Acinetobacter baumannii is a major global health threat. New drugs with novel chemical structures are needed to overcome a myriad of resistance mechanisms in A. baumannii. In this study, we screened an open-source Pathogen Box library for anti-A. baumannii compounds. Compound MMV675968  was the only non-reference compound found to inhibit the growth of all four A. baumannii test strains with IC50 of 0.6–2.7 μM, IC90 of 0.7–3.9 μM, and MIC of 1.6–10 μM. We showed that MMV675968 targeted A. baumannii dihydrofolate reductase (AbDHFR) as determined by an E. coli surrogate whose growth was dependent on AbDHFR function and by an in vitro DHFR activity assay. Additionally, chemical scaffolds of DHFR inhibitors that are effective as antibiotics against A. baumannii were identified using an in vitro DHFR activity assay and A. baumannii growth inhibition. MMV675968 was the most potent among DHFR inhibitors tested in inhibiting A. baumannii growth. This study shows for the first time that MMV675968 inhibits A. baumannii growth via selective inhibition of AbDHFR and is therefore a promising scaffold for further antibiotic development against A. baumannii.Antibiotic resistance in  Acinetobacter baumannii, a Gram-negative opportunistic pathogen, is a leading cause of nosocomial infection worldwide1. It has developed resistance to multiple classes of antibiotics with strains resistant to all commercially available antibiotics4. A. baumannii possesses an array of resistance mechanisms such as downregulation of porin, mutations in drug targets and altered expression of β-lactamases, aminoglycoside-modifying enzymes, and drug efflux pumps6. Treatment failure is common in A. baumannii infection owing to a lack of effective alternate antibiotics against this multidrug-resistant pathogen, responsible for up to 50% mortality among infected patients1. Discovery of new drug candidates and understanding of their mechanisms of action is direly needed to tackle the problem of resistance in this pathogenic organism.www.pathogenbox.org). Pathogen Box contains compounds with diverse chemical scaffolds distinct from currently available antibiotics, making the Pathogen Box library a promising source of compounds for novel antibiotics discovery. The mechanisms of action of these compounds are mostly unknown but crucial for hit-to-lead development in a drug discovery process, thereby worth investigating further8. In this study, we used Pathogen Box compounds to screen for those with anti-A. baumannii activity and investigated the mode of action of the most promising bioactive candidate.The Open-source Pathogen Box consists of 400 compounds known to be active against a number of pathogens responsible for neglected tropical diseases and have low toxicity (www.pathogenbox.org) for compounds that inhibit the growth of A. baumannii Mahidol and Naval-81 strains, an environmental and clinical strain, respectively. Using the Clinical and Laboratory Standards Institute (CLSI) microdilution method, the majority of the compounds exhibited no significant growth inhibitory activity at 10 μM , 0.7–3.9 μM (0.25–1.40 mg/L), and 1.6–10 μM (0.58–3.60 mg/L), respectively  and thymidylate synthase (TS) of thymidylate cycle required for thymidine synthesis hence making the strain a thymidine auxotroph15, as a model to test DHFR target specificity. The E. coli surrogate was co-transformed with plasmids expressing E. coli TS (EcTS) and either AbDHFR or human DHFR (hDHFR). When EcTS was expressed together with AbDHFR or hDHFR, E. coli PA414 was able to grow in the absence of thymidine , a bacterial DHFR inhibitor, or methotrexate (MTX), a known hDHFR inhibitor17. MMV675968 (10 µM) was able to inhibit the growth of the E. coli surrogate expressing AbDHFR but not that of surrogate expressing hDHFR  MMV675968 is less effective in inhibiting hDHFR than MTX and TMP, and 3) MMV675868 is likely to be more specific to AbDHFR than hDHFR since expression levels of AbDHFR and hDHFR in E. coli PA414 were comparable  harboring pET15b and that overexpressing AbDHFR or hDHFR were compared. Using equal amount of total protein, DHFR activity present in the lysate of E. coli BL21(DE3) containing pET15b accounted for only 3% and 6% of that detected in the lysate of E. coli BL21(DE3) overexpressing AbDHFR and hDHFR, respectively  expressing AbDHFR and hDHFR comes from overexpressed enzymes not endogenous EcDHFR. We found that MMV675968 inhibited AbDHFR and hDHFR in a dose dependent manner with IC50 values of 8.5 nM and 492.4 nM, respectively  inhibited the growth of two strains, namely, Mahidol and Naval-81 strains, but not the H72721 and 3–137 strains  not related to AbDHFR inhibition.Eight DHFR inhibitors were then tested for their ins Fig. . Even thtro Fig. , it did ted Fig. , implyinins Fig. . All founts Fig. , indicatAbDHFR in A. baumannii, we determined whether AbDHFR activity of each A. baumannii strain decreased upon treatment with DHFR inhibitors. A. baumannii strains were treated with 10 µM TMP, PYR, or MMV675968 for 90 minutes. Normalized lysate was then tested for DHFR activity. All four A. baumannii strains showed reduced DHFR activity upon MMV675968 treatment compared with untreated cells . The open source format is expected to jump-start the discovery of new antibiotics urgently needed to combat drug resistant organisms. From the Pathogen Box, compound MMV675968 was discovered to be an effective inhibitor of A. baumannii growth with IC50, IC90, and MIC values comparable to known effective antibiotics against A. baumannii  such as AcrAB/TolC efflux pump, which is shown to export MTX, rendering MTX ineffective against E. coli20.There are at least four major classes of DHFR inhibitors, namely, diaminopteridine, diaminodimethyltriazine, diaminopyrimidine, and diaminoquinazoline Fig. 19. MTX, ion Fig.  but did ate Fig.  and did nii Fig. , suggestPlasmodium DHFR inhibitors22, did not inhibit AbDHFR in vitro or show any growth inhibitory activity against A. baumannii  inhibitors, e.g. trimethoprim-sulfamethoxazole combination (Bactrim) and pyrimethamine-sulfadoxine combination (Fansidar). Using DHPS inhibitors in combination with MMV675968 or its analogs should help reduce the required doses and forestall resistance.E. coli surrogate expressing exogenous AbDHFR or hDHFR constructed in this study can be used for screening other compound libraries for selective inhibitors of AbDHFR in an antibiotic discovery research program. Model organisms lacking endogenous DHFR activity have been used to study the activity of exogenously expressed DHFR from pathogenic species and for inhibitor screening29. The advantages of such surrogate models are the ease and safety of handling model organisms rather than the pathogenic organisms of interest. However, one caveat of using model organisms for studying target enzymes is the vast differences in cell permeability between species. For example, compounds that are active against a Plasmodium enzyme expressed in yeast may not be able to penetrate Plasmodium membrane. However, since E. coli and A. baumannii are both Gram-negative bacteria, they share similar cell wall/cell membrane structure, making the results from the surrogate assay developed in this study easier to translate from one organism to the other.The A. baumannii. Further comparative studies to understand enzyme kinetics and structures of AbDHFR and hDHFR and studies on in vivo efficacy, toxicity, pharmacokinetics, and pharmacodynamics can provide insights into improving the starting compound into more effective and/or more selective antibiotic.In summary, we identified a diaminoquinazoline MMV675968 that could serve as a starting compound for drug development against multidrug-resistant A. baumannii Mahidol strain was isolated from soil as described previously30, and multidrug-resistant clinical Naval-81, 3–137 (OIFC137), and H72721 isolates were from BEI Resources, NIAID, NIH, USA32. E. coli PA414 strain carrying null mutations in folA and thyA encoding dihydrofolate reductase (DHFR) and thymidylate synthase (TS), respectively was used as a host for expressing DHFR from other organisms and TS from E. coli15. E. coli and A. baumannii were grown in Luria Bertani (LB) broth supplemented as necessary with 50 μg/ml thymidine, 100 μg/ml ampicillin, 10 μg/ml chloramphenicol, 0.2% arabinose, or their combination and incubated with shaking at 37 °C.33. In brief, compounds were solubilized and diluted in dimethyl sulfoxide (DMSO). Approximately 5 × 104 colony forming units (CFUs) were incubated with 10 μM (unless otherwise indicated) of each compound in cation adjusted Mueller Hinton broth (CAMHB)  at 37 °C for 18 hours. Optical density at 600 nm (OD600) was measured using SpectraMax M5 . OD600 of wells containing A. baumannii (either compound-treated or untreated) was subtracted with OD600 of blank. Percent growth of compound-treated A. baumannii of the same strain was calculated using the following formula:We performed bacterial growth inhibition assay according to the Clinical and Laboratory Standards Institute (CLSI) microdilution methodA. baumannii strain was therefore 100%. Hits are defined as compounds that inhibit bacteria growth >50%. For IC50 and IC90 (50% and 90% inhibitory concentration) determination, A. baumannii strains were treated with various concentrations of test compound and cultured according to the microdilution method above. Minimum inhibitory concentration (MIC) is defined as the lowest concentration that completely inhibits bacteria growth as determined by unaided eye33.Percent growth of each DMSO-treated Abdhfr was amplified from clarified cell lysates of four strains of A. baumannii using Phusion DNA polymerase  and primers 5′ctggtgccgcgcggcagccatATGGCATGGCAAAATGTAG3′ and 5′tcgggctttgttagcagccggatccTTATTTTTTATAAGTGGCAAATTCG3′ (lower case denoting sequence homologous to pET15b plasmid), and human DHFR from pL003534 using primers 5′ctggtgccgcgcggcagccatATGGTTGGTTCGCTAAAC3′ and 5′tcgggctttgttagcagccggatccTTAATCATTCTTCTCATATACTTCAAATTTG3′. Amplicons were inserted into NdeI and BamHI digested pET15b plasmid using a Gibson Assembly kit (New England Biolabs). The thymidylate synthase gene  was amplified from lysate of E. coli DH5α strain using Phusion DNA polymerase  and primers 5′ctagcggagctcggagtgaaacgATGAAACAGTATTTAGAACTGATGC3′ and 5′ gcctgcaggtcgactctagaTTAGATAGCCACCGGCGC3′ (lower case denoting sequence homologous to pBAD33 plasmid). Amplicon was inserted into SacI and XbaI sites of pBAD33 using the Gibson Assembly kit . Cloned plasmids were sequenced by 1st Base DNA sequencing service (Singapore) and nucleotide sequences were deposited at GenBank, accession numbers: MH152688, MH152689, MH152690, and MH152691.E. coli PA414 strain carrying various combination of expression plasmids was grown overnight at 37 °C with shaking in LB broth supplemented with 50 μg/ml thymidine, 10 μg/ml chloramphenicol, and 100 μg/ml ampicillin. Cells from overnight cultures were pelleted by centrifugation, washed with fresh medium and the OD600 adjusted to 4.0. Ten microliters of undiluted, 10-fold and 100-fold serially diluted cells were spotted onto LB agar plates supplemented with necessary antibiotics and 50 μg/ml thymidine or 0.2% arabinose and incubated at 37 °C overnight.E. coli BL21(DE3) carrying pET15b, pET15b-AbDHFR, and pET15b-hDHFR were diluted into fresh LB supplemented with 100 µg/ml ampicillin and grown with shaking at 37 °C for 3 hours. After addition IPTG to final concentration of 40 mM, the cultures were grown overnight with shaking at 16 °C. Bacteria cells were harvested by centrifugation and lysed by sonication. Protein concentration of clarified bacteria lysate was determined using Bradford assay  according to manufacturer’s protocol.Overnight cultures of 26. Briefly, 0.2 µg of total protein from lysate of E. coli containing pET15b or overexpressing AbDHFR or 5 µg of total protein from lysate of E. coli containing pET15b or overexpressing hDHFR was added to reaction mixture containing 50 mM TES, pH 7.0, 75 mM β-mercaptoethanol, 1 mM EDTA, 1 mg/ml BSA, 0.1 mM NADPH, 0.1 mM dihydrofolate, and various concentrations of inhibitors to initiate the enzymatic reaction. Absorbance at 340 nm (A340) was monitored over 100 seconds. DHFR activity was measured as changes in A340 per minute.The assay was performed as previously describedAbDHFR activity in A. baumannii cells is affected by DHFR inhibitors, overnight cultures of A. baumannii strains were harvested, OD600-adjusted to 5.6 OD/ml and treated with 10 µM inhibitors for 90 minutes. To determine DHFR activity in treated A. baumannii, cells were harvested, washed once with and resuspended in DHFR buffer . A. baumannii suspension was lysed by sonication, normalized for protein content, and used to initiate DHFR activity assay. DHFR activity in untreated A. baumannii was set to 100% activity. DHFR activity of inhibitor-treated A. baumannii was calculated relative to untreated control.To determine whether Supplementary InformationSupplementary Information


