To evaluate variability of central corneal thickness measurement (CCT) devices using a hitherto unprecedented number of CCT devices.CCT was measured consecutively in 122 normal corneas of 61 subjects with seven different devices using three distinct measurement technologies: Scheimpflug, Ultrasound, and Optical Coherence Tomography (OCT). Per device deviation from the mean CCT value per eye was used to determine which of the devices performed best, compared to the mean value.Cirrus OCT yielded the lowest deviation. Deviations of the individual devices from the mean CCT of each eye were (OS/OD) 12.8±5.0/14.9±9.4 μm for Topcon noncontact specular microscopy (NCSM), 11.3±5.9/10.6±7.3 μm for Pentacam, 10.7±5.2/10.4±4.8 μm for Spectralis OCT, 6.0±3.9/6.2±4.9 μm for Topcon DRI OCT, 5.1±3.4/5.9±10.3 μm for AngioVue OCT, 4.8±4.1/5.7±4.6 μm for US pachymetry, and 4.2±3.2/5.7±4.6 μm for Cirrus OCT. The maximum differences between US pachymetry and the other devices were very high (up to 120 μm).Central corneal thickness may be under- or overestimated due to high interdevice variations. Measuring CCT with one device only may lead to inappropriate clinical and surgical recommendations. OCT showed superior results. Clinical decision making, whether for the Laser-in-situ-Keratomileusis (LASIK), or for monitoring and screening of various diseases, often relies on the results of CCT measurement. It has been found, that a thinner central corneal thickness (CCT) may represent a higher risk in primary open-angle glaucoma, and patients with a thinner CCT tend to exhibit a more severe glaucomatous defect. , 2CCT measurements helped diagnose subclinical keratoconus , identifHowever, Doughty & Zaman reported that ultrasonic measurements tend to overestimate the cornea thickness than optical measurements.  RotatingSwept-source OCT (SSOCT) has shown a higher reliability than Scheimpflug imaging and allows for monitoring corneal thickness after photorefractive keratectomy. , 15To improve surgical procedures or treatment, this study compares hitherto unprecedented number of devices for central corneal thickness measurements. From seven (7) devices we calculated the lowest deviation from the mean CCT value.Subject age, race, sex and diurnal fluctuations were not considered in this study as our goal was to collect the most possible CCT data from many devices in short time.Sixty-one subjects who were visiting the clinic were randomly selected for CCT measurements using seven different devices. Inclusion criteria were as follows: Subjects with normal visual acuity (20/20), good and steady fixation, and clear cornea. Exclusion criteria included a history of corneal refractive surgery, corneal scarring, degenerations, and dystrophies. Written informed consent was obtained from all subjects, and the study was approved by the Ethics Committee Zentral und Ostschweiz, Switzerland (EKNZ UBE-15/881).The order of measurements using different devices was random, apart from the US pachymetry that was always done last in order not to disturb the corneal surface and impair the non-contact measurements. All 14 measurements were conducted at the University of Basel, Switzerland, within a timeframe of 60 minutes. It was proposed to use central thickness measurements to increase comparability between devices. CCT was measured using the following seven devices: Oculus Pentacam version 1.19r11 , noncontact specular microscopy SP-1P version 1.21 , spectral-domain OCT with Cirrus HD-OCT 5000–2997 version 7.0 , Spectralis HRA 2 version 6.0.10.0 , AngioVue OCT software version 2014.2.0.93 , swept-source OCT with prototype OCT DRI OCT-1 Atlantis software version 9.12.003.04 , and US pachymetry ALCON RxP version 1.15 .Automatic acquisition mode was used for the rotating Scheimpflug camera and for NSCM to capture CCT. For SDOCT, additional anterior lens systems were used in the Spectralis and AngioVue devices. The OCT scan patterns were: Cirrus anterior segment 5-line raster scan protocol without anterior segment lens , Spectralis anterior segment protocol with anterior segment lens , AngioVue cornea pachymetry protocol with anterior segment lens , and Topcon SSOCT volume scan patterns over a 6.0 mm x 6.0 mm area centered on the corneal apex (256 cross scans with a scan density of 512 x 128 pixel). The manufacturer caliper tools were used to measure the CCT. The US pachymetry measurement of the cornea was performed as the last test a few minutes after the local application of preservative-free oxybuprocaine (0.4%). Three consecutive measurements were taken from the central cornea. For all measurements the thinnest corneal point was selected.The axial resolution is 1μm for both the ultrasound device Alcon RxP  and for Gi) of data, one group for each measurement device. Each group Gi thereby consists of 61 data points, one point per eye. Our statistical analysis assumes that the value of each of these 61 data points Gi,n corresponds to the following sum: the device-independent true CCT value tn of the eye, the device-dependent bias bi, and the device-dependent measurement error ei:ei is assumed to have a normal distribution N. In order to determine a possible between-group difference, paired differences of Eq  and i ≠ j. For un-replicated data, the mean and standard deviation of the between-group differences Di usually convey the most useful information. Paired device-by-device differences between US pachymetry and the other six devices were computed and analyzed. As a summary statistic of inter-device variation, the mean absolute deviation from a central point, the mean CCT over all seven devices per eye, was calculated asxi,k are the 7 measurements  for the k-th eye , and m(X) is the mean of the 7 values, to investigate deviation of the measurements. Mean and standard deviation, calculated asi.Statistical analysis was performed using Microsoft Office Excel 2013 . Analysis of variance (ANOVA) and Chi-Square(Х2)-test were performed but not included in this manuscript as this study represents a method comparison study ranking pairs of methods on the basis of their extent of agreement. There were seven groups . No study subject was excluded. Men represented 34% of the study subjects .The overall mean CCT values were as follows (OS/OD): Pentacam 552±35/550±34 μm (SD), Topcon 529±34/530±30 μm (SD), Cirrus 544±34/546±33 μm (SD), AngioVue 540±32/540±32 μm (SD), Spectralis 552±34/553±35 μm (SD), DRI 537±34/537±33 μm (SD), and US pachymetry 539±35/539±36 μm (SD), .CCT ranges measured by different devices in the left eyes are Pentacam 481–619 μm, Topcon 463–595 μm, Cirrus 470–608 μm, AngioVue 467–606 μm, Spectralis 474–622 μm, DRI 462–595 μm, and US pachymetry 465–610 μm.CCT ranges measured by different devices in the right eyes are Pentacam 470–620 μm, Topcon 460–595 μm, Cirrus 470–618 μm, AngioVue 465–654 μm, Spectralis 473–626 μm, DRI 461–600 μm, and US pachymetry 461–604 μm.Di were analyzed by looking at the mean and standard deviation of paired CCT difference values between each device and the US pachymetry device (OS/OD). A “-”indicates that a device measured thicker, and a “+” indicates that a device measured thinner CCT values than the ultrasound device: US-Pentacam -14.5±7.8/-10.9±12.5 micron (SD), US-NSCM +9.4±8.0/+9.5±15.6 micron, US-Cirrus -5.3±7.5/-6.2±8.9 micron, US-AngioVue -1.3±8.9/-2.6±15.3 micron, US-Spectralis -14.1±7.6/12.8±8.9 micron, and US-DRI +1.9±8.6/+2.6±8.5 micron. These values and the histogram representations in Between-device differences NCSM is similar to US pachymetry for small values, but underestimates higher values. Cirrus slightly overestimates lower values with respect to US pachymetry. For higher values, the two devices are in good agreement. AngioVue overestimates low values and underestimates higher values with respect to US pachymetry. Spectralis overestimates low values and is in good agreement with US pachymetry for higher values. DRI SSOCT overestimates low and underestimates higher values with respect to US pachymetry. Even more surprisingly, there are differences of up to 120 μm between US pachymetry and the NCSM device.All devices were compared to the US pachymetry device, because US pachymetry has formerly been considered the gold standard in CCT measurement, and most ophthalmologists are familiar with this device. The 95% limit of agreement (LoA) of the differences between devices was (OS) -31.6μm to +2.7μm, -6.2μm to +25.0μm, -20.0μm to +9.5μm, -18.7μm to +16.2μm, -28.9μm to +0.7μm, and -14.8μm to 18.7μm; and (OD) -35.3μm to +13.6μm, -21.1μm to +40.1μm, -23.6μm to +11.2μm, -32.6μm to +27.4μm, -30.2μm to +4.6μm, and -14.0μm to 19.3μm, for US minus Pentacam, US minus NSCM, US minus Cirrus, US minus AngioVue, US minus Spectralis, and US minus DRI. Further, Bland-Altman difference plots were used to represent these comparisons , becauseDeviation of the individual devices from the mean CCT value of each eye was (OS/OD) for Pentacam 11.3±5.9/10.6±7.3 μm μm(SD), Topcon NCSM 12.8±5.0/14.9±9.4 μm μm(SD), Cirrus OCT 4.2±3.2/5.7±4.6 μm μm(SD), AngioVue OCT 5.1±3.4/5.9±10.3 μm μm(SD), Spectralis OCT 10.7±5.2/10.4±4.8 μm μm(SD), Topcon DRI OCT 6.0±3.9/6.2±4.9 μm μm(SD), and US pachymetry 4.8±4.1/5.7±4.6 μm μm(SD).This study compared an unprecedented number of devices for central corneal thickness measurements. The aim was to define the lowest deviation from the overall mean CCT value.CCT measurement results in this study were in good agreement with previous reports. , 24 WithIn contrast, previous studies have found that OCT devices underestimated CCT compared to US pachymetry., 27 But This study could be limited because no repeated measurements were performed to assess device precision. However, repeatability of CCT measurements has already been reported earlier. , 30, 31 Another limitation may be that this study did not compare corneal pachymetry values, because the US device does not provide thinnest pachymetry, as the decentered point cannot easily be detected by punctual measurements. Still it was crucial to include the US device, as clinical practice has traditionally been relying on that method for decision-making. Furthermore, the results may not be applicable to patients with pathological corneas or eyes that have had prior corneal surgery because of potentially change in biomechanical variables and corneal shape. Finally, this study demonstrates that the CCT measurement by OCT showed slightly superior results compared to the other measurement methods.S1 Table(PDF)Click here for additional data file.


