In this trial, 24 subjects were selected in the fasting and postprandial states, respectively. Enrolled subjects randomly accepted a single dose of 0.125 g Cefaclor granule or Cefaclor suspension. The washout period was set as 2 days. Blood samples were collected within 8 h after administration in the fasting state and within 10 h after administration in the postprandial state. Plasma concentrations were determined by Liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pharmacokinetic parameters  were used to evaluate bioequivalence of the two drugs. In the fasting trial, the geometric mean ratios  for Cmax, AUC0-t, and AUC0-∞ were 93.01% (85.96%–100.63%), 97.92% (96.49%–99.38%) and 97.95% (96.52%–99.41%), respectively. The GMR (90% CIs) for Cmax, AUC0-t, and AUC0-∞ in postprandial state were 89.27% (81.97%–97.22%), 97.31% (95.98%–98.65%) and 97.31% (95.93%–98.71%), respectively. The 90% CIs of AUC and Cmax in the fasting and postprandial states were within the 80–125% bioequivalence range. Therefore, Cefaclor granule and Cefaclor suspension were bioequivalent and displayed similar safety profiles. Furthermore, food intake affected the pharmacokinetic parameters of both drugs.We conducted a phase I bioequivalence trial in healthy Chinese subjects in the fasting and postprandial states. The goal of this trial was to compare the pharmacokinetics and safety of the test preparation Cefaclor granule  and the reference preparation Cefaclor suspension (Ceclor Haemophilus influenzae and Klebsiella. It has a strong inhibitory effect on certain anaerobic microorganisms including Propionibacterium acnes  studies have shown that cefaclor is well absorbed from the intestinal tract after oral administration . A singlHere, we conducted a phase I clinical trial to compare the pharmacokinetics and safety of Cefaclor granule and Cefaclor suspension in healthy Chinese subjects in the fasting and postprandial states. Meanwhile, we also focused on the effect of food intake for Cefaclor granule and Cefaclor suspension absorption.The volunteers were healthy Chinese men and women between the age of 18 and 55 years. The following screening protocols were performed in all volunteers: medical history, physical examination, blood cell count, general biochemistry , urine pregnancy test for women, HIV, hepatitis B and C virus serological tests, electrocardiography and imaging examinations. Excluded volunteers with one of the exclusion criteria, and the volunteers who met all of the inclusion criteria were enrolled the trial. The detailed inclusion and exclusion criteria were listed in Supplementary materials. 1.This clinical trial was performed at the Phase I Clinical Trial Laboratory, Affiliated Hospital of Changchun University of Chinese Medicine (China). It was conducted according to the requirements of Good Clinical Practice (GCP), the Declaration of Helsinki and the relevant domestic laws and regulations and was approved by the Ethics Committee of the Affiliated Hospital of Changchun University of Chinese Medicine (Number: CCZYFYLL2019 review-006). The study was registered at Drug Clinical Trial Registration and Information Disclosure Platform (Registration No. CTR20190515). All subjects had written informed consents before the initiation of the investigation.®, specification: 0.125 g, manufacturer: Eli Lilly and Company, Indianapolis, United States, batch number: C873035). After 2 days washout period, subjects orally administrated another formulation cefaclor with a single dose of 0.125 g. In each administration period, blood samples were collected to assay PK parameters at the following times: within 60 min (pre-dose), 5, 10, 20, 30, 45 min, 1, 1.25, 1.5, 1.75, 2, 3, 4, 6, 8 h after drug administration. The same administration protocol was used in the postprandial state, but blood samples for PK analysis were collected at the following times: within 60 min (pre-dose), 5, 10, 20, 30, 45 min, 1 h, 1 h 20 min, 1 h 40 min, 2 h, 2 h 20 min, 2 h 40 min, 3, 3.5, 4, 6, 8, 10 h after drug administration. 