MSA is a fatal neurodegenerative disease characterized by autonomic failure and severe motor impairment. Its main pathological hallmark is the accumulation of α‐synuclein in oligodendrocytes, leading to glial and neuronal dysfunction and neurodegeneration. These features are recapitulated in the PLP‐hαSyn mouse model expressing human α‐synuclein in oligodendrocytes. At present, there is no effective disease‐modifying therapy. Previous experiments have shown that the aggregation inhibitor, anle138b, reduces neurodegeneration and behavioral deficits in mouse models of other proteinopathies.To test the therapeutic potential of anle138b in a mouse model of MSA.Two‐month‐old PLP‐hαSyn mice were fed over a period of 4 months with pellets containing anle138b at two different doses (0.6 and 2 g/kg) and compared to healthy controls and PLP‐hαSyn mice fed with placebo pellets. At the end of the treatment, behavioral and histological analyses were performed.We observed a reversal of motor function to healthy control levels when PLP‐hαSyn mice were treated with both doses of anle138b. Histological and molecular analyses showed a significant reduction in α‐synuclein oligomers and glial cytoplasmic inclusions in animals fed with anle138b compared to nontreated mice. These animals also present preservation of dopaminergic neurons and reduction in microglial activation in SN correlating with the α‐synuclein reduction observed.Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.Anle138b reduces α‐synuclein accumulation in PLP‐hαSyn mice, leading to neuroprotection, reduction of microglial activation, and preservation of motor function supporting the use of anle138b in a future clinical trial for MSA. © 2018 The Authors.  Multiple system atrophy (MSA) is a rare and progressive neurodegenerative disorder characterized by autonomic failure and severe motor impairment leading to death a few years after symptom onset.+] neurons) in the SNc compared to healthy control animals. This neuronal loss is already present at 4 months of age and is followed by a significant reduction in density of dopaminergic terminals and in number of medium spiny neurons in the striatum at 12 months of age.Microglial activation and neuroinflammation constitute important pathological features of MSA.According to several studies, under physiological conditions α‐syn is mainly located in the pre‐synaptic terminals of the neurons predominantly as a monomer.PLP‐hαSyn mice overexpressing wild‐type (WT) human α‐syn under the PLP promoter, an oligodendroglial‐specific promoter, and generated in a C57/BL6 backgroundAt the end of the treatment, animal cages were randomly numbered by the animal caretaker and behavioral analyses were performed by the researcher blinded to genotype and treatment. Motor performance and coordination were analyzed with a modified version of the traditional beam test adapted from a previously published method.After behavioral analyses were performed, animals were perfused intracardially with phosphate buffered saline  under deep thiopental anesthesia and brains were extracted. For molecular analyses, brains were snap frozen in liquid nitrogen and stored at –80 °C. For histological analyses, brains were postfixed overnight in 4% paraformaldehyde  at 4 °C. After fixation, brains were washed in PBS and then transferred to 30% sucrose (in PBS) until they sank. Finally, brains were frozen using 2‐methylbutan (Sigma‐Aldrich) and stored at –80 °C for further analyses. In order to perform histological analyses, brains were serially cut in 40‐μm‐thick coronal sections using a freezing microtome  and stored free‐floating in a cryoprotectant buffer at –20 °C. One series was directly mounted on slides and stained with cresyl violet.+ neurons) in the SNc, serial sections were stained with rabbit anti‐TH antibody . For microglial activation analysis, representative sections of SN were stained with rat anti‐CD68 antibody . Sections were then incubated with biotinylated secondary antibodies, followed by Vectastain ABC reagent  and 3,3′‐diaminobenzidine (Sigma‐Aldrich), to visualize the immunohistochemical binding sites. Stained sections were mounted on slides, dehydrated, and coverslipped with Entellan . For immunofluorescence, suitable immunoglobulin Gs, conjugated with Alexa 488 or Alexa 594 , were applied, followed by nuclear staining with 4′,6‐diamidino‐2‐phenylindole  and finally coverslipped with mounting medium Fluromount‐G .Free‐floating sections were stained following standard protocols. To analyze the number of GCIs, representative sections, including striatum and SN, were stained with the following antibodies: rat antihuman α‐syn 15G7  and mouse antioligomeric human α‐syn 5G4 . To analyze the number of dopaminergic neurons (TH+ and cresyl violet positive (CV+) neurons in the SNc was measured by applying the optical fractionator workflow. For microglial activation assessment, images were acquired with a fluorescence microscope , and the CD68‐positive area was estimated using ImageJ software . Results are presented as percentage of CD68 area per SN area.After immunostaining, and preceding the acquisition of images/stereological counting, all immunofluorescence/immunohistochemistry slides were randomly numbered by the laboratory technician. All measurements were therefore performed by the researcher blinded to genotype and treatment. Neuroanatomy was assessed using a Mouse Brain Atlas. Stereological analysis was performed using the Nikon E‐800 microscope equipped with a Nikon digital camera DXM1200  and Stereoinvestigator software  as described previously.2, 0.1 mM of phenylmethylsulfonyl fluoride, 1 mM of N‐ethylmaleimide, 0.1% Nonidet P‐40 Substitute, ethylenediaminetetraacetic acid–free cOmplete Mini protease inhibitor , and PhosSTOP phosphatase inhibitor (Roche). Aliquots of the homogenate were frozen in liquid N2 and stored at –80 °C. Total protein concentration was determined by bicinchoninic acid assay. For the sucrose gradient analysis, 300 μg of protein in a final volume of 200 μL were used. To this end, the brain homogenate was thawed on ice and diluted in a buffer containing 50 mM of Tris (pH 7.4), 175 mM of NaCl, 0.1% N‐lauroylsarcosine sodium salt (sarcosyl), and 0.5% sodium deoxycholate. Samples were agitated at 1,200 rpm  at 4 °C for 30 minutes. Cellular debris was removed afterward by centrifugation for 1 minute at 16.000 g and 4 °C. Sucrose gradients were prepared in a 4‐mL 11 × 60 mm polyallomer tube . To this end, sucrose solutions containing 50 mM of Tris (pH 7.4), 0.1% sarcosyl, and 10%, 20%, 30%, 40%, 50%, or 60% of sucrose were pipetted into the tube, starting with 200 μL of the 60% sucrose solution and followed by 400 μL of the 50% to 10% sucrose solutions. Two hundred microliters of the centrifugation supernatant was pipetted as the uppermost layer onto the gradient. Samples were ultracentrifuged in a SW 60 Ti rotor (Beckman Coulter) at 40,000 rpm and 4 °C for 1 hour. Twelve fractions of 200 μL each were collected from the top to the bottom of each tube and subjected to trichloroacetic acid (TCA) precipitation (10% TCA) overnight at –20 °C. After thawing, samples were centrifuged at 25,000 g and 4 °C for 15 minutes. Precipitates were washed once with acetone (–20 °C), centrifuged, and finally resuspended in Laemmli sample buffer. Samples were boiled at 96 °C for 5 minutes and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. Antibodies against total α‐syn  and phosphorylated α‐syn  were used to stain the blots. Images were acquired using the Fusion FX system for western blot and gel imaging, and quantified with FUSION CAPT software .For continuous gradient centrifugation, a 10% (w/v) brain homogenate of the midbrain was prepared using a buffer composed of 50 mM of Tris (pH 7.4), 175 mM of NaCl, 1 mM of MgClP value <0.05 was considered statistically significant. Correlations were studied using linear regression analysis.All statistical analyses were conducted using the software Graph‐Pad Prism . Mean ± standard error of the mean (SEM) was used to present the results. One‐way analysis of variance (ANOVA) with a post‐hoc Bonferroni test was used to compare groups if not indicated otherwise. A To assess the effect of anle138b on motor function, motor coordination and balance were analyzed using the beam challenging test+ cells was quantified in this region. Stereological counting showed a clear preservation of dopaminergic neurons in the SNc of PLP‐hαSyn mice treated with both doses of anle138b compared to healthy controls  Research Project: A. Conception and Design; B. Acquisition of Data; C. Analysis and Interpretation of Data; (2) Manuscript: A. Writing of the First Draft, B. Review and Critique; (3) Other: A. Final Approval of the Submission Statistical Analysis; B. Obtaining Funding; C. Technical Support; D. Supervision of Data Collection.A.H.G.: 1B, 1C, 2A, 2B, 3A.D.W.: 1B, 1C, 2A, 2B, 3A.S.R.: 1C, 2B, 3A.A.L.: 1C, 2B, 3A.C.G.: 1A, 1C, 2B, 3A, 3D.A.G.: 1A, 1C, 2B, 3A, 3D.G.W.: 1A, 1C, 2B, 3A, 3B, 3D.N.S.: 1A, 1C, 2B, 3A, 3B, 3D.A.H.‐G. has been employed by Medical University of Innsbruck. D.W. has been employed by MODAG GmbH. S.R. holds intellectual property rights with EP patent application 17170855 and has been employed by Max Planck Institute for Biophysical Chemistry. A.L. holds intellectual property rights withEP Patent Application 17170855 and has been employed by Max Planck Institute for Biophysical Chemistry and MODAG GmbH. C.G. holds stock ownership in medically related fields: AMGEN, Bayer, Eli Lilly, Fresenius Healthcare, GlaxoSmithKline, Medtronic, Merck, Novartis, Pfizer, Procer Gamble, and Sanofi; patents of the last 12 months: compounds for the treatment and prevention of melanoma: PCT/EP2018/062236; consultancies: MODAG GmbH, Wendelsheim, Germany; expert testimony: ERC, Norwegian Science Foundation; advisory boards: TIFR, uNMR‐NL, and TGIR RMN THC; has been employed by Max Planck Society; Partnerships: KBSI, Daejon, Korea and MPLbioR, Rosario, Argentina; honoraria: Sanofi Aventis: 200€ for a lecture (2018); and Grants: DFG, FP7 health, and BMWi. A.G. stock ownership in medically related fields: shareholder of MODAG GmbH; intellectual property rights: several patents on anle138b and related compounds; employment: Ludwig‐Maximilians‐University Munich; and grants: Michael J Fox Foundation, FP7 health, BMBF. G.K.W. consultancies: Astra; advisory boards: Biogen; employment: Medical University of Innsbruck; honoraria: Novartis, Prana, Lundbeck, Affiris, and AbbVie; royalties: Springer, Cambridge University Press; grants: FWF, FP7 health, and MSA Coalition. N.S. has been employed by Medical University of Innsbruck and has received grants: FWF, FP7 health, and MSA Coalition.+) neurons in SNc . n = 5 per experimental group. Error bars indicate SEM. ANOVA, CV+ neurons/genotype: ****P < 0.0001; CV+ neurons/treatment: ####P < 0.0001 (Bonferroni's test). (B) Correlation analysis of number of neurons in the SNc and number of slips per step. P < 0.0001; R2 = 0.6478.  Correlation analysis of the number of CV+ neurons in the SNc and the density of GCIs (15G7 and 5G4 respectively) in the SNc. P = 0.0010, R2 = 0.6076 and P = 0.0079, R2 = 0.4884 respectively. (E) Correlation analysis of number of CV+ neurons in the SNc and CD68+ area in the SN. P = 0.0002; R2 = 0.6051.Supporting Information FIG. S1. Anle138b prevents neuronal loss in the SNc of PLP‐hαSyn mice. (A) Stereological counting of the total number of cresyl violet positive . (C) Correlation analysis of density of GCIs (5G4) in the SNc and number of TH+ neurons in the same region. P = 0.0368; R2 = 0.3672. (D) Correlation analysis of density of GCIs (5G4) in the SNc and number of slips per step. P = 0.0026; R2 = 0. 3194. (E) Correlation analysis between the CD68+ area in the SN and the density of GCIs (5G4) in the same brain region. P = 0. 0079; R2 = 0. 3477.Supporting Information FIG. 2. Anle138b reduces α‐syn accumulation and microglial activation in PLP‐hαSyn mice.  GCI density in SNc and STR of PLP‐hαSyn mice determined by stereological counting of brain sections stained with the 5G4 antibody against oligomeric human α‐syn and expressed in GCI/mmClick here for additional data file.