MEF2C (Myocyte-specific enhancer factor 2C) has been associated with neurodevelopmental disorders. This study aimed at delineating the clinical profiles of MEF2C gene mutations.In total, 112 Chinese patients with intellectual disability (ID) were recruited, including 44 patients presented with Rett syndrome (RTT) or RTT-like syndrome, and 68 patients with non-syndromic ID. Targeted next-generation sequencing (NGS) was performed. Detailed clinical information was collected.MEF2C gene mutations were identified, of which three were novel. The MEF2C mutant rate was 4.5% (5/112) in total, and 6.8% (3/44) in the RTT (−like) cohort. All patients with MEF2C gene mutation presented with cognitive impairment, gross motor delay, speech disorder and autistic features. Four patients had epilepsy, which responded well to antiepileptic drugs. One female was diagnosed with classical RTT, two females with RTT-like syndrome, and two males with non-syndromic ID. Generally, the phenotype of two males with relatively downstream mutations  was milder than that of three females with upstream mutations .Five heterozygous MEF2C dysfunctions, especially MEF2C point mutations. MEF2C mutations are associated with a broad clinical spectrum, ranged from classical RTT to non-syndromic ID. Through our study, it can be inferred that there is correlation between the phenotype and MEF2C-genotype, the mutation site. Overall, the MEF2C gene mutational analysis should be performed in ID cohort, especially in patients with features overlapped with RTT.Our findings expanded the current understanding of the consequences of The online version of this article (10.1186/s12881-018-0699-1) contains supplementary material, which is available to authorized users. MEF2C (Myocyte-specific enhancer factor 2C), a member of MEF2 subfamily, is a transcriptional activator binding specifically to the MEF2 element, which has pivotal role in myogenesis, hematopoiesis, neurogenesis and synaptogenesis [MEF2C is crucial for normal neuronal development, distribution, and electrical activity in the neocortex [MEF2C haploinsufficiency caused by either large deletions encompassing MEF2C, or intragenic mutations of MEF2C, is associated with neurodevelopmental disorders [ogenesis , 2. Espeeocortex . MEF2C hisorders .MEF2C mutations were identified in five patients, including three females with RTT or RTT-like features, and two males with non-syndromic ID. The phenotypes and genotype of MEF2C mutation in Chinese ID patients were reported here.In this study, we performed targeted next-generation sequencing (NGS) in Chinese patients with intellectual disability (ID) of unknown causes. MECP2, CDKL5 and FOXG1 have been ruled out in the patients of RTT (−like) cohort. The other 68 patients presented with non-syndromic ID. Metabolic or mitochondrial disorders, central nervous system infection or other known etiologies were excluded.In total, 112 Chinese patients with ID were enrolled into this study from August 2015 to March 2018, including 68 females and 44 males, aged from 13 months to 12.5 years. Among them, 25 girls were initially diagnosed with RTT , 16 females and 3 males with RTT-like syndrome. Mutations in Detailed clinical information including clinical manifestation, electroencephalogram (EEG), magnetic resonance imaging (MRI), and family history, etc., was collected. Genomic DNA was extracted from peripheral leukocytes of the patients and their parents. This study was approved by the Medical Ethics Committee, Peking University First Hospital. Written informed consent was obtained from the parents of the patients.A genetic panel which is related to ID was designed, containing 512 genes , assessment of the variants were carried out following the ACMG guidence. Common sites with population allele frequency above 5% according to dbSNP 138, 1000 Genome Project, esp6500si and ExAC databases were excluded. Rare loss-of-function variants were selected for follow-up analyses. Selected variants with depth lower that 20X and allelic fraction lower than 30% were manually reviewed with IGV. Functional variants annotated by HGMD, Clinvar or predicted to be deleterious by SIFT and Polyphen2 were regraded as candidates.Variants were annotated using ANNOVAR  were identified with pathogenic mutations, including 2 MEF2C mutations. The wild type and mutant allele of MEF2C is approximately at 1:1 (ranging from 39.1 to 47.2%), so mosaicism were not considered.Causative gene mutations were identified in 14 patients of our RTT (−like) cohort, with detection rate of 31.8% (14/44), incluing 3 MEF2C were identified in three females with RTT (−like) syndrome and two males with non-syndromic ID. The MEF2C mutant rate was 4.5% (5/112) in total, whereas 6.8% (3/44) and 2.9% (2/68) in Rett (−like) cohort and non-syndromic ID group, respectively. These five mutations included three nonsense mutations, one splicing site mutation, and one missense mutation. Three MEF2C mutations  arose de novo, and the other two were of unknown origin, as the parental DNA was not available. Among these mutations, two mutations, c.565C > T, p.Arg189* and c.766C > T, p.Arg256*, were reported pathogenic, the other three mutations, c.48C > G, p.Asn16Lys; c.334G > T, p.Glu112*; and c.403-1G > T, were novel. They were predicted as pathogenic by Mutationtaster, the missense mutations, c.48C > G, p.Asn16Lys, was predicted as pathogenic by PolyPhen-2 and SIFT as well. A schematic representation of the MEF2C protein, including the mutations identified in this study as well as the mutations previously reported, is shown in Fig. Five heterozygous mutations in s 4.5% 5/12 in totMECP2, CDKL5 and FOXG1 was negative. Through this study, a de novo missense MEF2C mutation, c.48C > G, p.Asn16Lys, was identified, which was a novel mutation.Patient 1 is a girl aged 5 years and 9 months, the first child of a healthy nonconsanguineous couple. Her psychomotor milestones were profoundly delayed, with raising head at 8 months, sitting alone at 1 year old, and still unable to walk by herself at 5 years and 9 months. Poor eye contact, hand clapping, hand wringing and bruxism were observed at 1 year old, followed by deterioration of hand skills. Epileptic attack occurred at 20 months old, and she responded to valproate (VPA), oxcarbazepine (OXC), and topiramate (TPM) combined therapy. Seizure free was achieved at 5.5 years old. EEG demonstrated spike-slow waves at right medial and posterior temporal, with generalization. MRI (1 year old) revealed enlargement of frontal subarachnoid space. Above manifestations led to the diagnosis of typical RTT. However, mutational analysis of 1 and T2 signal at posterior horn of bilateral ventricle. A nonsense mutation, c.565C > T, p.Arg189*, of MEF2C was discovered, a known disease causing mutation (https://www.ncbi.nlm.nih.gov/clinvar/).Patient 2 is a 2.5 years old girl presented with RTT-like ID. she displayed profound psychomotor retardation, with controlling head at 5 months, sitting alone at 8 months. She remained unable to walk independently and still had no speech at 2.5 years old. She developed stereotypic hand movements and bruxism at 2 years of age. No seizure was reported, but there was epileptiform discharge on EEG at age of 2 years. Brain MRI revealed high TMEF2C was identified.Patient 3 is a 23-month-old girl who presented with RTT-like features. Feeding difficulties caused concern at 3 months of age. No epilepsy was observed, though she had medical history of febrile convulsions at 9 months old. There was significant delay in her developmental milestones, without obvious retrogression. She could sit alone at 1 year old, and walk at 23 months of age, with abnormal gaits. Unmeaningful language began at 12 months. She also presented with poor eye contact, stereotypic actions, breathing disturbance, and sleeping abnormalities. She suffered from recurrent respiratory infections frequently after 15 months of age. MRI revealed hypomyelination at 1 year and 10 months of age. A novel nonsense mutation, c.334G > T, p.Glu112*, in MEF2C was identified, which arose de novo.Patient 4 was a boy aged 7 years and 8 months. He achieved the gross motor developmental milestones somewhat delay, with rising head at 1 year old, sitting alone at 1 year and 2 months, and walking at 1.5 years. Lack of speech was another problem, that he still cannot speak a single word so far (7 years and 8 months). He was always immersed in his own world, showed little interest to the others, and lacked eye-contact. Febrile seizures attacked at 1 year, which turned into afebrile seizures at 2 years old. Partial seizures occurred 1~ 2 times per month, lasted few minutes to more than half an hour. The epilepsy was fever-sensitive. VPA was used at 2.5 years of age, and no seizures occurred after 4 years of age. EEG at 2.5 years displayed (multi-) spike and slow waves at right occipital region, with slow rhythm on the background. MRI at 3 years of age was normal. A novel mutation, c.403-1G > T, of 1 and T2 signal around bilateral ventricle, and a septum pellucidum cyst. EEG was normal at 4 years of age. A known pathogenic de novo mutation, c.766C > T, p.Arg256*, in MEF2C was discovered.Patient 5 was a boy of 6 years and 4 months. He presented with developmental delay, with rising head at 7 months, sitting alone at 1 year, and walking at 2 years old. Language delay was a main problem, that he could only speak one to two words, such as “give me”, “I want”. He also displayed some autistic behaviors, including occasionally repetitive hand movements, no eye-contact and no interest to others. Febrile seizures initially occurred at 8 months of age, then attacked 2–3 times per year. Seizure-free was achieved for 4 months with levetiracetam (LEV) and VPA. MRI at 4 years old revealed long TMEF2C mutations were summarized in Table The genetic data and clinical information from the five patients with MEF2C mutations were identified in RTT (−like) syndrome or non-syndromic intellectual disability patients, of which three mutations were novel. The MEF2C gene was originally identified as the phenocritical gene in the 5q14.3 microdeletion syndrome [MEF2C were discovered in patients with neurodevelopmental disorders, including intellectual disability and epilepsy [MEF2C, the phenotype of MEF2C gene mutation is not well recognized [MEF2C have been described in patients with neurological disorders [MEF2C point mutations, as well as the genotype-phenotype relationship.In this study, five syndrome . Consecuepilepsy . As mostcognized , 9. Up tisorders , 10–13, MEF2C gene, located at 5q14, encodes myocyte enhancer-binding factor 2C, a transcription factor that is indispensable in early neuroprogenitor development [MEF2C protein consists five core structural domains, including MADS , myocyte enhancer factor 2 (MEF2), transcriptional activation domain 1 (TAD1), transcriptional activation domain 2 (TAD2), and nuclear localization signal (NLS) [MEF2C domain is to mediate dimerization and DNA binding, whereas the rest domains act as the transcriptional