We compared the estimated BF% with other ethnicity, and also with an earlier study on Singapore population from 20 years ago. Comparing among ethnicities, Singapore Chinese were found to have higher BF% compared to Caucasian. This supports the findings from the Singapore study in 2000  and otheOur finding updates the 2000 report  in that The population-adjusted prevalence of overweight and obesity of Singaporeans varied according to the classification used. Prevalence of overweight was 34.4 and 41.8%, obesity was at 12.9 and 26.6%, when using WHO international BMI classification and MOH classification respectively. When adopting BF% criteria, population-adjusted obesity prevalence was substantially higher at 82.0% (Table 2, with mortality rate significantly increased at BMI ≥26·0 kg/m2 for non-smokers [2 equates to BF% of 20·9–23·2% in Chinese men and 32·8–34·8% in Chinese women aged 50 years. At BMI 26 kg/m2, BF% equates to 30·4% in Chinese men and 40·9% in Chinese women, which is about 5% higher than the WHO BF% cut-off. Using the criteria of BF% ≥30·4% in men and ≥ 40·9% in women, 45% of men and 44% of women have increased mortality risk. These estimates are much higher than the prevalence based on BMI ≥30 kg/m2, but much lower than using the WHO BF% cutoff. Differences in prevalence based on BMI is expected because the relationship between BMI and body fat content varies according to body build and proportion [A previous study reported that the lowest all-cause mortality rate in Singapore Chinese was at BMI 18·5–19·9 kg/m-smokers , which i-smokers . Using ooportion , but ouroportion . The smaoportion . Physicaoportion . Given tThe strengths of this study are its population-based, random selection of participants and hence representativeness and validity of data. The ideal method to determine body composition is the multi-compartment model , howeverIn conclusion, our study found a large discrepancy between BF% and BMI measurement in Singaporean adults. The results confirmed that Singaporean adults have higher BF% at lower BMI compared to Caucasians and that BF% in our population have also increased over two decades. Further investigation into the body build, nutrition intake, physical activity level among the different ethnic groups may help understand the relationship between BF% and BMI.Additional file 1 : Supplementary Table. Comparing estimated BF% at various BMI point of Singaporean Chinese in this study versus the 1998 National Health Survey.