Cissus polyantha (Vitaceae) is a plant used in Cameroonian traditional medicine for the treatment of diabetes. The aims of this study were to evaluate the in vitro antioxidant and antidiabetic activities of the aqueous extract of Cissus polyantha leafy stems. The enzyme inhibitory activity was determined in vitro on α-amylase and α-glucosidase enzymes, followed by confirmative study in vivo on normal rats . The ferric reducing antioxidant power and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) antiradical activity of the extract were examined to evaluate the antioxidant potential of the extract. The total content of phenols, flavonoids, and tannins of the extract were also determined. The results showed an inhibitory effect of the extract on the α-amylase and α-glucosidase activities with IC50 values of 216.14 and 182.40 μg/mL, respectively. The extract at doses of 222 and 444 mg/kg induced a significant decrease in postprandial glycaemia during the starch and sucrose tolerance tests. A remarkable antiradical activity of the extract was obtained although lower than that of the standard product. The aqueous extract of leafy stems of Cissus polyantha has an interesting inhibitory activity on the α-amylase and α-glucosidase enzymes, as well as an antioxidant potential, thus validating its use in traditional medicine for the treatment of diabetes mellitus and its complications. It is responsible for the nonregulation of postprandial hyperglycemia which is a major factor of the cardiovascular complications . One of  enzymes .Oxidative stress is potentially involved in many diseases including diabetes, as a triggering factor or associated complications during its evolution . Antioxiα-amylase and α-glucosidase enzymes, such as Retama raetam [Picralima nitida [Combretum molle [Cissus polyantha (Vitaceae) is used in northern Nigeria as an antimicrobial and analgesic [C. polyantha leaves contains quinoids, sterols, terpenes, saponins, phenols, tannins, and flavonoids [Cissus polyantha, to determine its antioxidant potential and to evaluate its inhibitory properties on the α-amylase and α-glucosidase activities.The natural plant extract is used as the alternative therapeutic agents. Several plants have already shown their antioxidant and/or inhibitory potentials on the activity of a raetam , Picralia nitida , and Comum molle . Cissus nalgesic , in Libenalgesic , and in avonoids . The preα-glucosidase, α-amylase, 3,5-dinitrosalicylic acid, p-nitrophenyl-D-glucoside (pNPG), and DPPH were purchased from Sigma-Aldrich . Starch and sucrose were purchased from Edu-Lab Biology Kit . All others chemicals and drugs used were of analytical grade available commercially.Acarbose, Male Wistar rats weighing between 220 and 250 g and 8 to 12 weeks of age were provided by the animal house of the Department of Biological Sciences at the University of Ngaoundere (Cameroon). These animals were housed in plastic cages with free access to water and food at room temperature and in a natural environment. All rats were acclimated for 7 days before starting each test. The animal protocol was approved by the Institutional Animal Ethics Committee (IAEC) before starting the study.Cissus polyantha were collected in September 2017 from Mémé, Far North Region-Cameroon, authenticated in the National Herbarium of Yaoundé-Cameroon where the voucher specimen was deposited under the number 44346/NHC. The leafy stems were dried and ground to obtain a fine powder.The leafy stems of C. polyantha leafy stems was obtained.One-hundred gram (100 g) of the dried powdered plant was infused in 500 mL of distilled water for 30 minutes. After filtration and evaporation in an oven, 18.8 g of the crude extract of C. polyantha were estimated using the Folin–Ciocalteu method. The absorbance was measured at 640 nm using a spectrophotometer. The concentration of total phenols was calculated using a calibrated curve of gallic acid. The results were expressed as mg GAE/g dry wt. .Total phenols of aqueous extract of Total flavonoids were estimated using the aluminium colorimetric method of Zhishen et al.  using quTannin content was measured by the method of Bainbridge et al.  using caAll the analysis were carried out in triplicate.α-amylase enzyme was carried out according to the method described by Apostolidis et al. [μL) of extract or acarbose  was added to 25 μL of buffer phosphate , containing the solution of α-amylase (0.5 mg/mL), and the mixture was incubated at 25°C for 10 minutes. Subsequently, 25 μL of 0.5% starch solution in phosphate buffer  was added to each tube to begin the reaction. The reaction mixtures were then incubated at 25°C for 10 minutes. The reaction was stopped by addition of 50 μL of 3,5-dinitrosalicylic acid (96 mM) in each tube. The microplate was then incubated in a water bath for 5 minutes and then cooled to room temperature. Absorbance (A) was measured at 540 nm using a spectrophotometer. The inhibition percentage was calculated as follows: I% = (Ac − As)/Ac × 100, where Ac is the absorbance of the control and As is the absorbance of the sample. Distilled water was used as control and represents 100% enzymatic activity. The concentration of the extract necessary to inhibit the activity of the enzyme by 50% (IC50) was determined from the regression curve.The in vitro study of the inhibition of s et al. . Twenty-α-glucosidase inhibition study was carried out according to the method described by Kim et al. [μL of the solution of the extract or acarbose  was introduced into 100 μL of tris buffer  containing 100 μL of the solution of α-glucosidase (0.01 mg/mL) and preincubated at 25°C for 10 min. After preincubation, 50 μL of 5 mM pNDG (p-nitrophenyl-α-D glucopyranoside) was added to each tube to begin the reaction. The mixture was then incubated at 37°C for 15 min. The reaction was stopped after the addition of 2 mL of 500 mM Na2CO3 to each tube. Absorbance of the solution was read at 400 nm using a spectrophotometer. The inhibition percentage (I%) and the mean inhibition concentration (IC50) of the extract were calculated as in the case of α-amylase.The in vitro m et al. . In factGroup 1 (control) received 10 mL/kg of distilled waterGroup 2 (standard) received acarbose solution at dose of 10 mg/kg b.w.Group 3 received the extract at the dose of 111 mg/kg b.w.Group 4 received the extract at the dose of 222 mg/kg b.w.Group 5 received the extract at the dose of 444 mg/kg b.w.Twenty-five (25) rats were fasted for 16 hours and randomly divided into 5 groups of 5 rats each as follows :Group 1 th, and 120th minutes after the hyperglycemia induction.Ten minutes after the administration of different treatments, the rats were given orally 4 g/kg b.w. of sucrose or 3 g/kg b.w. of starch. The blood glucose level was measured before the treatment and at the 30th, 60th, 90μL of extract at different concentrations (0 to 800 μg/mL), 500 μL of phosphate buffer (2 mM), and 500 μL of a solution of K3Fe (CN) 6 (1%) were mixed. After incubation at 50°C for 20 min, 500 μL of trichloroacetic acid (10%) was added to stop the reaction. After that, 1.7 mL of distilled water and 200 μL of 0.1% (w/v) FeCl3 were added to the mixture. Absorbance was read against blank at 700 nm. The results were used to calculate the effective concentration (EC50) from the linear regression curve (DO = f (C)) and compared to those of ascorbic acid used as a positive control. All tests and analyses were carried out in triplicate.The reducing capacity of Fe3+ to Fe2+ by plant extract was determined by the method of Chaouche et al. . 200 μL C. polyantha extract was determined by the method of Sayyed et al. [μg/mL). The mixture was stirred well for 5 min at room temperature (25°C). For the control tube, distilled water was used in place of the extract. Butylhydroxyanisole (BHA) was used as positive control. Absorbance was read at 517 nm. The antioxidant activity of the extract was expressed as percent inhibition (%) according to the following equation: inhibition (%) = (Ac − As)/Ac × 100, where Ac is the control absorbance and As is the absorbance of the sample. The concentration of the plant extract and the positive control required to inhibit 50% of DPPH radicals (IC50) was calculated.The DPPH  antiradical effect of d et al. . A DPPH 50 and EC50 values were calculated using nonlinear regression analysis. p < 0.05 was considered to be statistically significant.The results were expressed as mean ± standard error of the mean (SEM). Data obtained were analyzed and means were compared using one-way or two-way ANOVA followed by Tukey and Bonferroni tests, respectively using Graph Pad Prism Version 5.03. ICCissus polyantha contains phenols, flavonoids, and tannins. The amount of total phenols in the extracts was determined using gallic acid  as standard. The results indicated that the aqueous extract content was 167.89 ± 0.1 mg GAE/g dry wt. of total phenols. The flavonoid content (7.54 ± 0.15 mg QE/g dry wt.) was determined using quercetin as standard . The tannin content (0.82 ± 0.5 mg CE/g dry wt.) was evaluated using catechin as standard . These results revealed that the aqueous extract of Cissus polyantha contains phenols, flavonoids, and tannins at varying proportions.n = 3). GAE: gallic acid equivalent; QE: quercetin equivalent; CE: catechin equivalent; dry wt.: dry weight.Values are means of three analyses of the extract ± standard deviation , 68.11% (p < 0.01), and 51.31% (p < 0.05) at 60th, 90th, and 120th minutes, respectively (p < 0.001) (p < 0.01), 60.10% (p < 0.01), and 53.11% (p < 0.05) at the 60th, 90th, and 120th minute, respectively. A significant (p < 0.05) decrease in blood glucose levels of approximately 55.04% and 48.82% was also recorded with the dose of 222 mg/kg of extract at the 60th and 90th minute, respectively. The AUC level was significantly reduced at doses of 111 (p < 0.01), 222 (p < 0.001), and 444 mg/kg (p < 0.001) of extract  . Similar extract .The results of the sucrose tolerance test are shown in Figures p < 0.05), 48.21% (p < 0.01), 41.93% (p < 0.01), and 37.33% (p < 0.05) at the 30th, 60th, 90th, and 120th minutes, respectively. The extract (444 mg/kg) caused a significant drop in blood glucose of about 39.68% (p < 0.05), 57.61% (p < 0.001), 66.85% (p < 0.001), and 48.00% (p < 0.01) at the 30th, 60th, 90th, and 120th minutes, respectively. This decrease was less significant at a dose of 222 mg/kg of extract, i.e., approximately 36.56% and 33.93% at 60th and 90th minutes, respectively. However, the dose of 111 mg/kg had no effect on the hyperglycemia of the animals during the 2 hours of observation.The regulation of postprandial glycaemia was greater in the rats given the reference product (acarbose) and in those treated with the extract at a dose of 444 mg/kg. In fact, acarbose resulted in a significant decrease in blood glucose of approximately 35.29% (p < 0.001) the AUC level. Doses of 111 and 222 mg/kg of extract also resulted in a significant  reduction in the AUC level.In addition, acarbose and the extract at a dose of 444 mg/kg significantly reduced (50) of the reference substance (87.63 ± 3.22 μg/mL) was largely higher than that of the extract (323.33 ± 2.46 μg/mL)  .50: effective concentration which corresponds to 0.5 of absorbance under the assayed conditions. SE: standard error.Data are expressed as mean ± SE. EC50 value of 80.88 ± 1.33 μg/mL, higher than the activity of the extract whose IC50 value was 393.25 ± 0.65 μg/mL  revealed a concentration-dependent increase in percentage inhibition of free radical DPPH. It appears that BHA has a strong antiradical activity with an IC65 μg/mL .50: inhibitor concentration to inhibit 50% of his activity under the assayed conditions. SE: standard error.Data are expressed as mean ± SE. BHA: butylhydroxyanisole. ICα-Amylases and α-glucosidases are two enzymes involved in the digestion of carbohydrates, the main source of glucose in the body. Their inhibition may significantly reduce the increase in postprandial glucose level and may therefore be a therapeutic potential for the treatment of obesity and diabetes [Cissus polyantha is one of those herbs traditionally used to treat diabetes. Phytochemical screening revealed that its leaves contain phenols, flavonoids, tannins, alkaloids, anthraquinones, steroids, carbohydrates, and glycosides [Cissus polyantha on the activity of two digestive enzymes and to evaluate its antioxidant potential. Determination of phenols, flavonoids, and tannins indicated that these three metabolites are present in leafy stems of Cissus polyantha, in significant proportions. The leaves have been shown to contain quercetin flavonoids [diabetes . Some codiabetes . Howeverdiabetes . Cissus ycosides . The objavonoids .