activator [MEF2C mutations are scattered through the whole MEF2C protein, and there’s no hot mutant region  . The MADctivator . The MEFMEF2C gene mutations encompass global developmental delay, intellectual disability, absent speech, poor eye-contact and various minor brain anomalies on MRI [MEF2C haploinsufficiency phenotypically resembles RTT, including psychomotor stagnation, stereotypical behavior, poor hand skills, etc., [Typical clinical manifestations in patients with s on MRI . A subses, etc., , 7, 16 OMEF2C gene, cannot walk independently. However, including 3 of our cases (patients 3–5), 7  patients with MEF2C point mutations acquired the walking abilities [MEF2C point mutations might cause milder phenotypes than large intragenic deletions or completely deletions. Additionally, the phenotype of patient 4  and patient 5  was milder than that of patient 1 , patient 2  and patient 3 (c.403-1G > T). As it was shown in Fig. MEF2C protein, which suggested that the phenotypic severity may be associated with the mutation site.The overwhelming majority of patients who had macro-deletions partially or entirely encompassing the bilities , 17. It MEF2C mutations [MEF2C gene mutation need to be further studied.Stereotypic behaviors, such as head rocking, hand-mouthing, hand clapping and wringing were reported in some patients with utations . In our MEF2C haploinsufficiency experienced epilepsy [More than half patients with epilepsy . Seizureepilepsy . In thisMild to severe hypotonia was reported in most of the patients . In our MEF2C mutations were founded in patients with heart disorders, including patent ductus arteriousus (PDA), double outlet right ventricle (DORV), ventricular septal defect (VSD) and dilated cardiomyopathy (DCM), accompanied with or without neurological symptoms [MEF2C mutations. There was no relationship between the genotype and phenotype with or without heart disorders and/or neurological symptoms. The clinical heterogeneity might indicated the partly incomplete penetrance.Recently, symptoms –21. All MEF2C gene, and also showed some relationship between phenotype and MEF2C-genotype. The different clinical features in male and female patients with MEF2C gene mutation need to be verified in a larger sample. MEF2C gene mutational analysis was recommended in patients with ID, especially in the RTT (−like) cohort without mutations in traditional RTT genes  .Our findings expanded the spectrum of point mutations of Additional file 1:Table S1. List of 512 candidate genes. (DOCX 20 kb)


Reactions were performed in the presence of the ruthenium-alkylidene catalysts (PCy3)2(Cl)2Ru=CHPh (I) and  (PCy3)Cl2Ru=CHPh (II), respectively. Catalyst II bears an N-heterocyclic carbene ligand (NHC) bounded to the ruthenium atom, which has a strong basic character; therefore it is more active toward trisubstituted olefins in comparison with catalyst I. In both cases, isolated monoterpene-terminated isoprene oligomers were obtained as products of the cross-metathesis degradation of NR. In the presence of catalyst II molecular weight values around nM × 102 and yields of 80% were obtained; whereas with catalyst I, the molecular weights of products were about nM × 104 with yields ranging 70 to 74%. The composition and yield of NR degradation products were determined by GC/MS (EI) analysis and it was found that the oligomers obtained have primarily one vinyl group and one terpene-monocyclic group at the chain end, with isoprene units mA = 2, 3 y 4.This study reports on the degradation of natural rubber (NR) via cross-metathesis with mandarin oil and  Mandarin oil  was used as received.Guatemala Natural Rubber was obtained from fresh field latex of AGROS and used without further purification.  was used as internal standard. FT-IR spectra were obtained on a Nicolet 5700 using a diamond tip as dispersing agent. Molecular weights and molecular weight distributions were determined with reference to monodisperse polystyrene standards on a waters 2695 ALLIANCE Separation Module GPC at 30 °C in tetrahydrofuran (THF) using a universal column and a flow rate of 0.3 mL/min. GC-MS chromatograms were registered using a GC-2010/MS-QP2010 system equipped with an AOC-20i autosampler, with an injector temperature of 335 °C, a split ratio of 1:5 and an injection volume of 1 μL. Capillary column separation was carried out using a 0.25 μL thick film [30 m × 0.32 mm ID Rtx-5MS (RESTEK) with a 5m integraguard column] at a flow rate of 1.22 mL/min and 68 kPa helium pressure, using helium as carrier gas. The initial temperature of the column was the 45 °C, isothermal for 3 min then heated at a rate of 10 °C/min to 150 °C, ramp at 15 °C/min to 340 °C, and finally isothermal for 14 min. The interface and ion source were set at 340 and 220 °C, respectively. The chromatograms were acquired in the electron impact (EI) scan mode at 70 eV with a mass range of 40–600 (m/z) at a rate of 0.1 scan s−1 [Metathesis degradation of NR was carried out in a glass vial under a dry nitrogen atmosphere at different temperatures. After terminating the reaction by addition of a small amount of ethyl vinyl ether, the solution was poured into an excess of methanol. The oligomers obtained were dried under a vacuum.d-limonene or mandarin oil as CTAs . 1H-NMR : δ 5.38 ; δ 5.13 ; δ 4.68, 4.70 ; δ 2.04 ; δ 1.68–1.59 . 13C-NMR : δ 139.5 ; 135.2, 133.6 ; 125.0 ; 120.9 ; 109.8 ; 38.1 ; 32.2 ; 30.7, 29.1, 27.9 ; 26.4 ; 23.8 ; 23.4 ; 22.5 . FT-IR ; 2953 ; 2920 ; 2851 ; 1375 , 888 ; 835 . GC/MS (EI) min : 17.3 , 19.6 , 21.7 , 12 , 16 , 13.7 .Metathesis degradations of NR (3.0g) using  as CTAs  were card-limonene were successfully used as CTAs in the metathesis degradation of NR. Mandarin oil was also found to be very suitable as a green solvent and it can be used instead of 1,2-dichloroethane and other non-green solvents. Catalyst II depolymerized NR completely in comparison with catalyst I when the reactions were conducted in the same conditions. Molecular weights of products were controlled primarily by the NR/CTA molar ratio giving values around nM × 102. The main products of the cross metathesis degradation of NR with mandarin oil were monoterpene terminated oligomers of series mA = 2, 3 and 4. Metathesis transformations of NR in the presence of mandarin oil allowed the synthesis of desired monoterpene-terminated products with higher selectivities than previously reported metathesis degradation procedures using β-pinene as a cross-metathesis partner.Mandarin oil and


Aspergillus, Penicillium and Fusarium (among others), which produce adverse health effects on humans and animals . In addition, mycotoxins negatively affect the productive parameters of livestock . Epidemiological studies are considered necessary to assist stakeholders with the process of decision-making regarding the control of mycotoxins in processing environments. This study addressed the prevalence in feed ingredients and compound feed of eight different types of toxins, including metabolites produced by Fusarium spp.  and two additional toxins  and aflatoxin M1 (AFM1)) from different fungal species, for over a period of five years. On the subject of Fusarium toxins, higher prevalences were observed for fumonisins  and DON , whereas, for OTA, a prevalence of 40.56% was found (n = 146/360). In the case of raw material, mycotoxin contamination exceeding recommended values were observed in cornmeal for HT-2 toxin , T-2 toxin , and ZEA . In contrast, many compound feed samples exceeded recommended values; in dairy cattle feed toxins such as DON , ZEA , T-2 toxin , and HT-2 toxin  were observed in high amounts. OTA was the most common compound accompanying Fusarium toxins . This study also provided epidemiological data for AFM1 in liquid milk. The outcomes unveiled a high prevalence of contamination  and several samples exceeding the regulatory threshold. Statistical analysis exposed no significant climate effect connected to the prevalence of diverse types of mycotoxins. Mycotoxins are secondary metabolites, produced by fungi of genera  Fusarium toxins are among the most commonly monitored as they are acknowledged to present serious health concerns [Mycotoxins are toxic fungal metabolites that can be found in feed ingredients and compound feeds ,2. Due tconcerns ,10. Undeconcerns ,12,13. In feed production, ca. 60% of the formulation consists of cornmeal, soybean meal, and their derivates ,15. In C−1 for cattle, pork, and chicken, respectively), 215 L, and 218 units per capita, individually [Mycotoxin metabolites retain toxicity and thus must be surveilled ,25. MycoIn this regard, epidemiological information tends to be more comprehensive when exploring data from several toxins simultaneously . AccuratFusarium spp. , T-2/HT-2 toxins, zearalenone (ZEA) and fumonisins (FB1 and FB2)), but also ochratoxin A (OTA), during five years are provided. Finally, in the same period, we analyzed the behavior of AFM1 in liquid milk. Herein, the prevalent data from feed and feed ingredient samples of eight different toxins, mainly produced by Fusarium toxins during the analyzed period (2012–2017) was observed for fumonisin and DON in 70.8% (n = 80/113) and 58.4% (n = 212/363) of the cases, respectively. For FB1 + FB2 the prevalence ranged from 27.8% (n = 5/18) in 2013 to 85.2% (n = 23/27) in 2014, with a maximum concentration of 53,580 µg kg−1 observed in 2015. The prevalence for DON ranged from 42.0% (n = 40/94) in 2016 to 79.3% (n = 69/87) in 2014, with a maximum concentration of 151,060 µg kg−1 presented in 2013 (n = 45/212) and 36.1% (n = 97/269) with a maximum mycotoxin level of 16,100 µg kg−1 (in 2015) and 12,500 µg kg−1 (in 2014) were observed for 3-acetyldeoxynivalenol and HT-2, respectively , T-2 toxin , and ZEA   was detected in this situation, and just one sample of wheat had an excessive amount of DON   . In a so, 12.5%) . With re, 12.5%) . n = 2/63, 3.2%). Dairy cattle feed presented the highest number of samples that surpassed the recommended levels of mycotoxins , specifically DON , ZEA , T-2 toxin  and HT-2  , FB1 , HT-2 toxin , and OTA . Cat and dog food also showed values above legal thresholds for fumonisins , with a maximum of 18,910 µg kg−1  with the mycotoxins ZEA , FB1 , and DON  infringing the respective recommended guidelines . Finally, in horse feed, Fumonisin B2 was found   . Poultry µg kg−1 . The secidelines . Fish fe6, 3.8%) .Fusarium species  (n = 66/155) 42.6% of prevalence, followed by FB1/FB2  and DON/3-ADON   . Therefo, 50.5%) . In the , 10.2%) .n = 146/360), ranging from 16.3% (n = 8/49) in 2013 to 76.6% (n = 49/64) in 2015. The maximum OTA reported level was 1810 µg kg−1, in 2016 , co-occurrence with other metabolites is a possibility. The most prevalent Fusarium toxins present in feed (different from OTA), in decreasing order of incidence, were ZEA, DON + FB1, FB1, and T-2 toxin with (n = 17/102) 16.7%, (n = 14/102) 13.7%, and (n = 12/102) 11.8% of incidence, respectively  milk samples (n = 2) were reported below the limit of quantification  and butter (n = 