4 mL blood was taken in tubes containing heparin sodium anticoagulant for separation. Blood samples were centrifuged for 10 min at 2000 ± 10 g (4°C) to take plasma using a Beckman Allegra X-15R , 800uL plasma was added into the detected tube. All plasma was stored at −80°C within 24 h for preservation until PK analyzed.In the fasting state, subjects were randomly divided into two groups (1:1) to receive a single dose of 0.125 g Cefaclor granule  or Cefaclor suspension  methods using Shimadzu LC-30AD . The linear quantification ranged from10 ng/mL to 8,000 ng/mL, and the lower limit of quantification was 10 ng/mL. The compounds were separated on a Acquity Uplc Hss T3 column  at 40°C. Chromatographic and mass spectrometric conditions were as follows. Chromatographic separation was carried out by gradient elution with mobile phase A  and mobile phase B . The flow rate was 0.6 mL/min and the column pressure was 6,000 psi. The injection volume of the sample was 10 µL. Plasm samples were quantified on an API 4000 mass spectrometer using ESI in positive ion mode and multiple reaction monitoring (MRM) using characteristic parent. The mass spectrometry conditions: ion source temperature was 550°C; curtain gas (CUR) was 10 psi; ion source gas 1 (GAS1) was 40 psi; ion source gas 2 (GAS2) was 40 psi; collision gas (CAD) was 6 uint; ion spray voltage (IS) was 5500 V; entrance potential (EP) was10 V; and collision cell exit potential (CXP1) was15 V. The dwelling time was set at 100 msec. The MRM transitions of cefaclor and Cefaclor-d5  were m/z 368.1 to 174.1 and m/z 373.1 to 179.1.Standard solutions and the pre-sample were prepared for the determination of plasma cefaclor concentrations. The standard working solution was used to spike blank plasma samples for the calibration standards at concentrations of 10, 20, 50, 200, 1000,4800, 7200, and 8,000 ng/mL or 10, 30, 300, 4000, 6,400, 32000 ng/mL quality control samples.max), area under the curve (AUC) from time zero to the last measurable concentration (AUC0-t) and AUC from time zero to observed infinity (AUC0-∞). Further endpoint parameters were the time of the maximum plasma concentration (Tmax), terminal half-life of the analyte in plasma (t1/2), terminal rate constant (λz).This clinical trial was a bioequivalence study with pharmacokinetic parameters as the endpoint. The primary endpoint PK parameters were the peak concentration . All AEs were recorded throughout the trial and followed until the AEs were eliminated or stabilized.Based on Guidance for industry bioavailability and bioequivalence studies of drug administration in an account of previous clinical trials, 18–24 subjects can meet the sample size requirements for most drugs, but for some drugs with high variability, the number of subjects should be increased appropriately . Previoumax, AUC0-t, AUC0-∞, Tmax, λZ, t1/2. The plasma drug concentration curve was log-transformed by Prism software 8.0 . All data statistical analysis were performed using SAS software 9.4 . Major pharmacokinetic parameters Cmax, AUC0-t and AUC0-∞ were transformed natural logarithm and tested for significance by analysis of variance (ANOVA). Statistical analysis using two-side test and 90% confidence interval was used to evaluate the bioequivalence of the test and reference drug. When the 90% CI of Cmax, AUC0-t and AUC0-∞ GMR for two drugs were in the bioequivalent interval of 80.00–125.00%, the two drugs were considered to be bioequivalent.Plasma concentration data were analyzed using Phoenix WinNonlin software 7.0  and PK parameters were calculated including Cmax. The blood samples were collected prior to the first dosing period. NO. K024 withdrew from the trial due to adverse events . The blood samples were collected within 2 h after Cefaclor suspension administration. Besides, the two subjects were still enrolled