α-amylase and α-glucosidase enzymes showed a strong concentration-dependent inhibitory activity of acarbose and C. polyantha extract. However, the extract exhibited the highest inhibition potential for α-glucosidase activity with an IC50 of 182.40 ± 0.54 μg/mL compared to that of the α-amylase enzyme whose IC50 was of 216.14 ± 0.83 μg/mL. Acarbose showed an overall inhibitory activity greater than that of the extract. These results have been confirmed by in vivo studies on starch and sucrose tolerance tests. During the starch tolerance test, the doses of 222 and 444 mg/kg of the extract produced a significant decrease in AUC (p < 0.001) and postprandial glycaemia at the 60th, 90th, and 120th minute. Concerning the sucrose tolerance test, a significant decrease in AUC and blood glucose level was noted after the 60th, 90th, and 120th minutes at the dose of 444 mg/kg of the extract. These results show that the aqueous extract of C. polyantha and acarbose delayed the digestion and absorption of starch and sucrose along the digestive tract, thus reducing postprandial hyperglycemia. From these results, it is observed that there is a good correlation between in vitro and in vivo tests. Acarbose contains compounds with structures similar to those of disaccharides and monosaccharides resulting from the digestion of carbohydrates, which allow them to bind to the α-glucosidase enzymes located on the brush border of the intestine, thus competitively inhibiting these enzymes [α-amylase and α-glucosidase. Quercetin and catechin act as competitive inhibitors of α-amylases [C. polyantha could inhibit the activity of the α-amylase and α-glucosidase enzymes, thereby decreasing postprandial hyperglycemia.The in vitro inhibition tests of the amylases . By the 50 of the order of 393.25 ± 0.65 and 80.88 ± 1.33 g/mL, respectively. The antioxidant activity of a compound is inversely proportional to its IC50 value [50) for iron reduction were 323.33 ± 2.46 g/mL for the extract and 87.63 ± 3.22 g/mL for ascorbic acid. The antioxidant activity of the extract was lower than that of the reference substances. These iron-reducing properties are attributed to phenolic compounds, especially flavonoids and tannins, whose hydroxyl groups can trap radical species [Cissus polyantha would therefore be a source of these bioactive molecules.The study of the antioxidant power of the extract revealed a DPPH antiradical activity and a concentration-dependent iron-reducing potential. The antioxidant potential of a substance is attributed to its ability to break the chain of free radicals by giving a hydrogen atom . In the  species  and chel species , 28. The species . Free ra species . It is e species . The leaCissus polyantha has a remarkable antioxidant potential and inhibitory activity of the α-amylase and α-glucosidase enzymes. These effects would be due to its important phenolic composition whose quantitative study has revealed the varied presence of polyphenols, flavonoids, and tannins. These results could justify the use of C. polyantha leafy stems in traditional medicine for the treatment of type 2 diabetes and complications.The present study has shown that the aqueous extract of the leafy stems of


Bound phenolics slightly (p > 0.05) decreased after 30 min fermentation (7% to 9%) compared to the dough after mixing, but increased significantly (p < 0.05) during 65 min fermenting and baking (16% to 27%). Their antioxidant activities followed a similar trend as observed for total phenolic content. The bread crust demonstrated increased free (103% to 109%) but decreased bound (2% to 3%) phenolic content, whereas bread crumb exhibited a reversal of these results. Total anthocyanin content (TAC) significantly (p < 0.05) decreased by 21% after mixing; however, it gradually increased to 90% of the original levels after fermenting. Baking significantly (p < 0.05) decreased TAC by 55%, resulting in the lowest value for bread crust (0.8 to 4.4 mg cyn-3-glu equiv./100 g). p-Hydroxybenzoic, vanillic, p-coumaric, and ferulic acids were detected in free-phenolic extracts, while protocatechuic, caffeic syringic, and sinapic were additional acids in bound-phenolic extracts. Cyanidin-3-glucoside was the detectable anthocyanin in purple wheat. Bread-making significantly (p < 0.05) increased the phenolic content and antioxidant activities; however, it compromised the anthocyanin content of purple wheat bread.Phenolic profiles and antioxidant properties of purple wheat varieties were investigated to document the effects of bread-making. Bread crust and crumb along with samples collected after mixing, 30 min fermenting, 65 min fermenting, and baking were examined. Free phenolic content (105.4 to 113.2 mg FAE/100 g) significantly ( Wheat, as one of the staple foods worldwide, is not only regarded a source of protein and carbohydrates, but is also recognized for its potential in reducing the risk of oxidative-stress related chronic diseases and age-related disorders, such as cardiovascular diseases, neurodegeneration, type II diabetes, obesity, and some cancers ,2. The pin vitro antioxidant properties of phenolic acids and anthocyanins. However, their specific changes, at each critical step of the bread-making process, have not been adequately investigated. Therefore, the present research aimed to investigate the changes of phenolic acids and anthocyanins as well as their antioxidant activities during bread production.Generally whole wheat, as harvested, is not ingested directly by humans, but requires some processing prior to consumption. Bread, as one of the processed cereals made from wheat, is an important food product in human diets and therefore acts as a suitable carrier for health-promoting compounds. There is growing consumer interest in ingesting whole-meal products because of the concentrated dietary fiber and phytochemicals in the outer layers of the grains ,7. Even et al. +), being the molecular weight of anthocyanin, and fragment ion m/z 287 arising from a loss of glucose residue m/z 162. This was in agreement with the study of Hosseinian et al. [et al. [To examine the presence of anthocyanins, an HPLC chromatogram with MS and MS/MS was recorded at a wavelength of 520 nm. Anthocyanin composition detected by HPLC-QTOF-MS/MS is shown in n et al. . Liu et p-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, and sinapic acid) were purchased from Sigma-Aldrich Chemical Co. . HPLC grade methanol and ethyl acetate were purchased from Fisher Scientific Co. . Deionized water (Milli-Q) was used in the HPLC analysis.Analytical grade acetic acid, Folin-Ciocalteu reagent, 2,2′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox), and phenolic acid standards  were received from Mørdrupvej 5 DK-3540 Lynge , including Indigo (97% purple kernels), Konini (84% purple kernels), and Öelands hvede (yellow kernels). These three wheat grains   with somAt the end of mixing, 30 min fermenting, 65 min fermenting, and baking, samples were removed immediately into a −20 °C freezer to terminate the chemical changes. All the samples were freeze-dried along with the raw flour, bread crust (5 mm thick outside the whole bread), and crumb (crust removed bread inner-side) and ground to pass through a 0.42 mm sieve. All the samples were stored at 4 °C before analysis.w/v) . Each time, the mixture was shaken for 1.5 h using a wrist action shaker  and then sonicated for 0.5 h  under nitrogen and dark condition at ambient temperature. The mixture was centrifuged at 11,963 g and 4 °C for 15 min . The supernatants were combined and filtered with a φ 90 mm filter paper (Whatman™ Cat No. 1004-125). The collected crude extracts were stored at −20 °C until the analysis for free phenolic content, DPPH• scavenging activity, and ABTS•+ decolorization capacity. To identify and quantify the soluble phenolic acids, the crude extracts were filtered with 0.45-μm syringe filters  prior to the injection into HPLC. The residues were dried in a fume hood at room temperature and kept in sealed bags at 4 °C before alkaline hydrolysis. Extraction was done in duplicate.A finely ground sample (2 g) was extracted twice with 80% methanol at a ratio of 1:5 . Every time after mixing, ethyl acetate was separated from the aqueous layer by centrifuging at 11,963 g and 4 °C for 5 min . The supernatant was collected into an Erlenmeyer flask using a pipette. Further dehydration was performed by adding 1 g of Na2SO4 and then filtering with a filter paper . The combined ethyl acetate extracts were evaporated and dried at 35 °C under a vacuum using a rotary evaporator  with a water bath . The dried residue was redissolved in 2 mL of 50% methanol and stored at −20 °C. The extract was used for the determination of bound phenolic content, DPPH• scavenging activity, and ABTS•+ decolorization capacity. To investigate the presence of insoluble phenolic acids, HPLC analysis was also performed. The samples were filtered with 0.45-μm syringe filters  before injection. Extraction was done in duplicate.The residue collected after 80% methanol extraction was subjected to alkaline hydrolysis. Insoluble phenolic compounds were released using the method described by Qiu et al.  with somv/v) at a ratio of 1:8 (w/v). The pH of the mixture was adjusted to 1. The mixture was shaken for 1.5 h , followed by sonication for 0.5 h  under nitrogen and in the dark at ambient temperature. The mixture was then centrifuged at 11,963 g and 4 °C for 15 min . The supernatant was filtered with a φ90 mm filter paper (Whatman™ Cat No. 1004-125) and evaporated to dryness at 35 °C under a vacuum using a rotary evaporator  with a water bath . The residue was then redissolved in 2 mL of 80% methanol. The crude extracts were kept at −20 °C for two days to precipitate large molecules and then filtered through a 0.45-μm syringe filter . The extracts were stored at −20 °C prior to the determination of total anthocyanin content (TAC) with pH differential method, DPPH• scavenging activity, and ABTS•+ decolorization capacity. Extraction was done in duplicate.Extraction of anthocyanins was performed according to a method summarized by Young and Abdel-Aal  with modet al. [The free, bound, and total phenolic contents  were determined using a Folin-Ciocalteau method as described by Li et al. . Brieflyet al. [t = 30 min. To determine the absorbance at t = 0 min, measurement was taken by adding 3.9 mL of DPPH• solution to 0.1 mL of 80% or 50% methanol . The antioxidant activity was calculated as: % DPPH• scavenging activity =  × 100. A plot of trolox concentration vs. % DPPH• scavenging activity was used as a standard curve. DPPH value was expressed as μmol TE /100 g.The DPPH method was used according to the modified method used by Beta et al. . A 60 μm+ reagent was made by mixing ABTS•+ stock solution (7 mM) (38.4 mg ABTS in 10 mL distilled water) and potassium persulfate stock solution (2.45 mM) (6.62 mg potassium persulfate in 10 mL distilled water) at a ratio of 1:1 for 2 min vortex. The mixture was stored for 12 to 16 h in the dark at room temperature prior to use. Then, 5 mL of ABTS•+ reagent was diluted with approximately 495 mL of distilled water and absorbance at a wavelength of 734 nm was adjusted to 0.7 by diluting the solution further with distilled water. The antioxidant activity of soluble or insoluble phenolic extracts and anthocyanin extracts (50 μL) was evaluated by adding 1.85 mL of freshly made ABTS•+ reagent. The absorbance was determined at t = 30 min. For absorbance at t = 0 min, 1.85 mL of freshly made ABTS•+ reagent was added to 50 μL of 80% or 50% methanol . The ABTS•+ decolorization (%) was