3) ranged from 0.021 to 0.024 µg kg−1. Even though 2016 was the year with the lowest number of analyzed samples, it was also the year when fewer samples surpassed the 0.05 µg kg−1 threshold . It was studied/monitored that, consistently, higher concentrations of AFM1 were obtained during March, August, and September  had prevalences higher than 40% during the five years. The average concentrations found in the different toxins in animal feed did not vary between one year and another, except for ZEA and T-2. The drastic increase of ZEA concentrations during 2017 was observed in corn meal and sorghum silo. There is a prior documented avidity of Fusarium spp. to produce ZEA when using moderately alkaline cereals  as substrates [Fusarium graminearum has demonstrated that conditions of pH 9 and incubation temperature of 15.05 °C are required to favor ZEA production [1 + FB2 and DON), the positive samples possessed comparatively low concentrations  ,46,47. F for DON . The dat for DON . Convers for DON . −1 neither affect feed intake nor are biotransferred to the milk [In both dairy and meat cattle, forage, hay, and silage input must not be underplayed, especially in countries where extensive feeding systems based on grazing cattle predominate. Considering Costa Rica a particular case, 85% and 95.9% of the dairy and beef cattle are based on grazing farming, respectively . Relativthe milk ,52.Mycotoxin effects over monogastric animals are varied, depending on the species and physiological and productive stage . For exaFusarium and Penicillium toxins have been described [1  [Mycotoxins in pet foods have already been reported by other countries, including industrialized ones  . Mainly,escribed . An elevctively) . Mycotoxctively) . Findingctively) ,59. Myco−1 [−1 feed have already confirmed adverse effects in productive parameters and increased mortality in some fish. even in a relatively short period [Presence of mycotoxins in fish feed is another proof of an industry which has progressively substituted animal protein sources for vegetable ones ,61. In t−1 . Levels t period . 1 and OTA, which are not produced by Fusarium species. Fusarium species have the potential of simultaneously producing the remainder of the toxins assayed [Aspergillus ochraceus, A. carbonarius, A. niger and Penicillium verrucosum [1 is not only produced by Aspergillus species but it is also a product of metabolism [A different spatial distribution profile was observed for AFM assayed ,64. OTA rrucosum . On the tabolism . Our dat1 is the most studied toxin in milk, other toxins have been described as well [−1), a Costa Rican citizen could be exposed up to 210 µg AFM1 per year. Similarly, a Jersey calf weighing 25–30 kg at birth would be fed with 10% of its live weight with contaminated milk (from 2.5 to 3 kg of milk per day) [1 per day. Milk weaning can occur at ten weeks old [1 per kg body weight per day when a maximum level of 0.5 μg kg−1 is used.Milk is not only a staple commodity by itself, but it can accompany other potentially contaminated products . Additionally, although AFM as well . Other d as well ). During as well . Assuminper day) . Reiteraeeks old . Milk co1 have been documented in other Latin-American countries [1 (the parent compound of AFM1) has been reported to be present in milk samples [1 and AFM1 have in the liver, recent evidence suggests that kidney toxicity is a certainty [−1) AFM1 levels in milk have been recently reported, although prevalence rates are also relatively high , [Much higher average concentrations of AFMountries . Interes samples ). Besideertainty . On the  38.8%), . Other L 38.8%), ,73,74,75 38.8%), ,77,78,79 38.8%), ,81,82 ar1 prevalence [1 in bovine milk during spring [1 occurrence during seasons [1 exposure for a human can be twice as much as breast milk contamination, as has also been well documented [1 contamination are available [Aflatoxin-contaminated feed must also be monitored to avoid feeding dairy cows with contaminated batches . For insevalence , probablg spring . Seasonag spring . Other r seasons . It has  seasons . Finallycumented . Althougvailable , pre- anvailable . Fusarium metabolites must be stridently monitored as it is clear that contamination in feed and feed ingredients is unfortunately common; this is especially true for fumonisins and T-2. Feed manufacturers, farmers (both in the field and storage facilities) and pet owners alike should be educated as to the proper conditions for food storage to avoid mycotoxin-producing fungal colonization. Toxin metabolite analysis and co-occurrence are paramount for complete surveillance of toxin feeds, and efficiently execute systems for the control and reduction of mycotoxins, as well as their metabolites in feeds. In addition, a strict control of AFM1 in milk is necessary, because the prevalence of AFM1 in milk is considerable and several samples exceeded the regulatory thresholds. It must be remembered that milk is the raw material for a wide variety of dairy products , therefore, the exposure of the population to this mycotoxin is increased. Toxicologically relevant concentrations were found during the five-year survey as some sample concentrations exceeded the regulatory guidelines. Fumonisin and deoxynivalenol feed contamination is worrisome since these toxins have the capacity of being found in significant levels in these matrices, and, in our case, higher levels of toxins are found in the Central Valley of the country. Therefore, surveillance programs should be expanded to the outermost productive regions of the country to suppress sampling bias, if existing any. Thermopluvial conditions do not seem to have a considerable effect on toxin levels, although some metabolites actually seem to behave concurrently. 1, FB2 (TSL-202), and OTA (TSL-504) was purchased from Trilogy® Analytical Laboratory Inc . All standards have an initial concentration of 100 mg L−1, except for FB2 that was at 30 mg L−1. Additionally, a naturally contaminated reference material  was used as a quality control sample . Acetonitrile (ACN) and methanol (MeOH), both chromatographic grade, were purchased from J.T. Baker . Ultrapure water  was obtained using an A10 Milli-Q Advantage system and an Elix 35 system .An analytical standard with certified concentrations, dissolved in acetonitrile, for DON, 3-ADON, T-2 (TSL-314), HT-2 (TSL-333), ZEA (TSL-401), FBn = 487 different feedstuffs of ca. 5 kg were collected during 2013–2017 by government inspectors from n = 107 Costa Rican feed manufacturers, as part of a countrywide surveillance program. Sample collection was composed of compound feed and feed ingredients, as follows: dairy cattle feed 28.9% (n = 141), cornmeal 9.9% (n = 48), citrus pulp 5.5% (n = 27), cattle feed 5.5% (n = 27), pig feed 5.3% (n = 26), calf feed 4.3% (n = 21), palm kernel meal 4.1% (n = 20), fish feed (Tilapia) 3.7% (n = 18), poultry feed 3.5% (n = 17), distillers dried grains 3.5% (n = 17), hay 3.3% (n = 16), dog food 3.3% (n = 16), wheat middlings 2.9% (n = 14), soybean meal 2.7% (n = 13), layer hen feed 2.0% (n = 10), horse feed 1.8% (n = 9), forage 1.8% (n = 7), pineapple byproducts 1.2% (n = 6), cassava meal 1.2% (n = 6), sorghum meal 0.6% (n = 3), rodent feed 0.6% (n = 3), ground roasted coffee 0.6% (n = 3), banana peel 0.6% (n = 3), rice bran 0.4% (n = 2), chamomile flowers 0.4% (n = 2), soybean hulls 0.2% (n = 1), shrimp feed 0.2% (n = 1), rice meal 0.2% (n = 1), rabbit feed 0.2% (n = 1), hydrolyzed feather meal 0.2% (n = 1), fish feed , fish feed , corn silage (n = 1), and corn gluten (n = 1). Selection of feed and feed ingredients to be tested, number of samples, sampling sites, and specific toxins to assay (per matrix) were chosen by feed control officials. The selection considered the most common feedstuffs used in Costa Rica, import and export regulations, contamination risk factors, the productivity of the feed industry, and the risk for human and animal health associated with each feed or feed ingredient. Sampling was performed following the Association of American Feed Control Officials (AAFCO) recommendations for mycotoxin test object collection [n = 180 dairy samples (mostly liquid bovine milk) from n = 13 different Costa Rican dairy farms were assayed; 50 mL subsamples were processed from 500 mL samples. A total of llection , and samllection . Additio1 was assayed according to the methods in [Mycotoxins were assayed using the following methods: DON/3-ADON , T-2 andthods in ,92 for m18 column  except for T-2/HT-2 toxin analyses for which a Luna® Phenyl-Hexyl column  was used . All analytes, except AFM1, were extracted using Immunoaffinity columns . All analytes were assayed using HPLC. Equipment consisted of an Agilent 1260 Infinity series HPLC with a quaternary pump (G1311B), a column compartment (G1316A), a variable wavelength and fluorescence detector (G1314B and G1321B) and an autosampler system (G1329A) . Peak separation was accomplished using a 5 mm Agilent Zorbax Eclipse C® (R-biopharm) columns were used for sample extraction. Briefly, 200 mL of purified H2O was added to 25 g of test portion. The mixture was dispersed using an Ultra-Turrax®  at 8000 rpm. The supernatant was filtered by gravity over an ashless filter paper . Subsequently, an exact 2 mL aliquot from the supernatant was transferred to the IAC column and passed at 1 mL min−1 using an SPE 12 port vacuum manifold  at 15 mm Hg vacuum. After a washing step using 2× 10 mL water, the columns were left to dry and then four MeOH fractions of 500 µL were passed through the IAC. The total volume recovered was concentrated to dryness under vacuum at 60°C. The sample was reconstituted with MeOH to 300 µL and transferred to an analytical HPLC conical vial insert  before injection into the chromatograph.DONPREP2O, Solvent A/CH3OH, Solvent B as per chromatographic conditions. The rest of the program was as follows: at 0.5 min 80% A, at 5.50 min 90% A, at 10 min 90% A, at 11 min 80% A, and at 15 min 80% A. DON and 3-ADON absorption at 220 nm was exploited for detection purposes. Linear calibration curves ranging from 1.25 to 10.00 µg mL−1 were prepared during quantification. The limit of quantification for DON/3-ADON was 10.00 and 40.00 μg kg−1.Gradient mode starting at 80:20 H® T-2 and HT-2 IAC (R-biopharm). Extraction solvent consisted in 125 mL of MeOH/H2O (90:10) and 2.5 g of NaCl. An aliquot of 5 mL 10-fold diluted in PBS (1.37 mol L−1) was passed through the column. Precolumn derivatization was performed after the evaporation step using 50 µL of 4-dimethylaminopyridine  and 50 µL of 1-anthroyl cyanide  both at 1 mg mL−1 in toluene . Gradient mode started at 70:30 CH3CN, Solvent A/H2O, Solvent B as per chromatographic conditions. The rest of the program was as follows: at 5 min 70% A, at 15 min 70% A, at 25 min 85% A, at 27 min 100% A, at 32 min 100% A, and at 35 min 70% A. Flow rate was set at 1 mL min−1. Adduct fluorescence was measured at λex = 381 and λem = 470 nm. Linear calibration curves ranging from 125.00 to 1000.00 µg L−1 were prepared during