in the full analysis set (FAS) and safety analysis set (SS), the No. K014 was not enrolled in pharmacokinetics analysis set (PKPS) and bioequivalence analysis set (BES), the No. K024 was enrolled in PKPS but not in BES. In the postprandial state, 24 subjects were all included in the safety analysis set, and no adverse events led subject withdrawn from the trial. The detailed process for this trial is shown in In the trial screening for fasting and postprandial states, 62 and 64 volunteers participated, respectively. Ultimately, the trial regarding the fasting state and the postprandial state enrolled 24 subjects, respectively. Two subjects belonging to fasting state have withdrawn from the trial. NO. K014 discontinued the trial due to vomiting within the double times of medium Tmax values for Cefaclor granule and Cefaclor suspension were 6,432.73 ± 1645.99 ng/mL (25.59%) and 7006.52 ± 2032.34 ng/mL (29.01%), respectively; the AUC0-t values were 5421.56 ± 1013.57 h*ng/mL (18.70%) and 5548.92 ± 1116.70 h*ng/mL (20.12%), respectively; and the AUC0-∞ values were 5446.43 ± 1013.11 h*ng/mL (18.60%) and 5573.02 ± 1118.52 h*ng/mL (20.07%), respectively. The median Tmax of the two drugs were 0.3333 h and 0.3331 h, respectively. The mean ± SD of the t1/2 values for Cefaclor granule and Cefaclor suspension were 0.78 ± 0.10 h and 0.79 ± 0.08 h. The mean ± SD of the λz values for Cefaclor granule and Cefaclor suspension were 0.91 ± 0.12 and 0.89 ± 0.09. Other detailed PK parameters are listed in max for drug formulations (Cefaclor granule and Cefaclor suspension), administration periods and sequences (p > 0.05). Although AUC0-t and AUC0-∞ were not statistically different during the administration periods (p > 0.05), there were statistical differences between drug formulations and administration sequences (p < 0.05)  .max values for Cefaclor granule and Cefaclor suspension were 1982.50 ± 601.31 ng/mL (30.33%) and 2276.25 ± 831.71 ng/mL (36.54%), respectively; the AUC0-t values were 5253.26 ± 669.95 h*ng/mL (12.75%) and 5396.25 ± 661.68 h*ng/mL (12.26%), respectively; and the AUC0-∞ values were 5299.69 ± 691.82 h*ng/mL (13.05%) and 5442.40 ± 671.45 h*ng/mL (12.34%), respectively. The median Tmax of the two drugs were 1.9997 h and 2.168 h, respectively. The mean ± SD of the t1/2 values for Cefaclor granule and Cefaclor suspension were 0.98 ± 0.34 h and 1.05 ± 0.56 h. The mean ± SD of the λz values for Cefaclor granule and Cefaclor suspension were 0.78 ± 0.23 and 0.76 ± 0.24. Other detailed PK parameters are listed in max, AUC0-t and AUC0-∞ for administration periods and sequences (p > 0.05). However, both Cmax, AUC0-t and AUC0-∞ had statistical differences with drug formulations (p < 0.05)  .max and Tmax but not AUC. Meanwhile, there were no differences in the PK parameters values between Cefaclor granule and Cefaclor suspension (p > 0.05).The above PK parameters suggested that food intake might affect Cmax, AUC0-t, and AUC0-∞ for Cefaclor granule and Cefaclor suspension were 93.01% (93.78%), 97.92% (>99.99%) and 97.95% (>99.99%), respectively. The 90% confidence intervals (CIs) of GMRs for Cmax, AUC0-t, and AUC0-∞ were 85.96%–100.63%, 96.49%–99.38% and 96.52%–99.41%, respectively. The primary PK parameters were within the bioequivalence range of 80.00–125.00%. The above results indicated that Cefaclor granule was bioequivalent to Cefaclor suspension in the fasting state (The comparisons of the geometric mean ratios (GMRs) for the main pharmacokinetic parameters between Cefaclor granule and Cefaclor suspension in the fasting state are listed in ng state .max, AUC0-t, and AUC0-∞ for Cefaclor granule and Cefaclor suspension were 89.27% (68.92%), 97.31% (>99.99%) and 97.31% (>99.99%), respectively. The 90% CI of GMRs for Cmax, AUC0-t, and AUC0-∞ were 81.97%–97.22%, 95.98%–98.65% and 95.93%–98.71%, respectively. The primary PK parameters were within the bioequivalence range of 80.00–125.00%. These