calculated as  × 100. A standard curve was generated based on different trolox concentrations vs. % ABTS•+ decolorization and the antioxidant activities of extracts were expressed as μmol TE/100g sample.The ABTS•v/v) at a ratio of 1:8 (w/v). The mixture was shaken for 0.5 h  under nitrogen and dark condition at ambient temperature. The mixture was then centrifuged at 11,963 g and 4 °C for 15 min . The supernatant was filtered with a φ90 mm filter paper (Whatman™ Cat No. 1004 125). The absorbance of the collected supernatant was scanned at wavelengths from 230 to 700 nm using an Ultrospec 1100 Pro UV/Visible spectrophotometer  controlled by Biochrom Data Capture Software (version 2.0) . A plot of absorbance vs. wavelength was created.A finely ground sample (1 g) was extracted with acidified methanol/HCl (1 N)  with pH = 1 was added to 1 g of finely ground sample. The mixture was shaken for 1.5 h , followed by sonication for 0.5 h  under nitrogen and in the dark at ambient temperature. The mixture was centrifuged at 11,963 g and 4 °C for 15 min . The supernatant was filtered with a φ 90 mm filter paper (Whatman™ Cat No. 1004-125). The absorbance at 535 nm was measured without further dilution of the crude extracts. Cyanidin-3-glucoside was used as a standard, which was the primary anthocyanin in wheat . The total anthocyanin content of each sample was calculated as:Total anthocyanin content was measured according to the method reported by Liu et al.  with som3CO2Na∙3H2O was dissolved in 960 mL distilled water and the pH was adjusted to 4.5 with acetic acid. Anthocyanin extracts that had been reconstituted into 2 mL of 80% methanol were used in this assay. Samples were diluted five times with both buffers to a final volume of 2 mL, respectively (0.4 mL of anthocyanin extracts in 1.6 mL of either potassium chloride buffer or sodium acetate buffer). The absorbance of each sample was measured at 520 nm against a blank of distilled water after 30 min of preparation. The haze and sediment were corrected by measuring the absorbance again at 700 nm. The concentration (mg/L) of each anthocyanin was calculated according to the following formula and expressed as mg C3GE/100 g sample:520nm − A700nm) pH 1.0 − (A520nm − A700nm) pH 4.5, MW is the molecular weight for cyanidin-3-glucoside = 449.2 g/mol, DF is the dilution factor = 5, L is the path length = 1 cm, and ε is the extinction coefficient = 26,900 L × cm−1 × mol−1 for cyanidin-3-glucoside.A second procedure for determination of total anthocyanin content is based on a pH differential method . TheoretIdentification and quantification of phenolic compounds were carried out on an HPLC  equipped with a photodiode array (PDA) detector  and an autosampler . Analysis was performed on a Gemini 5 μ C18 110 A column (150 mm × 4.60 mm)  and the mobile phase consisted of 0.1% acetic acid in water (solvent A) and 0.1% acetic acid in methanol (solvent B). A 70 min linear gradient was programmed as follows: 0–11 min, 9%–14% B; 11–14 min, 14%–15% B; 14–17 min, 15% B; 17–24 min, 15%–16.5% B; 24–28 min, 16.5%–19% B; 28–30 min, 19%–25% B; 30–36 min, 25%–26% B; 36–38 min, 26%–28% B; 38–41 min, 28%–35% B; 41–46 min, 35%–40% B; 46–48 min, 40%–48% B; 48–53 min, 48%–53% B; 53–65 min, 53%–70% B; 65–66 min, 70%–9% B; 66–70 min, 9% B. The injection volume was 10 μL and the flow rate was 0.9 mL/min. Identification of phenolic acids was achieved by comparison to the retention time of phenolic acid standards and their maximum UV absorption. A sample spiked with external standards was used for further identification. Phenolic acid quantitation was based on the standard curves of the corresponding phenolic acids at a wavelength of 280 nm and peak area was used for calculations. The HPLC analyses were done in duplicate for each sample.et al. [The anthocyanin composition was evaluated following a method described by Bicudo et al. . The chrp < 0.05. Pearson’s correlation test was used to evaluate the correlation among variables at significant levels of p < 0.05 and p < 0.01.The results were reported as mean ± standard deviation (SD) of duplicate determinations. Analysis of variance (ANOVA) for main factors  was determined using the GLM procedure with SAS version 9.3 . Least significant differences (LSD) were employed to define significant differences among varieties at a level of In summary, the changes of FPC, BPC, TAC as well as their AOA during the production of bread were first systematically investigated. Individual phenolic acids were also identified and quantified in fractions from each stage of bread-making. Mixing, fermenting, and baking significantly increased FPC and BPC. Bread crust contained the highest FPC, while bread crumb contained the highest BPC. AOA were correlated to their respective phenolic content. Different phenolic acids behaved differently in the bread-making process, which might be ascribed to their difference in chemical natures, sensitivities towards heat, and ease of liberation from plant cell walls. TAC was significantly reduced through mixing and baking, but fermentation elevated the level of TAC. Anthocyanin extract of purple wheat exerted higher AOA than the common wheat, indicating the potential of purple wheat to be used as a functional ingredient in baking.


In this paper, a new kind of graphene double-ribbon bend structure, which can support two edge graphene surface plasmons (EGSPs) modes, is proposed. In this double-ribbon bend, one edge mode can be partly converted into another one. We attribute the mode conversion mechanism to the interference between the two edge plasmonic modes. Based on the finite element method (FEM), we calculate the transmission and loss of EGSPs propagating along this graphene double-ribbon bend in the mid-infrared range under different parameters. Graphene, due to its unique mechanical, electrical and optical properties ,2,3,4, i2O3 substrates [The graphene ribbons can support both waveguide GSPs (WGSPs) modes and strongly confined edge GSPs (EGSPs) modes. EGSPs are the fundamental modes, which are strongly localized along the graphene edge, show a larger effective refractive index and stronger field confinement . EGSPs mbstrates . The widbstrates . Conventbstrates , ribbon bstrates  and wrinbstrates , are stubstrates . Furtherbstrates .In the paper, we propose a new kind of double-ribbon bend that supports two EGSPs. Based on the finite element method (FEM), we firstly discuss the dispersion relation of EGSPs for this double-ribbon bend. We calculate transmission and loss of edge modes with different bending angles under Fermi level, separation distance, incidence wavelength, bending radius and double-ribbon width. The mode conversion is due to interference between the two EGSPs, and the loss of double-ribbon originates from the absorption loss and bending loss. The period of the mode conversion by simulation is in accordance with the theoretical results.D. When the two edges of two ribbons come closer to each other, they will bring out two of edge modes with opposite parity [The SPs supported by individual graphene nano-ribbons has been discussed very frequently. Plasmon interaction and hybridization in pairs of neighboring aligned ribbons are shown to be strong enough to produce dramatic modifications in the plasmon field profiles . What wie parity .D is deposited on the SiO2 substrate, and these two bending ribbons are of equal width. 2 substrate is 2.25. The radius and the width of the double-ribbon bend are R and W, respectively. The thickness of the graphene double-ribbon bend is 1 nm. The bending angle of the structure is denoted by θ. Here, we only discuss the condition when D is smaller than 20 nm, where the ribbons structure supports the two EGSPs modes.As illustrated in W >> D), we can consider the two graphene ribbons as two semi-infinite sheets, and the coupling happens at the internal edge of the double-ribbon bend σG=ieħ, e and Bk represent reduced Planck’s constant, the electron charge and Boltzmann’s constant respectively. Equation (1) shows that Gσ depends on the Fermi levels fE, the momentum relaxation time τ, temperature T, and the photon frequency ω. Fermi energy level is shown in Equation (2)n is the charge carrier concentration, the Fermi velocity fV is 106 m/s. The permittivity Gε of graphene is governed byη0 (≈377 Ω) is the impedance of air, τ is chosen as 0.5 ps, the thickness of graphene d is 1 nm, and the temperature T = 300 K. Due to graphene’s particular characteristics, Gε can be tuned by modifying the gate voltage or doped by chemical doping.In Equation (1), z-component of magnetic field in the x-z plane, when the incident wavelength is 6 μm, fE = 0.2 eV, D = 10 nm and W = 200 nm. Z-component of magnetic field in We choose COMSOL Multiphysics based on the finite element method (FEM) to conduct modes analysis of GESPs mode for our double-ribbon bend and all simulation in the paper will be conducted by FEM . Figure The differences between the real parts of effective refractive indexes for the two edge modes can be obtained byn1) and Re(n2) represent the real part of two EGSPs’ effective refractive index respectively. n1), Re (n2), and Δeffn is related to the following parameters: Fermi levels fE, wavelengths λ, and separation distance D. effn)) of the SEM (black line) and AEM (red line) mode both decrease with increasing fE; but the exact opposite is for the difference Δeffn between two EGSPs’ modes (blue line), when λ = 6 μm and D = 10 nm. As can be seen in effn firstly rises then decreases with increasing Fermi level fE under the same wavelength. And the Δeffn is larger when λ is increasing from 5 to 8 μm under the same fE. D = 10, 12 and 14 nm , Δeffn firstly rises then decreases with increasing Fermi level fE under the same separation distance D. The Δeffn is larger when D is decreasing from 14 to 10 μm under the same fE. In Equation (4), Re. The SEM’s transmission is plotted in the black line and AEM’s is in the red line. The blue ones represent the conversion efficiency P, in fE. The variation amplitude of SEM’s declines with bending angle, but those of AEM and P are nearly unchanged. tθ is the conversion period, when the bending angle is tθ/2, P reaches the maximum; and when the bending angle is tθ, P reaches the minimum. It is found that when fE is 0.3, 0.4, 0.6, 0.8, and 1 eV, tθ is 54.6°, 54°, 57.8°, 62.4°, and 67° respectively, which is accordance with Equation (5). The theoretical value Tθ of the conversion period is 54.5°, 54.8°, 58.6°, 64°, and 69°, respectively. The maximum of conversion efficiency P is 15.40%, 13.92%, 11.86%, 8.68%, and 5.80%, respectively, and the conversion efficiency P decreases with increasing fE.TL, which is calculated byR1 and R2 are the reflectivity of SEM and AEM for our proposed structure. TL consists of absorption loss and bending loss. Here, we define this absorption loss of per unit length for a straight double-ribbon waveguide as normalized absorption loss La. The normalized total loss Lt is derived from the total loss TL over the actual propagation length . The normalized bending loss Lb is defined by the difference between Lt and La.Because of graphene absorption loss and bending radiation loss, SEM’s transmission decreases gradually. Here, we define the total losses TL vs. bending angle under different fermi levels. TL increases with increasing bending angles under the same fE, because larger bending angles mean larger propagation loss of EGSPs. TL decreases with increasing fE under the same bending angles. The three normalized losses Lt, La and Lb are plotted by cyan, orange, and olive lines respectively in fE. effn) decreases with increasing fE for both SEM and AEM. Larger Im(effn) causes a bigger normalized absorption loss La, which can explain that La decreases with increasing fE in Hz are shown in fE = 0.3 eV. It is found that when bending angle is 27.3°, SEM can be partly converted into AEM (P = 15.40%), so magnetic field distributions Hz at the output is the superposition of AEM and SEM; when bending angle is 54.5°, SEM can propagate through the bending structure without mode conversion, so magnetic field distributions Hz at the output is