quantification. The limit of quantification for T-2 and HT-2, was 5.00 and 3.00 μg kg−1, respectively. The extraction was similarly performed as detailed for DON/3ADON using an EASI-EXTRACT3CN/H2O 60:40 and an EASI-EXTRACT® ZEARALENONE IAC (R-biopharm). Isocratic mode using a 40:10:50 CH3CN/CH3OH/H2O mixture at a flow rate of 0.7 mL min−1 was used as per chromatographic conditions. ZEA natural fluorescence  was exploited for detection purposes. Linear calibration curves ranging from 300.00 to 1200.00 µg L−1 were prepared during quantification. The limit of quantification was 0.072 μg kg−1.Extraction was performed using 100 mL of CH3CN/MeOH/H2O (25:25:50) and FUMONIPREP® IAC (R-biopharm). Fumonisin derivatization was based on the reaction with o-phthalaldehyde  and 2-mercaptoethanol  as stated on the reference method. However, pre-column derivatization was performed in situ in the autosampler injector, according to Bartolomeo and Maisano (2006), but increasing the sample and OPA reagent volume 5-fold. Adduct fluorescence was measured at λex = 335 and λem = 440 nm. Isocratic mode using MeOH/0.1 mol L−1 NaH2PO4 (77:23), adjusted to apparent pH 3.3 with H3PO4, was used at a 0.8 mL min−1 flow rate. The limit of quantification was 0.05 μg kg−1 for both FB1 and FB2.Extraction was performed using 100 mL of CH3CN/H2O 60:40 and an OCRAPREP® IAC column. OTA elution from column and resuspension after evaporation was achieved using a 98:2 MeOH and acetic acid solution to ensure OTA protonation. Isocratic mode using a 50:50 H2O/CH3CN mixture using 0.2 mol L−1 trifluoroacetic acid, pH = 2.1 (74564 Millipore Sigma) at a flow rate of 0.7 mL min−1 was used as per chromatographic conditions. OTA natural fluorescence  was exploited for detection purposes. Linear calibration curves ranging from 2.50 to 40 µg L−1 were prepared during quantification. The limit of quantification was 0.011 μg kg−1.Extraction was performed using 100 mL of CH® M1  columns were used for sample extraction. An exact 50 mL of raw or processed milk, previously homogenized and filtered by gravity over an ashless filter paper, was transferred to the IAC column. After a washing step using 3× 10 mL of water, the columns were left to dry and eluted using MeOH and concentrated as described above in 5.4.1. Isocratic mode using a 10:35:55 CH3CN/CH3OH/H2O mixture at a flow rate of 0.6 mL min−1 was used as per chromatographic conditions. AFM1 natural fluorescence  was exploited for detection purposes. Linear calibration curves ranging from 0.50 to 2.00 µg L−1 were prepared during quantification. The limit of quantification was 0.014 μg kg−1.AflaStarIn the case of the butter samples, the preparation was performed according to the method in . BrieflyTM, Redlands, CA, USA). For each contaminant, Spearman Rank Order tests were applied to assess the association among the toxin concentration and climatic variables . In this particular case, toxin levels below the limit of detection were considered zero for association purposes; this analysis was performed using SigmaPlot 14 . Sampling date was linked to mean monthly values and data were retrieved from the closest climatological station to the sampling region. Meteorological data were provided by the Costa Rican National Weather Service (https://www.imn.ac.cr/boletin-meteorologico). For


Dual-site transcranial magnetic stimulation to the primary motor cortex (M1) and dorsolateral prefrontal cortex (DLPFC) can be used to probe functional connectivity between these regions. The purpose of this study was to characterize the effect of DLPFC stimulation on ipsilateral M1 excitability while participants were at rest and contracting the left- and right-hand first dorsal interosseous muscle. Twelve participants were tested in two separate sessions at varying inter-stimulus intervals  at two different conditioning stimulus intensities (80% and 120% of resting motor threshold). No significant effect on ipsilateral M1 excitability was found when applying a conditioning stimulus over DLPFC at any specific inter-stimulus interval or intensity in either the left or right hemisphere. Our findings suggest neither causal inhibitory nor faciliatory influences of DLPFC on ipsilateral M1 activity while participants were at rest or when performing an isometric contraction in the target hand muscle. Motor and cognitive function depends on neural activity in a number of spatially distributed but interconnected brain regions. The dorsolateral prefrontal cortex (DLPFC), which includes Brodmann’s areas (BA) 46 and 9 ,2, closedsTMS) is a method to probe cortico-motor pathways both at rest and during different functional contexts in the motor system  was randomized for each experiment. Stimuli were applied every 5 seconds. For each participant, twelve responses were collected for each ISI and CS intensity  and averaged . Measurements were repeated for each of the four experiments. at rest . For alln = 12, x = −21.90 ± 7.31, y = −18.79 ± 10.57, z = 55.43 ± 5.66) and right M1  sites overlapped with the ‘hand knob’ [We used methods previously described elsewhere . Brieflynd knob’ . To targnd knob’ ,60. FiguRM) were performed for the left (Experiments 1 and 2) and right (Experiments 3 and 4) hemispheres on the MEP amplitudes with CS-TS interval  as the within-subject factor for both CS intensities (80% and 120% RMT) and contexts conditions (rest and 20% MVC). Next, we measured and expressed peak-to-peak amplitude of each MEP as a ratio of baseline control MEP  for each participant. Normalized MEP amplitudes were then used in separate 2x11 ANOVARM for the left (Experiments 1 and 2) and right (Experiments 3 and 4) hemispheres using within-subject factors CS intensity (2 levels: 80% RMT or 120% RMT) and ISI  at rest and during the 20% MVC isometric contraction. The threshold for statistical significance was set at p ≤ 0.05.To determine whether DLPFC stimulation modulates MEPs at rest or during an isometric contraction of the target muscle, separate one-way repeated measures analysis of variance  for RMT with the CS coil and intensity required to elicit a MEP amplitude of ∼1 mV in the FDI muscle with the TS coil for each experiment is reported in RM. In Experiment 1, we found a non-significant main effect of ISI on MEP amplitude for right FDI muscle for 80% RMT  = 0.45, p = 0.93, ηp2 = 0.04) and 120% RMT  = 0.45, p = 0.93, ηp2 = 0.02). In Experiment 2, we found a non-significant main effect of ISI on MEP amplitude for right FDI muscle for 80% RMT  = 0.23, p ≥ 0.99, ηp2 = 0.02) and 120% RMT  = 0.30, p = 0.97, ηp2 = 0.02). In Experiment 3, we found a non-significant main effect of ISI on the MEP amplitude for left FDI muscle for 80% RMT  = 0.21, p ≥ 0.99, ηp2 = 0.02) and 120% RMT  = 0.22, p ≥ 0.99, ηp2 = 0.02). In Experiment 4, we found a non-significant main effect of ISI on the MEP amplitude for left FDI muscle for 80% RMT  = 0.32, p = 0.98, ηp2 = 0.03) and 120% RMT  = 0.33, p = 0.98, ηp2 = 0.03).For all four experiments we first performed a separate one-way ANOVARM revealed a non-significant main effect of CS intensity  = 1.38, p = 0.27, ηp2 = 0.11), ISI  = 0.59, p = 0.82, ηp2 = 0.05), and their interaction  = 1.43, p = 0.12, ηp2 = 0.61) on the MEP amplitude ratio for right FDI muscle at rest  = 0.60, p = 0.46, ηp2 = 0.11), ISI  = 0.38, p = 0.95, ηp2 = 0.03), and their interaction  = 1.23, p = 0.28, ηp2 = 0.10) on the MEP amplitude ratio for right FDI muscle while participants maintained a muscle contraction  = 1.18, p = 0.30, ηp2 = 0.17), ISI  = 1.48, p = 0.16, ηp2 = 0.71), and their interaction  = 1.39, p = 0.19, ηp2 = 0.67), on the MEP amplitude ratio for left FDI muscle at rest  = 1.22, p = 0.29, ηp2 = 0.17), ISI  = 1.07, p = 0.39, ηp2 = 0.10), and their interaction  = 0.47, p = 0.91, ηp2 = 0.23) on the MEP amplitude ratio for left FDI muscle during the 20% MVC  as a function of ISI (abscissa) for FDI muscle. traction B. For th at rest C. Same r 20% MVC D.dsTMS approach to examine the temporal and functional interactions of frontal cortico-cortical circuits, focusing on inputs to M1 from ipsilateral DLPFC while participants were at rest or contracting the target muscle. The current findings provide evidence for no causal inhibitory nor facilitatory influences of DLPFC (BA 46) on ipsilateral M1 activity while participants were at rest, irrespective of hemisphere. They also imply that the transmission of causal influence of DLPFC on ipsilateral M1 activity does not change during sustained isometric contractions in the target muscle. Recent dsTMS studies strongly suggest that the influence of frontal cortical areas on motor cortical excitability are associated with cognitive control of movements. For example, changes in functional connectivity between left DLPFC (BA 46) and ipsilateral M1 in the non-target and, to a lesser extent, the target hand muscle have been found during movement preparation of cued and free selection tasks [We used a conditioning-test on tasks . In parton tasks . In anoton tasks . In addion tasks . Importaon tasks , future on tasks  and bimaon tasks .dsTMS technique is unable to specifically target any of these potential pathways. Therefore, conditioning DLPFC stimulation could cause complex activity in these multiple polysynaptic pathways, which may be of particular importance to explain our lack of DLPFC-M1 resting-state connectivity findings with dsTMS. Interestingly, contralateral DLPFC stimulation (defined as 5 cm anterior to M1) has been shown to modulate M1 excitability at longer ISIs  across a wider range of CS intensities [Our study has several methodological limitations. First, we only examined two CS intensities. Future experiments should examine a wider range of CS intensities, similar to previous work ,51, to densities . Therefoensities , it is pensities .dsTMS approach described here to probe how DLPFC-M1 interactions dynamically change during higher-level aspects of motor control across the lifespan and how these interactions are affected by abnormalities of motor and cognitive function in pathological conditions such as stroke, Parkinson’s, and Alzheimer’s disease.In summary, our data show for the first time that stimulation of DLPFC does not influence ipsilateral motor cortex activity both while participants were at rest and contracting the target muscle. Although caution should be used when interpreting findings with TMS , it woul