results showed that Cefaclor granule was bioequivalent to Cefaclor suspension in the postprandial state  in 11 subjects caused by Cefaclor granule and Cefaclor suspension, such as anemias, vomited, sweating, slow pulse, dizziness, infection urinary tract and positive urinary occult blood. Five subjects had 5 AE cases regarding Cefaclor granule, 6 subjects had 8 AE cases in Cefaclor suspension group. The detailed AEs are shown in Cefaclor is a highly absorbable oral cephalosporin antibiotic widely used in outpatient treatment for community-acquired pneumonia and other mild to moderate infections . This trmax and AUC of cefaclor showing similar changes after different types of breakfasts  . SubjectAn interesting phenomenon emerged in max and Tmax values calculated in the fasting state were approximately 3-fold and 6-fold higher than that in the postprandial state. The PK parameters Cmax values for the two studies after meal and under an empty stomach status were 6,432.73 ± 1645.99 ng/mL (Cefaclor granule) and 7006.52 ± 2032.34 ng/mL (Cefaclor suspension), as well as 1982.50 ± 601.31 ng/mL (Cefaclor granule) and 2276.25 ± 831.71 ng/mL (Cefaclor suspension), respectively. The results were similar to the data published  in healthy Chinese subjects under the fasting and postprandial states. By evaluating the primary PK parameters, Cmax, AUC0-t, and AUC0-∞ all met the bioequivalence criteria, supporting the bioequivalence of the two drugs. Cefaclor granule and Cefaclor suspension were safe in healthy Chinese subjects in the fasting and postprandial states.This trial is a single-center, randomized, open, single-dose, two-period crossover phase I clinical trial to compare the bioequivalence and safety of Cefaclor granule  and Cefaclor suspension (Ceclor1) The results of this trial indicated that Cefaclor granule and Cefaclor suspension were bioequivalent and displayed similar safety profiles.2) Food intake reduced the maximum plasma concentration and prolonged the peak time of the two oral cefaclor.


Zingiber zerumbet, also known as ‘Lempoyang’, possesses various phytomedicinal properties, such as anticancer, antimicrobial, anti-inflammatory, antiulcer, and antioxidant properties. Secondary metabolites possessing such properties i.e., zerumbone and α-humulene, are found dominantly in the plant rhizome. Synergistic effects of plant growth hormones and elicitors on in vitro α-humulene and zerumbone production, and biomass growth, in adventitious root culture (AdRC) of Z. zerumbet cultivated in a two-stage culture are reported. The culture was induced by supplementation of 1.0 mg/L NAA and 2.0 mg/L IBA (dark), and subsequently maintained in medium supplemented with 1 mg/L NAA and 3 mg/L BAP (16:08 light-dark cycle), yielded the production of zerumbone at 3440 ± 168 µg/g and α-humulene at 3759 ± 798 µg/g. Synergistic elicitation by 400 μM methyl jasmonate (MeJa) and 400 μM salicylic acid (SA) resulted in a 13-fold increase in zerumbone , while 400 μM MeJa and 600 μM SA produced a 4.3-fold increase in α-humulene  compared to control. Kaempferia galanga L. [Curcuma longa [Elettaria cardamomum Maton. [Zingiber officinale [Zingiber zerumbet [Zingiber zerumbet Smith, commonly known as Lempoyang, is a small, perennial, medicinal herb plant that has been established and cultivated mainly in Asia and subtropical regions [Z. zerumbet plant mass could be stored only for a short duration due to fungal diseases that deteriorate the rhizome [Z. zerumbet [The family Zingiberaceae includes various species, such as langa L. , Curcumama longa , Elettarm Maton. , Zingibeficinale , and Zinzerumbet , that po regions . Its rhi regions , antican regions , anti-al regions . Hence,  rhizome . Further rhizome . Despite rhizome . Besideszerumbet .Morinda coreia Buck. and Ham. [Hypericum perforatum [Eurycoma longifolia [Alternatively, adventitious root culture (AdRC) could serve as natural biofactories that grow vigorously