symmetrical.Tθ and simulation value tθ for conversion period respectively, under different fE, in fE. It is found that the theoretical value Tθ matches very well with the simulation results.The solid line and dashed line represent the theoretical value D = 10, 12, and 14 nm) is shown in fE = 0.6 eV, R = 300 nm, λ = 6 μm, and W = 200 nm. It is also found that SEM is partly converted to AEM varying with different bending angles. The transmission of two EGSPs and P both have the same changing period with bending angle (ranging from 0° to 90°) under the same D when D is 10, 12, and 14 nm, tθ is 58°, 65.7°, and 74°. The theoretical value Tθ, which is obtained by Equation (5), is 59.2°, 68.0°, and 78.4°. tθ decreases with increasing D. That is because Δeffn increases with increasing D under the same fE in Pmax respectively are 6.1%, 9.2%, and 13.5%, and increases with increasing D.Periodical mode conversion between SEM and AEM under different separation distances  of SEM decreases slightly with increasing D, but the opposite is true for AEM, which is shown in D, but the opposite is true for AEM. The absorption loss mainly originates from SEM’s propagation loss. So when D is increasing, Lt, La and total loss TL decreased a little due to the absorption loss difference between SEM and AEM. The proposed double-ribbon bend has the same curvature under three separation distances D, so Lb is also the same.As illustrated in λ = 5, 6, 7 and 8 μm). Other parameters are as follows: fE = 0.6 eV, D = 10 nm, R = 300 nm, and W = 200 nm. The transmission of two EGSPs and the conversion efficiency P both have the same change period with bending angles under the same wavelength. It is can be seen in λ = 5, 6, 7 and 8 μm, tθ is 54.9°, 57.8°, 63.5°, and 69.1°. Theoretical value Tθ is 54.4°, 58.6°, 64.4° and 70.1° based on Equation (5). The maximum conversion efficiency Pmax is 15.40%, 11.86%, 8.40%, and 4.96%, respectively. As illustrated in TL is plotted in pink, yellow, blue, and green lines when the wavelength is 5, 6, 7, and 8 μm, respectively. It is found that total loss TL enhances with increasing bending angle under the same wavelength λ because of the absorption loss of EGSPs. TL decreases with increasing wavelength λ under the same bending angle. As shown in Lt, La and Lb are plotted in cyan, orange and olive lines, respectively. They all decrease with increasing λ. As shown in effn) of SEM decreases with increasing λ, AEM does just the opposite. It’s worth noting that the proportion of SEM is much larger than AEM’s, the downtrend for Im(effn) of SEM will play a dominating role in the absorption loss. Thus, La declines with increasing λ.As shown in fE = 0.6 eV, D = 10 nm, λ = 6 μm, and W = 200 nm. The transmission of two EGSPs and the conversion efficiency share the same converting period. When R is 300, 400, 500 and 600 nm, tθ is 57.8°, 43.3°, 35.0°, and 29.0°. The theoretical angel Tθ obtained by Equation (5) is 58.6°, 44.4°, 33.9°, and 29.6°. The maximum of conversion efficiency Pmax is 6.06%, 3.45%, 2.22%, and 1.55%, respectively.As shown in TL of the double-ribbon bend varying with different bending angles under different R: 300 nm (pink line), 400 nm (yellow line), 500 nm (blue line), and 600 nm (green line). TL increases not only with increasing bending angles under the same R, but also increases with increasing R under the same bending angle. Because a longer propagating length will be obtained by a larger bending angle under the same R and larger R under the same bending angle, so does the absorption loss. As can be seen in Lt and Lb both decrease with increasing R, but the normalized loss La is nearly unchanged with R. Because the fE, D, and λ is the same, Im(effn) of AEM and SEM is the same, the normalized absorption loss La is accordingly unchanged with R. Because a larger R has a smaller curvature and lower bending radiation loss, Lb decreases with increasing R and so does Lt. To improve the mode conversion, gain medium can be added to reduce the absorption loss [ion loss .In this paper, we propose a new kind of double-ribbon bend that supports two EGSPs and focus on the conversion between two EGSPs. Using FEM, we prove the period of the conversion between two EGSPs is determined by a separation distance, incidence wavelength, and bending radius, and the period of the conversion can be also tuned by the Fermi level. The mode conversion originates from the interference between the two edge plasmonic modes. The loss of double-ribbon bend consists of the absorption loss and bending loss, and the effects of parameters on loss are also discussed. Our double-ribbon bend may provide a new perspective to understand the conversion relationship between two EGSPs.


M and bicomponent fitting of short T2⁎ (T2⁎S), long T2⁎ (T2⁎L), short T2⁎ fraction (Frac_S), and long T2⁎ fraction (Frac_L) were performed within tendons and ligaments. Our results showed that biexponential fitting was superior to single-exponential fitting in PT, PCL, and ACL. For knee joint samples, there was no statistical difference among all data in PT, PCL, and ACL. For volunteers, all parameters of bicomponent fitting were statistically different across PT, PCL, and ACL, except for T2⁎S, T2⁎L, and T2⁎M resulting in flawed measurements due to the magic angle effect. 3D multiecho FS-UTE-Cones acquisition allows high resolution T2⁎ mapping in PT, PCL, and ACL of keen joint samples and PT and PCL of volunteers. The T2⁎ values and their fractions can be characterized by bicomponent T2⁎ analysis that is superior to single-component T2⁎ analysis, except for ACL of volunteers.The collagen density is not detected in the patellar tendon (PT), posterior cruciate ligament (PCL), and anterior cruciate ligament (ACL) in clinic. We assess the technical feasibility of three-dimension multiecho fat saturated ultrashort echo time cones (3D FS-UTE-Cones) acquisitions for single- and bicomponent T2⁎ analysis of bound and free water pools in PT, PCL, and ACL in clinic. The knees of five healthy volunteers and six knee joint samples from cadavers were scanned via 3D multiecho FS-UTE-Cones acquisitions on a clinical scanner. Single-component fitting of T2⁎ As a result, early stages of tendon and ligament degeneration may not be detected by traditional MRI.Many of the degenerate tendons and ligaments from cadavers and biopsies from patients had a decreased collagen concentration and this change may predispose the tendons and ligaments to rupture, as a reduction in the collagen density has been correlated with the tensile strength of tendons and ligaments. If we can detect the reduction in the collagen density in the degenerate patellar tendon (PT), posterior cruciate ligament (PCL), and anterior cruciate ligament (ACL) in clinic, some medical methods might be used to prevent PT, PCL, and ACL from being ruptured. However, tendons and ligaments typically have very short transverse relaxation times  techniques, which use nominal TEs about 10-200 times shorter than those of conventional clinical MR sequences, can directly detect signal from short T2 tissues and might be used for diagnosis of these diseases at early stages . Fat saturements . Previouy stages .∗ and is located between the network of interwoven collagen bundles, and bound water has a shorter T2∗ and is associated with collagen and/or proteoglycan [∗ values (T2∗M) alone is not able to discern short and long T2∗ components. By performing bicomponent UTE T2∗ analysis, “short” (T2∗S) and “long” (T2∗L) T2∗ values and fractions can be determined. The short T2∗ value, T2∗S, represents bound water while the long T2∗ value, T2∗L, represents free water [Most knee joint tissues, including PT, PCL, and ACL, have two components, namely, bound water (BW) and free water (FW). Free water has a longer T2eoglycan –8. Singlee water , 10.∗ mapping of free and bound water components for PT, PCL, and ACL in a clinical 3T scanner.However, bicomponent analysis typically requires a long scan time to allow acquisition of all images at different TEs , 11. HigFive heathy volunteers  were enrolled to investigate the clinical feasibility of 3D multiecho FS-UTE-Cones imaging techniques using a clinical whole-body 3T scanner. Written informed consent and approval from the institutional review board (IRB) of our hospital were obtained before the in vivo scans. The inclusion criteria for the volunteers were as follows: no history of knee joints pain, no nontraumatic joint pain history, and no metal implants or pacemakers.Six sets of PCL, ACL, and PT samples from cadaveric knees of six donors  were obtained from University California, San Diego morgue. A transverse cut at the proximal one-third of the samples and a longitudinal cut through the center of the ligament stored in -20°C refrigerator. A transverse slab of ~10 mm thickness and a longitudinal slab of ~5mm thickness were cut and stored in a phosphate buffered saline (PBS) soaked gauze at 4°C prior to MR imaging. After the ex vivo scans, the samples from the center of the PCL, ACL, and PT substance were immediately fixed in Z-Fix  for histology. Samples were embedded in paraffin, and five micrometer thick sections were cut and stained with hematoxylin and eosin (H&E).μs) followed by 3D spiral trajectories with conical view ordering , flip angle (FA) = 16°, bandwidth (BW) = 128 kHz, field of view (FOV) = 8×8cm, acquisition matrix of 256×256, 26 slices with a slice thickness of 2 mm, and a total scan time of 12 minutes. In clinic, except for the same acquisition matrix with knee joint samples, the acquisition was TR = 86.2 ms, four groups of four echoes were TEs = 0.032/4.4/20/40 ms, 0.4/6.6/25/50 ms, 0.8/1/30/60 ms, and 2.2/16/35/70 ms, flip angle (FA) = 14°, bandwidth (BW) = 125 kHz, field of view (FOV) = 20×20 cm, 30 slices with a slice thickness of 4 mm, and total scan time of 18 minutes. Image of axial level is obtained.To save scan time, a multiecho FS-UTE-Cones acquisition scheme was designed for mapping of T2∗ analyses were performed in MATLAB  using code developed in-house as previously described [∗ values for ROIs were recorded for analysis and evaluated by a musculoskeletal radiologist of twenty- year experiences.Both single- and bicomponent T2escribed . The ROIN(t) is the signal intensity, and A is the amplitude of the total component T2∗M.S is the amplitude of the short component, AL is the amplitude of the long component, T2∗S is the short component T2∗, and T2∗L is the long component T2∗. Apparent short component fraction (Frac_s) was defined as AS/(AS + AL), and long component fraction (Frac_L) was defined as AL/(AS + AL).Both single- and biexponential fitting procedures were performed on the selected ROIs, for all MR data sets. For single-exponential fitting, a three-parameter function (see ) was use∗ calculation, only the pixels that satisfied the following condition (For the T2ondition  were con created . The roo created .