Diplotaxis virgata D.C. and D. erucoides (L.) D.C. (Brassicaceae) offered to the isolation of two new flavonoids isorhamnetin-3-O-α-l-glucopyranoside (1) and rhamnetin-3,3ʹ-di-O-β-d-glucopyranoside (2), respectively. Their structures have been elucidated from the extended spectroscopic methods, including 1D- and 2D-NMR, UV and mass spectrometry analysis and by comparison with literature data. The fatty acid composition of the hexane extracts of the two species was also investigated by using GC-MS. The antioxidant activity of ethanol, ethyl acetate, n-butanol extracts and the isolated compounds from the two species was evaluated using DPPH and ABTS+ scavenging assays. All the tested samples showed an efficient radical scavenging ability, with IC50 values ranging from 16–40 µg/mL for the DPPH and from 17–44 µg/mL for the ABTS+ assays. In addition, the antibacterial activity of the prepared extracts and compounds 1 and 2, determined by well diffusion agar method against two Gram positive and five Gram negative bacteria, was evaluated and the results showed significant effects against all strains used.A phytochemical investigation of  Diplotaxis (Brassicaceae) is represented by more than 30 species mainly distributed in the north Mediterranean −) in accordance with the molecular formula of C22H22O12.The acquisition of the mass spectrum by using the ES-HRMS in a negative mode showed a peak at 1H- and 13C-NMR spectra of compound 1 , δH 7.48 ppm  and δH 7.73 ppm  were characteristic to the ring B of the isorhamnetin nucleus −).Compound 2  was isol1H- and 13C-NMR spectra of compound 2 , and δ 6.28 ppm , protons of a 5,7-disubstituted ring A from a flavonoid skeleton ..d-glucop13C-NMR spectrum revealed the presence of 28 carbon signals among which 16 correspond to the methoxylated flavonol [-unsaturated ketone at δC 180.6 (C-4) and 10 quaternary 2spcarbons. This assignment was supported by the literature data relative to flavonoid glycosides in plants, namely rhamnetin and isorhamnetin derivatives [The flavonol  and indiivatives ,15,16,17C 136.7) of the ring C of the flavonol moiety observed in the HMBC spectrum. On the other hand, the position of the second glucopyranosyl system (Glc II) was deduced without ambiguity from the same spectrum by the observation of the correlations H-1ʹʹʹ/C-3ʹ (δC 147.1), H-2ʹ/C-3ʹ and H-5ʹ/C-3ʹ (The position of the first sugar (Glc I) moiety was ascertained by the long range correlation between the anomeric proton H-1ʹʹ and the quaternary carbon C-3 , whereas only three acids were detected in the hexane extract of D. erucoides (HFPA-DE). They were identified to be palmitic acid (14.35%), linolelaidic acid (6.91%) and linoleic acid (29.10). In the two hexane extracts, the unsaturated acids constitute the main fraction (42.73% in HFF-DV and 36.01% in HFPA-DE). Moreover, linoleic acid was found to be predominant in the two fractions  and from 18.00 ± 0.01 μg/mL to 27.02 ± 1.53 μg/mL (for D. erucoides). Lower IC50 value indicated higher antioxidant activity. In both cases, the n-BuOH fractions exhibited a radical-scavenging activity slightly higher than that of the EtOAc ones. This finding could be explained by their richness in phenolic metabolites as compounds 1 (16.01 ± 0.13 μg/mL) and 2 (18.00 ± 0.01 μg/mL) which exhibited practically the same activity as that of BHT (18.00 ± 0.23 μg/mL). The antioxidant activity of our extracts was found comparable to that of extracts from Diplotaxis harra and D. simplex [1 and 2 is certainly due to the free phenol groups present in each structure. The higher activity of compound 1 compared to that of compound 2 is easily interpreted through the presence of an additional free hydroxyl group at the 7 position which could easily transfer its hydrogen atom to DPPH. These two flavonoids remain less active than their analogue quercetin (5.00 ± 0.12 μg/mL) used as positive control. The significant activity of these phenolic derivatives is in good agreement with literature data [1 was found to be more active against DPPH than its aglycon (isorhamnetin) showing an IC50 value of 24.5 μg/mL [2 exhibited a better antioxidant activity towards DPPH than its aglycon  [1 and 2 either by improving the solubility of these compounds in ethanol or by participating in the stability of the new radical that forms after the transfer of the hydrogen atom.According to the results given in  simplex . The higure data . However.5 μg/mL . Similar0 μg/mL) . These f+ is a well-known nitrogen-centered synthetic radical and is also used to evaluate antioxidant potential. The ABTS+ radical is generated by oxidation of ABTS with potassium persulphate and, when samples are added to the ABTS+ radical, it is converted into a non-radical form. The results from the radical scavenger assays using the ABTS+ radical for the prepared extracts, fractions and the isolates 1 and 2, expressed as IC50 (μg/mL), are shown in + radical as they have practically done with DPPH. All the samples exhibited interesting radical-scavenging activity towards the ABTS+ radical with IC50 values ranging from 17.03 ± 0.02 μg/mL (compound 1) to 44.00 ± 1.72 μg/mL (EtOAc fraction of D. virgata flowers) compared to BHT (50.00 ± 0.20 μg/mL).ABTSD. virgata flowers was the most potent according to ABTS radical-scavenging activity with IC50 value of 25.54 ± 1.32 μg/mL whereas the crude ethanolic extract of the non-flowering aerial parts of D. erucoides was found to be slightly more active (27.04 ± 1.53 μg/mL) than its fractions (EtOAc and n-BuOH). This result can be interpreted by a possible synergism effect due to the concentration of some phenolic components. On the other hand, the results showed that compounds 1(17.03 ± 0.02 μg/mL) and 2 (19.04 ± 0.02 μg/mL) are more than twice as active as BHT. According to ABTS radical-scavenging activity, compounds 1 and 2 remain also less active than quercetin (6.91 ± 0.42 μg/mL).Indeed, the n-BuOH fraction from Diplotaxis virgata and D.erucoides as well as of the isolated compounds 1 and 2 was assessed in vitro against a panel of Gram + (Listeria monocytogenes and Staphylococcus aureus) and Gram– bacteria by using the disc diffusion method. The results are given in Diplotaxis harra and D. Simplex [D. virgata flowers was found to be more effective (IZ = 15.00 ± 0.02–22.03 ± 0.02 mm) against all the used bacteria than the isolated compound 1 (IZ = 12.04 ± 0.05–16.55 ± 0.05 mm). This finding may be explained by a possible synergism effect due to the concentration of several antibacterial compounds present in this extract. E. coli appeared the most sensitive towards the n-BuOH extract of D. virgata flowers (IZ = 22.03 ± 0.02 mm) followed by A. hydrophila (IZ = 21.00 ± 0.05 mm)  .D. erucoides aerial parts (IZ = 16.00 ± 0.01–21.07 ± 0.05 mm) than compound 2 (IZ = 13.00 ± 0.02–17.60 ± 0.04 mm) except E. coli which was slightly more sensitive against compound 2 (IZ = 17.60 ± 0.04 mm). Additionally, compound 2 was slightly more efficient than compound 1 against all bacteria tested. This finding could be explained essentially by the additional sugar moiety in compound 2 at position C-3ʹ. Tsuchiya and colleagues sought to establish a structure–activity relationship for flavonoids by the isolation of a number of differently substituted compounds and testing their antibacterial activity towards a panel of bacterial strains. Their study demonstrated that 5,7-dihydroxylation of the A ring in the flavonoid skeleton was important for antibacterial activity [1 and 2, dihydroxylated, hydroxylated and methoxylated in those positions, respectively.The used bacteria were found to be more sensitive towards the n-BuOH extract of activity . This re-Vis Jenway 6505 spectrophotometer. NMR experiments were performed on a BRUKER AVANCE III HD 400 spectrometer operating at 400.13 MHz equipped with a QNP 1H/ 13C/ 19F/ 31P probe. Spectra were recorded at 30 °C in a mixture of deuterated solvents . Acquisition sequences were 1D-NMR 1H-, 13C-, DEPT 135 and 2D-NMR experiments COSY, HSQC, HMBC, and NOESY. The chemical shifts (μ) reported in ppm were referred to residual non deuterated CH3OH. Coupling constants were measured in Hz and signals are using the following abbreviations: s, singlet; d, doublet; dd, doublet of doublets; m, multiplet. HPLC analysis (Agilent 1100), was carried out on a YMC-Pack Pro C18 column  under the following conditions: injected volume 1 µL, flow rate 0.3 mL/min, column temperature 40 °C, wavelength detection at 210 nm ; 254 nm ; and 340 nm . A gradient elution was carried out using the following solvent system: mobile phase A, water formic acid (99.9: 0.1 (v/v)); mobile phase B, methanol. The gradient program (flow 0.3 mL/min) was from 10%–100% methanol as followed: 10% (0–1 min); 100% (1–10 min) as post run time 4 min. MS analysis was carried out using a quadrupole time of flight (QTOF) mass detector , equipped with an electrospray interface operating in positive and negative ion mode, using the following operation parameters: capillary voltage 3500 V, nebulizer pressure 0.6 bar, drying gas 4 L/min, gas temperature 180 °C. MS accurate mass spectra were recorded across the range 50–1000 m/z.UV spectra and Antioxidant activity was measured on a UVDiplotaxis virgata (L.) D.C. was collected from the region of Sousse (Tunisia) and Diplotaxiserucoides (L.) D.C. was collected from the region of Zaghouan (Tunisia) in March 2012, and identified by Prof. Nadia Ben BRAHIM . Flowers were separated from aerial parts before extraction. A voucher specimen from each species  has been deposited in the department of Botany of the same Institute.Diplotaxis erucoides (50 g) and flowers of Diplotaxis virgata (50 g) were extracted for three times with 80% ethanol at room temperature for 72 h. The ethanol extracts concentrated under reduced pressure, giving (10 g (20%), 2.5 g (5%), respectively). The residue was successively partitioned with hexane, chloroform, ethyl acetate, n-butanol and water. The extracted solutions were evaporated under reduced pressure to yield 320 mg (3.2%), 300 mg (12%), of a hexane-soluble extract, 140 mg (1.4%), 160 mg (6.4%), of CHCl3-soluble extract, 379 mg (3.79%), 180 mg (7.2%) of EtOAc-soluble extract, 1.179 g (11.79%), 0.950 g (38%) of butanol-soluble extract, respectively and the remaining aqueous solution were lyophilized to yield 7.98 g (79.8%), 0.91 g (36.4%) of water-soluble extract, respectively.The non-flowering aerial parts of -BuOH extract of flowers of D. virgata (250 mg) was fractionated by HPLC-UV on a C18 SPE cartridge using as solvent water formic acid 99.9:0.1 (v/v)  and MeOH . A total of 10 fractions were obtained. Fraction eluted by  constitutes compound 1 (25 mg). The fractionation of the n-BuOH extract of the non-flowering aerial parts of D. erucoides (250 mg) by HPLC-UV on a C18 SPE cartridge applying the same mobile phase, gives 10 fractions. The purified fraction using  corresponds to compound 2 (23 mg).The n-MS). The capillary column used was an agilent 19091N-205 . Helium was used as carrier gas at a flow rate of 0.8 mL/min with 1 µL injection volume. Samples were analyzed with the column held initially 60 °C for 2 min after injection then 5 °C/min to 250 °C for 20 min. The injection was performed in splitless mode. Dectector and injector temperature were respectively 280 and 250 °C. Run time was 80 min. MS scan range was (m/z) 35–450 atomic mass units (a.m.u) under electron impact (EI) ionization (70 eV).The process of converting free or esterified FAs into methyl esters is referred to as transesterification, with a methanolic solution of potassium according to the AOAC official method 969.33 . Individ50 values denote the concentration required to scavenge 50% of DPPH radicals. Results were