in a formulated media with great potential to produce beneficial plant-derived compounds. The formation of the adventitious root is a complex process influenced by endogenous- and exogenous factors . One of and Ham. , Hypericrforatum , and Eurngifolia .Oldenlandiaumbellata when 50 mg/L of pectin was added [Oplopanax elatus when MeJA was added to the culture medium after 30 days of culture [In recent years, medium optimization, combined elicitor supplementation, and bioreactor application have been studied for high-yield secondary metabolites production. Elicitors are defined as any chemicals that trigger a mechanism in living organisms to produce secondary metabolites. Elicitors can be categorized into two types: biotic elicitors, including bacteria, viruses, and fungi; and abiotic elicitors, including metal ions and inorganic compounds , such as jasmonic acid (JA), methyl jasmonate (MeJA), salicylic acid (SA), acetyl salicylic acid (ASA), and nitric oxide (NO), which create a stress-like environment in a culture system of plants, triggering secondary metabolites production. For instance, the highest elicitation of anthraquinones (24.33 mg/g dry weight) was obtained from adventitious roots of as added . Additio culture .15H24) that contains an 11-membered ring, and consists of three isoprene units containing three non-conjugated C=C double bonds -farnesyl diphosphate. In the pathway, the terpene synthase catalyzes the synthesis of α-humulene. Thereafter, α-humulene is converted to 8-hydroxy-α-humulene by α-humulene-10-hydroxylase, followed by conversion to zerumbone by zerumbone synthase. Therefore, the investigation focused on the enhancement of root culture growth, and the production of α-humulene and zerumbone in AdRC of Z. zerumbet Smith through the synergistic application of plant growth regulators and elicitors.Zerumbone is a sesquiterpene that can be identified by three double bonds , and a dle bonds . The IUPZingiber zerumbet Smith (herbarium number 041148) were collected from a nursery at Pusat Asasi Sains, Universiti Malaya, in April 2018. They were cleaned thoroughly by repeated washing and maintained in the laboratory for sprouting.Mature rhizomes of v/v) Clorox (5.25% w/w sodium hypochlorite) solution with a few drops of Tween 20. After 30 min, the outer layer of the shoot buds was removed and transferred aseptically into 30% (v/v) Clorox for 10 min. Subsequently, the shoot buds were rinsed with sterile distilled water, and air-dried in a laminar air-flow chamber.Young buds 1.5–2.0 cm in length were used as explants, and surface sterilized with 70% (w/v) sucrose. The media were solidified with 2 g/L gelrite. Explants grown in medium without hormones served as a control. The pH of the medium was adjusted to 5.7 prior to autoclaving. The cultures were maintained at 25 ± 1 °C under total dark conditions. For each treatment, the percentage of root response, root length, and the number of roots per explant were measured. All experiments were carried out in triplicate cultures with five explants in each replicate.The sterilized shoot buds were excised horizontally to 1.0 × 1.0 cm in length, and inoculated onto a petri dish containing Murashige and Skoog (1962) (MS) medium supplemented with a combination of naphthalene acetic acid (NAA) and indole-3-butyric acid (IBA) at a concentration of 0.5, 1, or 2 mg/L and 3%  and 8 h dark . For each treatment, the fresh weight (FW) and dry weight (DW) were measured at six-day intervals over 30 days of culture. The growth parameter, namely the specific growth rate (μ), was determined for the AdRC by Equation (1). All experiments were carried out in triplicate. Roots were maintained in the optimized media from this experiment, and subculture every 30 days for further elicitation experiments.After one month of induction in an optimized media (1.0 mg/L NAA and 2.0 mg/L IBA), the induced roots were transferred into shake flasks containing