∗s and fractions of the two components with a root-mean-square error (RMSE) value of less than 3%, providing a clinically achievable SNR of 60 or higher.As a result, the bicomponent fitting model can estimate T2∗M, T2∗S, T2∗L, Frac_s, Frac_L, and RMSE, were described as the means and standard deviation (SD) for normal PT, PCL, and ACL. An independent sample T-test with equal variances was performed to obtain the difference in RMSE between single- and bicomponent analysis of PT, PCL, and ACL, respectively. Pairwise differences across T2∗M, T2∗S, T2∗L, and their fractions were also analyzed for PT, PCL, and ACL. P less than 0.05 were considered statistically significant.All statistical analyses were analyzed in SPSS Statistics version 13.0 for Windows. Calculated values, including T2∗ analysis. RMSE error values (means ± SD) of bi- versus single-exponential fitting model were shown as follows: for knee joint samples, 0.08 ± 0.04% versus 0.91 ± 0.41% for PT; 0.12 ± 0.02% versus 1.38 ± 0.25% for PCL; 0.33 ± 0.09% versus 1.91 ± 0.71% for ACL; for volunteers, 1.63 ± 0.15% versus 2.56 ± 0.12% for PT; 1.34 ± 0.13% versus 2.57 ± 0.25% for PCL; 3.23 ± 0.33% versus 3.62 ± 0.33% for ACL. Biexponential fits were significantly different from single-exponential fits for PT and PCL (P = 0.01 in both cases), while there was no statistical significance between the single- and biexponential fits for ACL (P = 0.29) in volunteer. The single- and biexponential fits in ACL had the largest errors among RMSEs in PT, PCL, and ACL in volunteer.Simulation results are shown in ∗S, T2∗L, Frac_S, Frac_L, and T2∗M obtained in PT are similar to all data measured in PCL and ACL, which have been proved to be normal tissues using histology. ∗S, T2∗L, Frac_L, and T2∗M are smaller in PT and PCL than those in ACL. On the contrary, Frac_S was smaller in ACL than in PT and PCL   . P > 0.05); for volunteers, there are significant differences in Frac_S and Frac_L, for PT versus PCL, for PCL versus ACL, and for PT versus ACL, with the P value of 0.01 for all cases. Moreover, T2∗S, T2∗L, and T2∗M show statistical differences between PCL and ACL and between PT and ACL. P values for all the aforementioned cases were 0.01. On the contrary, no significant differences were found for T2∗S, T2∗L, or T2∗M between PT and PCL  or between PCL and ACL . ∗s (B), Frac_L (C), and T2∗L (D) in PT, PCL, and ACL of samples and volunteers. Except for measurements in ACL of volunteers, variations among all data were smaller. If we applied 0.01 < P values < 0.001, there was no statistic significance among all measurements.∗s and relative fractions in PL, PCL, and ACL. Previously, 2D UTE sequences have been employed for bicomponent analysis of bound and free water components in various knee joint tissues [∗ and longer T2∗ components [∗s and relative fractions.Our results suggest that the 3D Cones FS-UTE sequences, together with an interleaved multiecho acquisition strategy, allow mapping of bound and free water T2 tissues . 3D UTE  tissues . In addi tissues , 20. Themponents . Finally∗ fitting performance with very small RMSE values in all measurements, except for that in ACL of volunteers. This is consistent with the previous results published by Raya et al., showing that biexponential analysis can reduce errors to less than 4% with an SNR of 50 [∗ fitting in ACL is inferior to that in PT and PCL. These results might be explained by the fact that ACL is oriented closer to the magic angle (~54°) relative to the B0 field and thus exhibits greatly reduced the signal from short T2 components.As shown in NR of 50 . Single-∗M of 2.05 ± 0.27 ms in PT was consistent with the range of values from healthy volunteers shown by Kijowski et al. via a UTE-T2∗ mapping sequence in a 3.0T scanner  [∗M in ligament using 3D UTE before. Our preliminary results show that the T2∗M in PT were similar to ones in PCL and ACL of knee joint samples, and that the T2∗M was lower in PT (2.05 ± 0.27 ms) than in PCL (2.21 ± 0.96 ms), and much lower than in ACL (7.65 ± 1.29 ms) of volunteers. It is well known that the percent of water in PT is known to be 60-70%, which is similar to the 55–65% percent water in ligament [∗ values can be influenced not only by the water and collagen content but also the collagen fiber orientation, including longitudinal, transverse, and oblique directions [∗ values [∗M in ACL than in PT and PCL of volunteers.The T2ligament . Additioligament . Howeverligament . T2∗ valrections , 26. In ∗ values . So, the∗ bicomponent analyses in PT are consistent with results reported in previous studies. An example is a study performed by Kijowski et al., with T2∗S of 1.5 ms (CI: 1.3–1.8 ms) and Frac_S of 75.5% (CI: 74.7–78.9%) in PT [∗S of 1.8 ms (range 1.4-2.4 ms), Frac_S of 79% (CI: 67-93%), and Frac_L of 21% (range 8-33%) via 2D UTE sequences on a 3T MR scanner [S range from 47% to 79% and a T2∗L range from 7.9 ms to 31.8 ms in PT. However, T2∗L values in our study were intermediate between the T2∗L of 23.1 ms (95%CI 21.7–25.0ms) reported by Kijowski et al. and the T2∗L of 9.2 ms (range 5.6-16.4 ms) identified by Chang et al. This could be explained by the fact that collagen fiber organization affects the T2∗L more than the T2∗S in the knee. Also, measurements are susceptible to nearby tissues (such as fat and water in knee joints), which result in lengthened T2∗L in PT [Our results for T2∗L in PT .∗ bicomponent analysis of PCL and ACL by using a 3D Cones UTE sequence. Our study showed that all parameters were statistically different among PT, PCL, and ACL, except for T2∗S and T2∗L in PT versus PCL, as well as in PCL versus ACL of volunteers. The differences in parameters between PT and PCL were less than those between PT and ACL of volunteers in our results  and anteromedial (AM) bundles mean diameter of 9 mm (range 7–17 mm). For knee joint samples, collagenous fibers are parallel to the B0 field; there was no magic angle effect on the measurement in PT, PCL, and ACL. In addition, the relationship between magnetization fractions and matrix components may be affected by proton exchange between compartments. For example, Lattanzio et al. used a four-spin component exchange model in their study of articular cartilage [−1 between PG and collagen was found, which is intermediate to the corresponding nominal relaxation rates in our analysis of 1/T2,1 = 438 s−1 and 1/T2,2 = 39.6 s−1. Therefore, the T2∗s and associated fractions measured in our experiment might be influenced by this exchange. Moreover, as discussed by Zheng et al., T2∗ measurements could actually reflect a combination of molecular, structural, and procedural levels of complications [For the first time, we performed T2 results . Frac_S artilage . A magneications . Hence, ications , 33.∗ mapping. Volumetric analysis of T2∗ mapping would be for future study. Second, bicomponent T2∗ analysis can characterize the short and long T2∗ values and fractions for PT and ligaments. It seems likely that both water bound to collagen and water bound to proteoglycans contribute to the short T2∗ signal component. In particular, the short T2∗ signal component is mainly derived from the water bound to collagen. On the contrary, the long T2∗ signal component derived from the free water in ligament and tendon. So, the T2∗S and their fractions might provide information about water bound to collagen matrix, which would be used as biomarkers for early degeneration associated with injury of collagen matrix in PT and ligaments. 3D UTE Cones MR acquisition might be particularly useful for measurement of PT and PCL tissues, the distinction between abnormal from normal tissue. Finally, FS-UTE 3D Cones imaging can provide relatively shorter the total scan time of 18 min than UTE imaging in previous studies.In summary, our results suggest that multiecho 3D UTE Cones acquisitions have some advantages over existing technologies. First, the multiecho 3D FS-UTE-Cones acquisition allows highresolution 2D T2∗ mapping. Movement of the subjects was minimized by careful knee fixation, and the images were coregistered in postprocessing. Second, the number of volunteers was small, consisting of five knees from five healthy volunteers. With more volunteers, we expect that the RMSE of ACL would be decreased, that the difference between ALC and PCL would reach significance, and that clinical diagnostic guidelines for making decisions about disorders of PT, PCL, and ACL would be found using 3D UTE Cones MR acquisition. Finally, although the total scan times of 18 min were shorter than the scan times using UTE techniques in previous studies, in clinic MRI, this UTE scan time is still too long for clinical use. Further reduction in the total scan time will be explored in future studies via few TE, parallel imaging, and/or compressed sensing techniques.Our study has several limitations. First, 3D UTE requires longer scan times. The increased likelihood of patient movement increases susceptibility to motion artifacts and could introduce errors in biexponential T2∗ mapping in a clinical setting. For PT and PCL, the short and long T2∗ components and their fractions can be characterized by bicomponent T2∗ analysis, which is superior to single-component analysis, having reduced RMSE during fitting and greater information on both bound and free water components.This study confirms that interleaved multiecho 3D UTE Cones acquisitions allow T2


The half maximum inhibition concentration (IC50) of 17-DMAG on B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi was 77.6 ± 2.9, 62.4 ± 1.9, 183.8 ± 3.2, 88.5 ± 9.6, and 307.7 ± 7.2 nM, respectively. The toxicity assay on MDBK and NIH/3T3 cell lines showed that 17-DMAG affected the viability of cells with an IC50 of 15.5 ± 4 and 8.8 ± 2 μM, respectively. Since the IC50s were much lower on the parasites than on the host cell lines, the selectivity index were high for all tested species. Furthermore, the two-drug combination of 17-DMAG with diminazene aceturate (DA) and atovaquone (AV) showed synergism or addition on in vitro cultures of Babesia and Theileria parasites. In the mouse model, 17-DMAG at a concentration of 30 mg/kg BW effectively inhibited the multiplication of B. microti. Moreover, if combined with DA or AV, 17-DMAG showed a comparable inhibition at the half dose. Taken together, these results indicate that 17-DMAG is a potent drug for treating piroplamosis. The data warrant further evaluation of 17-DMAG as an antibabesial drug and as an option in combination with atovaquone for the treatment of human babesiosis.Heat shock protein 90 (Hsp90) is a chaperone protein that stabilizes cells during stress or non-stress responses. Previous reports have shown that Hsp90 is a potential drug target to suppress the multiplication of several protozoan parasites. In this study, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an Hsp90 inhibitor, was evaluated for its inhibitory effect on five   •in vitro multiplication of Babesia and Theileria parasites.17-DMAG inhibits the •Combination of 17-DMAG with diminazene aceturate or atovaquone were also effective.•B. microti in mice.17-DMAG also inhibits the multiplication of •17-DMAG is a new treatment option for babesiosis in animal and human. Heat shishmania . Furtherishmania .Streptomyces hygroscopicus. The inhibition of geldanamycin on Hsp90 led to the degradation of its client proteins. However, geldanamycin has poor solubility, induces liver damage, and is toxic to the erythrocyte , bovine Babesia that is also known to infect humans (B. divergens), equine piroplasms parasites , and rodent Babesia that also infects humans (B. microti). The evaluations were performed using in vitro cultures of bovine and equine species and, for B. microti, using a mouse model. The cytotoxicity of 17-DMAG was also evaluated using mammalian cells including Madin-Darby Bovine Kidney (MDBK) and mouse embryonic fibroblast (NIH/3T3) cell lines.This study aimed to evaluate the inhibition of 17-DMAG on the multiplication of bovine 22.1Babesia parasite cultures were maintained using a microaerophilic stationary-phase culture system (Babesia parasites were grown in bovine red blood cells (RBC) in the specific complete medium for each species. The medium for B. bovis (Texas strain) contained GIT medium supplemented with 10% bovine serum, while the medium for B. bigemina (Argentina strain) and B. divergens (Germany strain) was Medium 199 and RPMI 1640 medium, respectively, supplemented with 40% bovine serum  was grown using equine RBC in GIT medium supplemented with 10% equine serum. T. equi (USDA strain) was grown in equine RBC in M199 medium supplemented with 40% equine serum and hypoxanthine  at a final concentration of 13.6 μg/ml. All of the media included 60 U/ml penicillin G, 60 μg/ml streptomycin, and 0.15 μg/ml amphotericin B . The cultures were incubated at 37 °C in a humidified chamber with an atmosphere of 5% CO2, 5% O2, and 90% N2.e system . Brieflyne serum . B. cabaB. microti (Munich strain) was recovered from −80 °C stock in two 6-week female Balb/c mice . The parasitemia was monitored every 2 days. After parasitemia reached approximately 30%, mice were euthanized, and blood was collected by cardiac puncture to initiate the in vivo experiment , EDTA (10 mM), saponin , and Triton X-100 (1.6% v/v) was prepared, filtered through 0.22 μm of polyethersulfone, and stored at 4 °C. Prior to fluorescence measurement, the lysis buffer was mixed with 0.2 μl/ml