expressed in inhibition percentage at different sample concentrations (mg/mL). The inhibition of free radical DPPH in percentage (I%) was calculated as follows: blank is the absorbance of the control reaction  and A sample is the absorbance of the test compound. Extract concentration providing 50% inhibition percentage against extract concentration. Tests were carried out in triplicate.This test allows to measure the extracts capacity to scavenge the stable radical 2,2-diphenyl-1-picryl hydrazil (DPPH) formed in solution by donation of a hydrogen atom or an electron . If the + free radical decolorization assay developed by the method of [2S2O8. The mixture was allowed to stand for 15 h in the dark at room temperature. The solution was diluted with ethanol to obtain the absorption of 0.7 ± 0.2 units at 734 nm. Samples were separately dissolved in ethanol to yield the following concentrations: . In order to measure the extracts antioxidant activity, 10 µL of each were added to 990 µL of diluted ABTS+ at various concentrations. The absorption was read after 20 min. The synthetic BHT and quercetin were used as positive control. The antioxidant capacity of the tested samples was expressed as the inhibition percentage (%). The ABTS+ scavenging percentage was calculated according to the following formula: blank is the absorbance of the control reaction , and Asample is the absorbance of the test compound.The antiradical activity was done using the ABTSethod of , with soin vitro antibacterial activity of the tested samples was carried out by wells agar diffusion method [Staphylococcus aureus ATCC 25923 and Listeria monocytogenes ATCC 11120) and 5 Gram-negative bacteria . The nutrient agar was used as culture medium. One hundred microliters of each tested strain (about 107 CFU/mL) was added to 20 mL of molten medium and let to solidify in sterile petriplates. After solidification, wells were perforated using a sterile cork borer (6.0 mm diameter) and 50 µL of n-BuOH extract or pure compound (250 µg/mL) were added. The plates were incubated at 37 °C and examined after 24 h for clear zones showing the bacteria inhibition. The negative control plates had no product added to the filter paper whereas in the positive control plates, discs were impregnated with the same volume of ampicillin solution (5 mg/mL). The diameters of the inhibition zones were measured. The experiments were repeated in triplicates and the results were expressed as average values.The n method , againstDiplotaxis species, D. virgata and D. erucoides, which led to the isolation of two new flavonoids, isorhamnetin-3-O-α-l-glucopyranoside (1) and rhamnetin-3,3ʹ-di-O-β-d-glucopyranoside (2). Their structures were established by UV, HRMS and RMN analysis. The fatty acid composition of the hexane extracts from the two species was determined by gas chromatography (GC-MS) for the first time. The in vitro antioxidant activity of the prepared extracts and the isolates was investigated by using DPPH and ABTS+. The results demonstrated that the n-BuOH extracts and the isolated compounds exhibit potent free radical scavenging activities. These antioxidant activities were found to be in perfect harmony with the structure of the identified phenolic compounds. The n-BuOH extracts from the two species and the isolated compounds 1 and 2 were also tested for their inhibitory effects against a panel of bacterial agents and it was found that compounds 1 and 2 exhibited interesting antibacterial activity toward most bacteria tested (IZ = 12.04 ± 0.05–17.60 ± 0.04 mm). Finally, this research work reinforced the potential of these species as a possible source of novel bioactive compounds and allowed the discovery of new bioactive flavonoids.In summary, this work described the chemical investigation of two Tunisian


Between group comparisons using repeated measures ANOVA showed that the policosanol 20 mg group had a significant reduction of SBP at 12 weeks (p = 0.020) and a reduction of diastolic BP (DBP) at 8 weeks (p = 0.041) and 12 weeks (p = 0.035). The policosanol 10 mg and 20 mg groups showed significant reductions in aortic SBP of 7.4% and 8.3%, respectively. The policosanol groups showed significant reductions of total cholesterol (TC) of 9.6% and 8.6% and low-density lipoproteins (LDL-C) of 21% and 18% for 10 mg and 20 mg of policosanol, respectively. Between group comparisons using repeated measures ANOVA showed that the policosanol (10 mg and 20 mg) groups at 12 weeks had a significant reduction of TC (p = 0.0004 and p = 0.001) and LDL-C (p = 0.00005 and p = 0.0001) and elevation of %HDL-C (p = 0.048 and p = 0.014). In conclusion, 12-week consumption of policosanol resulted in significant reductions of peripheral SBP and DBP, aortic SBP and DBP, mean arterial pressure (MAP), and serum TC and LDL-C with elevation of % HDL-C.The current study was designed to investigate the short-term effects of policosanol consumption on blood pressure (BP) and the lipid parameters in healthy Korean participants with prehypertension. A total of 84 healthy participants were randomly allocated to three groups receiving placebo, 10 mg of policosanol, or 20 mg of policosanol for 12 weeks. Based on an average of three measurements of peripheral BP, the policosanol 20 mg group exhibited the most significant reduction, that is, up to 7.7% reduction of average systolic BP (SBP) from 136.3 ± 6.1 mmHg (week 0) to 125.9 ± 8.6 mmHg (week 12,  Hypertension is a key risk factor for the incidence of stroke and cardiovascular disease (CVD) and is often accompanied by dyslipidemia and diabetes ,2. It haSaccharum officinarum L.). These primary alcohols range from 24–34 carbon atoms, with octacosanol, triacontanol, dotriacontanol, hexacosanol and tetratriacotanol as main constituents. Consumption of policosanol can reportedly reduce TC and LDL-C with less oxidation of LDL [Policosanol is mixture of eight aliphatic primary alcohols purified from sugar cane wax  policosanol consumption on peripheral BP and central aortic BP along with any changes in the serum lipid profile in healthy Korean subjects who had prehypertension.24H49OH, 0.1–20 mg/g);1-hexacosanol ; 1-heptacosanol ; 1-octacosanol ; 1-nonacosanol ; 1-triacontanol ; 1-dotriacontanol ; 1-tetratriacontanol .The policosanol raw material  was obtained from Rainbow & Nature Pty, Ltd . The policosanol consisted of several alcohol chains of various lengths. More than 90% of policosanol contents were higher aliphatic wax alcohols. Individual alcohols present in policosanol are identical with our previous report  and otheThe placebo tablet had the same taste and smell with an identical colour. It contained the same basic pigment (Gardenia Blue color) and ingredients such as lactose, cellulose, glycerin fatty acid ester, magnesium stearate, etc, except for the policosanol.n = 84) were grouped randomly by dice casting as group 1 (placebo), group 2 (policosanol 10 mg), or group 3 (policosanol 20 mg). All participants in each group consumed policosanol for 12 weeks, according to the study design  were recruited by newspaper advertisement. All participants were pre-screened for suitability and the inclusion criteria were as follows: age 19–65 years old who had pre-hypertension without any history of endocrinological disorder. Heavy alcohol drinkers (>30 g ethanol (EtOH)/day) and those who had consumed prescribed drugs associated with hyperlipidemia, diabetes mellitus, or hypertension were excluded. All participants had unremarkable medical records without illicit drug use or past history of chronic diseases. On the first visit, all participants  or placebo, and film coated tablets were manufactured by CosmaxBio, Inc.  for this study. The ingredients, manufacturing process, and facility were approved by the Korean FDA. All participants were advised to avoid aggressive changes in dietary habits and excessive alcohol consumption (less than 30 g of ethanol per day). They were also instructed to avoid intense exercise (less than 30 min per day at 60–80% maximum capacity). If subjects had a sedentary lifestyle before the study, they were encouraged to balance their lifestyle during the policosanol consumption period to avoid bias due to exercise or other lifestyle habits.On the visit day, anthropometrical parameters such as height, body weight, body mass index (BMI), subcutaneous fat (kg), and visceral fat mass (kg) were individually measured for each participant. The parameters were measured at the same time of day at 4-week intervals using an X-scan plus II body composition analyzer .Measurement of BP was carried out between 8 a.m.–12 p.m. on visit day because the participants had to do overnight fasting for blood collection. BP was measured using three measuring instruments at each visit and the average was recorded at 4-week intervals. First, we used a mercury sphygmomanometer for manual measurements by a licensed technician (S.-J.K.). Second, digital BP device  was employed. Third, SphygmoCor system  was employed to measure peripheral and central aortic BP . Centralg) and stored at −80 °C until analysis. Total cholesterol (TC), triglyceride (TG), HDL-C, and glucose levels were measured in plasma using commercially available kits . Plasma aldosterone was measured by radioimmunoassay (RIA) using an instrument  at the Seegene Medical Foundation . After overnight fasting, blood was collected from each participant on the visit day. For plasma collection, blood was collected in a vacutainer  containing EDTA  at weeks 0 and 12 by low-speed centrifugation  in the tables. SBP and DBP and blood profile were distributed normally as assessed by a Shapiro-Wilk test in t-test  was usedt-test .p value of <0.05 were considered significant and the statistical software SPSS  was used for statistical analysis. In 2 and 1.6–2.1 kg of visceral fat. Based on the average of three methods of measurement of peripheral BP at week 8 (p < 0.001) and DBP was reduced from 84.2 ± 7.3 mmHg (week 0) to 79.5 ± 7.8 mmHg . The policosanol 10 mg group showed 4.5 % and 3.6% reduction of SBP and DBP, respectively, at week 8 compared with week 0. The placebo group showed no change in brachial SBP and DBP after 8 weeks. We also quantified the confidence interval  of blood pressure values in the different groups to measure the range that provides the variability of the observed population statistic (precision) and to report the probable relationship in the population from which the data were extracted (accuracy). For the placebo group, the measured CI at week 0 and week 8 for the peripheral and aortic pressure were SBP 95% ,  and DBP 95% , . For the policosanol 10 mg group, the measured CI at week 0 and week 8 for the peripheral and aortic pressure were SBP 95% ,  and DBP 95% CI , . For the policosanol 20 mg group, the measured CI at week 0 and week 8 for the peripheral and aortic pressure were SBP 95% ,  and DBP 95% , .As shown in t week 8 , the polp < 0.001). The policosanol 10 mg group showed a 6.1% reduction in SBP from 135.8 ± 12.3 mmHg at week 0 to 127.7 ± 9.6 mmHg at week 