different media formulations. The roots were cultured on MS media with different combinations of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) at concentrations of 1, 3, 5, and 7 mg/L, and supplemented with 3% μ = Specific growth rate (day−1)x0 = Initial fresh weightx = Final fresh weight after incubationt = period of incubation (days)ΔThe root cultures were maintained at 25 ± 1 °C in orbital shaker at 70 rpm. Elicitation by different combinations of methyl jasmonate (MeJA) and salicylic acid (SA) at the followings were carried out: (T1) 400 µM MeJa + 400 µM SA; (T2) 400 µM MeJa + 600 µM SA; (T3) 400 µM MeJa + 800 µM SA; (T4) 400 µM MeJa + 1200 µM SA; (T5) 600 µM MeJa + 400 µM SA; (T6) 600 µM MeJa + 600 µM SA; (T7) 600 µM MeJa + 800 µM SA; (T8) 600 µM MeJa + 1200 µM SA; (T9) 800 µM MeJa + 400 µM SA; (T10) 800 µM MeJa + 600 µM SA; (T11) 800 µM MeJa + 800 µM SA; (T12) 800 µM MeJa and 1200 µM SA; (T13) 1200 µM MeJa + 400 µM SA; (T14) 1200 µM MeJa + 600 µM SA; (T15) 1200 µM MeJa + 800 µM SA; (T16) 1200 µM MeJa + 1200 µM SA.The combination treatments were introduced on day 15 for a 26-day cultivation period. AdRC without elicitation was used as a control, and was harvested after 26 days. A stock solution of MeJA and SA was prepared separately by the dissolution of each substance in distilled water and filter sterilized using 0.22 μm filters prior to its addition to the root suspension cultures. Meanwhile, for the untreated sample, an equivalent volume of sterilized distilled water was added to the cultures as a blank treatment control for the different elicitor concentrations studied. All experiments were performed in triplicate, with each treatment repeated three times.Z. zerumbet.After each treatment, roots were harvested and washed with distilled water followed by drying at 60 °C  for 24 hHPLC analysis was performed using a Shimadzu HPLC system, which consisted of SIL-20A HT autosampler, LC-20 AT multisolvent delivery system, DGU-20A degasser, CTO-20 AC cooler, and SPD-M20A UV/Visible detector, and Chromolith RP-18 endcapped column (100 × 4.6 mm) was associated with the LC solution software. HPLC-grade organic solvents, i.e., acetonitrile, methanol, and phosphoric acid (BDH), were used. The mobile phase consisted of a mixture of ultrapure water with 0.1% phosphoric acid (A) and acetonitrile (B). Analyses of zerumbone and α-humulene were carried out by the following elution gradient: 0.01 min, 50% A, 50% B; 2.00 min, 15% A, 85% B; 7.00 min, 15% A, 85% B; 7.50 min, 50% A, 50% B; 11.00 min, 60% A, 40% B. The total running time was 20 min at a flow rate of 1.0 mL/min. The temperature of the column was set at 35 °C at UV detection wavelength 243 nm. All samples were filtered through 0.25 µm membrane filters prior to injection into the HPLC. The zerumbone and α-humulene compounds were identified by matching the retention times and spectral characteristics of a sample to a commercially available standard . Quantitative analysis was performed using the peak area calibrated to external standard concentration. Three replicates were prepared for each standard concentration. The injection was also performed in triplicate.p < 0.05.Data were analyzed by one-way ANOVA followed by Tukey’s HSD test at a significance level of Gynura procumbents, which appeared after seven days of inoculation [AdRC is a viable route for secondary metabolite production due to its high proliferation rate and stable production of valuable secondary metabolites . In thisculation . Even thOrthosiphon stamineus [Cichorium endivia in total darkness was also reported [Melissa officinalis [A significant increase in adventitious root growth was observed with an increasing concentration of IBA up to 5 mg/L in both light regimes, contrary to the treatment combination with BAP . The obstamineus , in whicreported . It alsoicinalis .