SYBR Green I .17-DMAG , diminazene aceturate , and atovaquone  were diluted in DMSO to make a 10 mM stock solution, which was stored at −30 °C until use in the 2.3B. bovis and T. equi parasitized RBC to obtain 1% parasitemia (infected RBC). Subsequently, 100 μl of infected RBC was added to 900 μl of complete medium in a 24-well plate. The untreated erythrocytes were used as a control. The parasitemia was monitored daily via microscopy observation of a Giemsa-stained blood smear for 4 days. Furthermore, the effect of 17-DMAG on mice was evaluated using 8-week-old BALB/c mice. Groups 1 and 2, consisting of three mice each, received intraperitoneal injections of 17-DMAG at a concentration of 15 and 30 mg/kg BW, respectively, for 5 days. One group (three mice) remained as an untreated control. The changes in body weight and hematology profiles, including RBC, hematocrit, and hemoglobin, were observed for 20 days.Prior to the subculture of parasites, bovine and equine RBC were incubated with 1 μM of 17-DMAG for 3 h. The RBC were washed three times with PBS and mixed with 2.4B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi with a parasitemia of 8%, 3%, 10%, 4%, and 9%, respectively, were harvested and adjusted to 1% parasitemia with fresh RBC before the inhibition assay. The 60 inner wells of a 96-well plate were used in the assay, while the peripheral wells were filled with sterile distillate water to reduce evaporation during incubation. To each well in triplicate, 2.5 μl of infected RBC  was added and mixed with a culture medium containing the drug to a total volume of 100 μl. The stock solution of 17-DMAG, DA, and AV was dissolved in a culture medium at various concentrations using two-fold dilution. A culture medium containing DMSO at a final concentration of 0.2% was added to the well with infected RBC as a positive control and to the well with uninfected RBC as a negative control. Furthermore, a drug combination assay was conducted in parallel with the single drug assay at the constant ratio in accordance with the Chou–Talalay method  with the excitation and emission wavelength at 485 nm and 518 nm, respectively. The fluorescence data were subtracted from the negative control and used to calculate the IC50.The assays were conducted in accordance with the protocol described previously with modification . Brieflyy method . In the 2.52 culture flasks with MEM medium  and DMEM , respectively, and cultured in a humidified incubator at 37 °C with 5% CO2. Each medium was supplemented with 10% fetal bovine serum, 0.5% penicillin/streptomycin , and an additional 1% glutamine. The medium was changed every 3–4 days and incubated until approximately 80% confluent. The cells were free from mycoplasma contamination after being checked by staining with DAPI . The detachment of cells from the culture flask was done using TrypLE™ Express  after washing two times with Dulbecco's phosphate-buffered saline (DPBS). Subsequently, the counting of viable cells was carried out using a Neubauer improved C-Chip  after staining with 0.4% trypan blue solution.MDBK and NIH/3T3 cells were maintained in 75 cm2.62. One hundred microliters of cells at a density of 5 × 104 cells/ml was seeded per well and allowed to attach to the plate for 24 h. Ten microliters of twofold drug dilutions was added to each well to a final concentration of 0.312–40 μM in triplicate. The wells with only a culture medium were used as blanks, while the wells containing cells in a medium with 0.4% DMSO were used as negative control, representing the highest concentration of drug solvent. The exposure of drugs was carried out for 24 h, followed by the addition of 10 μl of CCK-8. The plate was further incubated for 3 h, and the absorbance was measured at 450 nm using a microplate reader.The drug-exposure viability assay was performed in accordance with the recommendation of the Cell Counting Kit-8 . Briefly, the assay was carried out using a 96-well plate at 37 °C in a humidified incubator with 5% CO2.7B. microti mouse model according to the previous protocol , 17-DMAG + AV (15 + 10 mg/kg BW), and DA + AV (15 + 10 mg/kg BW), respectively, via a route similar to that for the single drug. The parasitemia was estimated from Giemsa-stained blood smears using microscopy in approximately 5000 RBC. Furthermore, the hematology profiles including RBC, hemoglobin, and hematocrit were measured from 10 μl of mouse blood using an automatic hematology analyzer . The rate of parasitemia, hematology profiles, and mouse body weight was monitored for 60 days. The experiment was repeated once so each group contained 10 mice.The inhibitory effect of 17-DMAG was also determined using a protocol . Briefly2.850 of 17-DMAG, DA, and AV was calculated from the percentage of inhibition on the in vitro multiplication of all tested species using non-linear regression (curve fit), available in GraphPad Prism . Combination index (CI) values for the drug combination were calculated using CompuSyn software (50) + (2 x IC75) + (3 x IC90) + (4 x IC95))/10 )/10 . The dif3in vivo observations during drug treatment including parasitemia, mice body weight, and hematology profiles are available at Mendeley Data (https://doi.org/10.17632/6sdnnh9hng.1).Original data of this study including percent inhibition of drug on each parasite, drug combination data, and 3.1B. bovis and T. equi. Microscopy observation and parasitemia counting showed that the multiplication of B. bovis and T. equi did not significantly differ between 17-DMAG-treated RBC and normal RBC for either species. Evaluation was also conducted using mice. Mouse body weight was reduced slightly in the group of 30 mg/kg BW on day 10 post treatment but was not significantly different from that of the control group, while the group of 15 mg/kg BW did not show any effect  for 3 h prior to the subculture of y effect . For thes tested . The IC5ectively . Compareivergens . It was 50 of AV .Fig. 1Do3.250, IC75, IC90, and IC95 , additive (similar effect), or antagonism (reduce or block the effect). Five dilutions of 17-DMAG, as recommended in the Chou–Talalay method , were coand IC95 . The weiand IC95 . The dru3.3in vitro culture of Babesia and Theileria parasites. The effect of 17-DMAG on the host was evaluated using MDBK and NIH/3T3 cell lines. The IC50 of 17-DMAG on MDBK cells was 8.8 ± 2 μM, while that on NIH/3T3 cells was 15.5 ± 4 μM. The result showed that 17-DMAG is more toxic than DA and AV. In a separate assay, DA and AV at a concentration of 40 μM did not show any inhibition on MDBK and NIH/3T3 cell viability. The selectivity index, defined as the ratio of cell line IC50 to the parasite IC50, is shown in 50 on B. bigemina, while the lowest was on T. equi with a selectivity index of 50. The highest and lowest selectivity index for MDBK cells was also determined on B. bigemina and T. equi, respectively.17-DMAG showed an effective inhibitory effect similar to that with currently used drugs, DA and AV, on the 3.4B. microti. The experiment was carried out using a mouse model, since the in vitro culture of B. microti is not well established. The mice were divided into eight groups (five mice/group), corresponding to single drug treatment, controls, and combinations of 17-DMAG, DA, and AV. The drugs were combined at approximately half the concentration of single drug treatment. The experiment was conducted two times, with each group containing 10 mice. In the DMSO control group, the multiplication of B. microti increased significantly and reached the highest parasitemia at 45.6 ± 10.5% on day 9 post infection (p.i). The parasitemia decreased gradually on the following days. On day 60, B. microti was still detected in four mice. B. microti in the 17-DMAG 30 mg/kg BW group reached peak parasitemia (4.7 ± 2.0%) on day 10 p.i., and no parasite was detected via microscopy starting on day 36 p.i. Similar inhibition was observed on the DA (25 mg/kg BW) and AV (20 mg/kg BW) groups, but the parasite had disappeared from blood smears by day 28 . Significant differences in mouse body weight were observed between the DMSO control group and all drug-treated groups except for the DA (25 mg/kg BW) group (P < 0.0001).The inhibitory effect of 17-DMAG was further evaluated on the multiplication of y day 28 A. Howevey day 28 B. HoweveW) group A. The RBW) group B, hemoglW) group , and hemW) group  were als4Trypanosoma evansi in cattle and a relapse of B. gibsoni in dogs (in vitro or in vivo multiplication of Babesia and Theileria parasites; however, all of them are in research and still far from clinical trial (Plasmodium, was also found to inhibit the multiplication of Babesia parasites (Babesia and Theileria parasites will provide more options for treatment of the disease both in humans and domestic animals.Treatment of babesiosis remains a significant challenge, especially in the livestock industries. Although diminazene aceturate and imidocarb dipropionate are still effective, current reports showed the development of resistance in  in dogs . Many inal trial . Furtherarasites . Since tBabesia species and Theileria equi in vitro. Its ability to bind Hsp90 causes a significant reduction in parasite survival. Hsp90 is conserved in most organisms, including Babesia and Theileria parasites. There are some isoforms of Hsp90 in B. bovis based on reported full genome sequences (Toxoplasma gondii (Plasmodium falciparum (Eimeria tenella (Trypanosoma brucei (Theileria annulata (Babesia and Theileria parasite Hsp90, causing insufficient binding of client proteins to Hsp90 and disrupting the normal multiplication of parasites.This study showed that 17-DMAG inhibits the multiplication of several equences . These pequences . Howevera gondii , Plasmodlciparum , Eimeria tenella , Trypanoa brucei , and Theannulata  that leaPlasmodium falciparum was the increasing ATPase activities in this species compared with human Hsp90. The high stress conditions during parasite invasion of host cells might increase the activity of their Hsp90 and evolved significantly (Babesia and Theileria Hsp90 than to the host Hsp90. Similar to Plasmodium falciparum, Babesia and Theileria parasites experience high stress during their invasion of host cells. Further study of the biochemical characterization of Hsp90 in Babesia and Theileria is required to better understand the potency of this protein as a drug target.Hsp90 is also conserved in the host cells, which means that the binding of the inhibitor to the host's Hsp90 is possible. For this reason, the inhibitor should specifically bind to the parasite Hsp90 to avoid toxicity to the host. The basis of Hsp90 as a drug target in ficantly . Our stuThe potency of 17-DMAG as an inhibitor was further evaluated in a combination study using DA and AV. The cytotoxicity assay showed that 17-DMAG was more toxic than DA and AV. The combination study aimed to enhance the potency of 17-DMAG while reducing the dose that led to reduced toxicity. Our results demonstrated that the combination of 17-DMAG and DA or AV against all species was range from synergism to additive. Therefore, the combination of 17-DMAG and DA might have potential as a novel regime for treating piroplasmosis in a wide range of animals. Furthermore, 17-DMAG might be used as a treatment option in combination with AV for the treatment of human babesiosis.B. microti in a mouse model. The combination of 17-DMAG with DA or AV at a half dose showed a multiplication inhibition comparable to that of those single drugs at full dose. Hematology profiles such as RBC count, hemoglobin, and hematocrit were also improved compared with the DMSO control group, as were parasitemia and mouse body weight. Taken together, these results indicate that 17-DMAG is a potent antibabesial and antitheilerial drug, as confirmed by the inhibition of five species, B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi on in vitro cultures and B. microti in vivo. Even though 17-DMAG showed toxicity in the host cells, its combination with diminazene aceturate and atovaquone could improve the effect while reducing the toxicity.The potent effect of 17-DMAG combined with DA or AV was also observed on the multiplication of