12 (p < 0.001), while the placebo group showed no change in BP between week 0 (134.4 ± 8.8 mmHg) and week 12 (132.1 ± 10.2 mmHg). For the placebo group, the measured CI at week 0 and week 12 for the peripheral and aortic pressure were SBP 95% ,  and DBP 95% , . For the policosanol 10 mg group, the measured CI at week 0 and week 12 for the peripheral and aortic pressure were SBP 95% ,  and DBP 95% , . For the policosanol 20 mg group, the measured CI at week 0 and week 12 for the peripheral and aortic pressure were SBP 95% ,  and DBP 95% , .As shown in p < 0.001) and the time and group interaction (p = 0.012). Further analysis with repeated measures ANOVA showed that both policosanol 10mg (p < 0.001) and 20 mg (p < 0.001) groups showed significant differences within the group, whereas the placebo group showed no significance within the group (p = 0.286). One-way ANOVA between the groups showed no significant difference among the three groups at 0 week (p = 0.753), 4 weeks (p = 0.770), and 8 weeks (p = 0.631), except for 12 weeks (p = 0.020). Using the least significant difference (LSD) method as a post-hoc test with the 12-week data, the policosanol 20 mg group showed a significant difference compared to the placebo group, whereas the 10 mg group showed no difference. As shown in p = 0.0004), 8 weeks (p = 0.007), and 12 weeks (p = 0.0002) compared to that at 0 week. The policosanol 10 mg group also showed a significant decrease in SBP at 12 weeks (p = 0.025) compared to that at 4 weeks. The policosanol 20 mg group showed a more significant reduction of BP at 4 weeks (p < 0.001), 8 weeks (p < 0.001), and 12 weeks (p < 0.001) compared to that at 0 week. The policosanol 20 mg group also showed a significant reduction of BP at 8 weeks (p = 0.021) and 12 weeks (p < 0.001) compared to that at 4 weeks. In addition, the policosanol 20 mg group showed a significant reduction of SBP at 12 weeks (p = 0.007) compared to that at 8 weeks.Within each policosanol group, multiple comparisons using the LSD method revealed the policosanol 10 mg group to show a significant reduction of SBP at 4 weeks (p < 0.001) and time and group interaction (p = 0.023). Further repeated measures ANOVA revealed significant differences between the policosanol 10mg (p = 0.028) and 20mg (p < 0.001) groups, while placebo group showed no significance within group (p = 0.270). One-way ANOVA analysis between the group showed that the three groups were similar at 0 week (p = 0.831) and 4 weeks (p = 0.532). On the other hand, the same analysis revealed a significant difference at 8 weeks (p = 0.041) and 12 weeks (p = 0.035). Using LSD method as a post-hoc test with the 8-week and 12-week data, the policosanol 20 mg group showed significant differences compared to the placebo group. From repeated measures ANOVA with DBP data , the polp = 0.021), 8 weeks (p = 0.042), and 12 weeks (p = 0.028) compared to that at 0 week. The policosanol 20 mg group showed a more significant reduction of DBP at 4 weeks (p = 0.002), 8 weeks (p < 0.001), and 12 weeks (p < 0.001) compared to that at 0 week. The policosanol 20 mg group also showed a significant reduction of DBP at 8 weeks (p = 0.002) and 12 weeks (p < 0.001) compared to that at 4 weeks. These results suggest that 20 mg of policosanol is significantly more effective in lowering the SBP and DBP than 10 mg of policosanol with dose responsiveness according to repeated measures ANOVA. Within the each policosanol group, multiple comparisons using the LSD method showed that the policosanol 10 mg group had a significant reduction of DBP at 4 weeks (p = 0.020) and a reduction of DBP at 8 weeks  and 12 weeks  compared with placebo. In contrast, the policosanol 10 mg group showed no significant difference compared with placebo at each time point, although the 10 mg group showed significant reduction of SBP and DBP from baseline of 0 week.Based on the SphygmoCor measurements, the placebo group did not show significant changes in aortic SBP and DBP during the 12-week consumption. However, between 0 and 8 weeks, the placebo, policosanol 10 mg, and 20 mg groups showed aortic SBP reductions of 4.8%, 6.9%, and 5.6%, respectively. Aortic DBP was reduced significantly in policosanol 10 and 20 mg groups by 4.0% and 4.7% respectively, at week 8. Mean arterial pressure (MAP) was reduced in the policosanol groups in a dose-dependent fashion. In the policosanol 10 mg and 20 mg groups, MAP was reduced by 4.1% and 6.0%, respectively, while the placebo group showed no change .p < 0.001). The policosanol 10 mg group showed also significant reductions by 7.4% in aortic SBP from 123.0 ± 12.5 mmHg (week 0) to 113.9 ± 8.6 mmHg . Although aortic DBP was significantly reduced only in the policosanol 20 mg group by 5.1% reduction, however, MAP was significantly reduced by 5.2% and 7.5% in the policosanol 10 mg and 20 mg groups, respectively. Interestingly, the peripheral SBP and DBP, aortic SBP and DBP, and MAP were lowered in a policosanol dose-dependent manner. In week 12, as shown in After 12 weeks, as shown in The percentage of HDL-C in TC (%HDL-C/TC) was also remarkably elevated in the policosanol groups in a dose-dependent manner. In the policosanol 10 mg group, %HDL-C/TC was increased to 25.9 ± 6.5% in week 12 compared with 20.2 ± 4.7% in week 0. In the policosanol 20 mg group, %HDL-C/TC was increased to 26.6 ± 6.0% at week 12 from 21.3 ± 8.4% at week 0 . TG/HDL-p = 0.009). Although there were no difference in group and time, however, the time × group interaction was significantly different. Further analysis with repeated measures ANOVA showed that both policosanol 10 mg (p = 0.010) and 20 mg (p = 0.024) groups showed significant differences within the group, whereas the placebo group showed no significance within the group (p = 0.203). One-way ANOVA between the groups showed no significant difference among the three groups at 0 week (p = 0.915), 4 weeks (p = 0.169), and 8 weeks (p = 0.167), except for 12 weeks (p = 0.0005). Using the least significant difference (LSD) method as a post-hoc test with the 12-week data, the policosanol 10 mg group (p = 0.004) and 20 mg group (p = 0.001) showed a significant difference compared to the placebo group. Within each policosanol group, multiple comparisons using the LSD method revealed the policosanol 10 mg group to show a significant reduction of TC only at 12 weeks (p = 0.001) compared to that at week 0. The policosanol 10 mg group also showed a significant decrease in TC at 12 weeks (p = 0.010) compared to that at 4 weeks. The policosanol 20 mg group showed a more significant reduction of TC at 4 weeks (p = 0.005), 8 weeks (p = 0.015), and 12 weeks (p = 0.029) compared to that at week 0. These results suggest that policosanol groups showed significant reduction of TC with time dependent manner, while placebo group was not. As shown in top panel of p = 0.035), time (p < 0.001), and time × group interaction (p < 0.001), suggesting that LDL-C was significantly and distinctly reduced in policosanol groups. Further analysis with repeated measures ANOVA showed that both policosanol 10mg (p = 0.001) and 20 mg (p < 0.001) groups showed significant differences within the group, whereas the placebo group showed no significance within the group (p = 0.103). One-way ANOVA between the groups showed no significant difference among the three groups at 0 week (p=.916), 4 weeks (p = 0.226), and 8 weeks (p = 0.148), except for 12 weeks (p < 0.001). Using the least significant difference (LSD) method as a post-hoc test with the 12-week data, the policosanol 10 mg group (p = 0.00005) and 20 mg group (p = 0.0001) showed a significant difference compared to the placebo group. Within each policosanol group, multiple comparisons using the LSD method revealed the policosanol 10 mg group to show a significant reduction of LDL-C at 12 weeks (p < 0.001) compared to that at week 0. The policosanol 10 mg group also showed a significant decrease in LDL-C at 12 weeks (p = 0.001) compared to that at 4 weeks. The policosanol 20 mg group showed a more significant reduction of LDL-C at 4 weeks (p = 0.008), 8 weeks (p = 0.0002), and 12 weeks (p = 0.0001) compared to that at week 0. These results suggest that policosanol groups showed significant reduction of LDL-C with time dependent manner, while placebo group was not.As shown in middle panel of p < 0.001), and time × group interaction (p = 0.003). Further analysis with repeated measures ANOVA showed that both policosanol 10 mg (p < 0.001) and 20 mg (p < 0.001) groups showed significant differences within the group, whereas the placebo group showed no significance within the group (p = 0.311). One-way ANOVA between the groups showed no significant difference among the three groups at 0 week (p=.684), 4 weeks (p = 0.918), and 8 weeks (p = 0.769), except for 12 weeks (p = 0.035). Using the least significant difference (LSD) method as a post-hoc test with the 12-week data, the policosanol 10 mg group (p = 0.048) and 20 mg group (p = 0.014) showed a significant difference compared to the placebo group. Within each policosanol group, multiple comparisons using the LSD method revealed the policosanol 10 mg group to show a significant elevation of %HDL-C at 8 weeks (p = 0.002) and 12 weeks (p < 0.001) compared to that at week 0. The policosanol 10 mg group also showed a significant elevation in %HDL-C at 8 weeks (p = 0.026) and 12 weeks (p = 0.00007) compared to that at 4 weeks. The policosanol 20 mg group showed a more significant elevation of %HDL-C at 8 weeks (p = 0.00001), and 12 weeks (p = 0.00004) compared to that at week 0. The policosanol 20 mg group also showed a more significant elevation of %HDL-C at 8 weeks (p = 0.0002), and 12 weeks (p < 0.001) compared to that at week 4. These results strongly suggest that policosanol groups showed significant elevation of %HDL-C with time dependent manner, while placebo group was not. Serum aldosterone was remarkably decreased in the policosanol groups by 35% and 24% for the 10 and 20 mg groups, respectively, while the placebo group showed no change after 12-week consumption.As shown in bottom panel of Pearson’s correlation analysis of peripheral and aortic blood pressure with lipid profile after 12 weeks of placebo and policosanol treatment is shown in the Hypertension is closely linked to the incidence of metabolic syndrome , which in = 76) had similar BP (around 135.6 ± 9.1 mmHg of SBP and 84.6 ± 7.5 mmHg of DBP), serum TC (188 ± 20 mg/dL) and LDL-C (130 ± 32 mg/dL). However, policosanol consumption for 12 weeks lowered the peripheral BP, aortic BP, MAP, and serum levels of TC and LDL-C in a dose-dependent manner. In particular, the policosanol 20 mg group showed a remarkable reduction in peripheral SBP and DBP in a time-dependent manner between each group of different doses compared with the placebo group after 12 weeks from repeated measurement ANOVA , and serum TC and LDL-C with elevation of %HDL-C. The BP-lowering efficacy of policosanol is also well correlated with reductions in serum levels of TG and TG/HDL-C.