−1). However, treatments F and K showed no significant difference when compared with treatment C in terms of FW, DW, and specific growth rate  concentrations  produced a significantly lower AdRC. In addition, the lowest specific growth rate and biomass of AdRC  were obsavonoids .Plumbago rosea when it was cultivated in media containing auxin and cytokinin [Catharanthus roseus root culture [Meanwhile, treatment F produced the highest levels of zerumbone (3440 ± 168 µg/g) and α-humulene (3759 ± 798 µg/g) with 1.90 ± 0.1 g of DW . Similarytokinin . In addi culture . Thus, tVicia faba, where concentrations above 10 µM SA inhibited root growth in some species, while treatment with 5000 µM SA resulted in a 25% reduction [In this study, the root yield decreased with increasing concentrations of MeJa and SA, as shown in eduction .Talinum paniculatum (Javanese ginseng) [However, the production of these compounds showed significant increase with treatments ranging from 400 µM to 800 µM of the elicitors’ combination compared to the control. Similarly, the combined treatment of MeJA and SA was found to enhance saponin production, which increased by 1.5- and 1.3-fold in the adventitious root culture of ginseng) .Z. zerumbet  and elicitors  at low concentrations enhanced zerumbone and α-humulene production relative to untreated control and in vivo rhizome of


Eu1–Eu3, have been synthesized and characterized with CHN analysis, mass-spectrometry and 1H NMR spectroscopy. The complexes display strong emission in dichloromethane solution upon excitation at 405 and 800 nm with a quantum yield from 18.3 to 31.6%, excited-state lifetimes in the range of 243–1016 ms at 20 °C, and lifetime temperature sensitivity of 0.9%/K (Eu1), 1.9%/K (Eu2), and 1.7%/K (Eu3). The chromophores were embedded into biocompatible latex nanoparticles (NPs_Eu1–NPs_Eu3) that prevented emission quenching and kept the photophysical characteristics of emitters unchanged with the highest temperature sensitivity of 1.3%/K (NPs_Eu2). For this probe cytotoxicity, internalization dynamics and localization in CHO-K1 cells were studied together with lifetime vs. temperature calibration in aqueous solution, phosphate buffer, and in a mixture of growth media and fetal bovine serum. The obtained data were then averaged to give the calibration curve, which was further used for temperature estimation in biological samples. The probe was stable in physiological media and displayed good reproducibility in cycling experiments between 20 and 40 °C. PLIM experiments with thermostated CHO-K1 cells incubated with NPs_Eu2 indicated that the probe could be used for temperature estimation in cells including the assessment of temperature variations upon chemical shock .Three novel luminescent Eu(III) complexes,  The thermosensors based on lanthanide complexes dihydrochloride , cetyltrimethylammonium bromide (CTAB), and acetone were used as received. Water was purified using a “Simplicity” (“Merck Millipore”) water purification system (type 1 water). 1,10-Phenanthroline-5,6-dione phenanthroline.https://doi.org/10.1002/ejic.202100189), 4-nitrobenzaldehyde , 1,10-phenanthroline-5,6-dione , aniline , and ammonium acetate  were added to 7 mL of acetic acid and refluxed overnight. The precipitated powder was centrifuged and washed with diethyl ether to give a yellow powder, which was then recrystallized through gas-phase diffusion of diethyl ether into dichloromethane solution of the product; yield 62%. 1H NMR  δ = 9.24 , 9.14 , 9.10 , 8.19 , 7.84–7.71 , 7.60 , 7.47 , 7.34  ppm. ES MS (m/z): [L + H]+ 417.1213 . Anal. calc. for C25H15N5O2·CHCl3 (%): C, 58.18; H, 3.00; N, 13.05. Found: C, 58.58; H, 3.03; N, 13.29.According to the previously published procedure , ligand L1 , tetrabutylammonium hydroxide , and Eu(OAc)3·3H2O  were dissolved in acetone (16 mL). The solution was stirred at 50 °C overnight. The resulting mixture was extracted with dichloromethane (3 × 50 mL). Combined organic phases were dried over calcium(II) chloride and evaporated under a vacuum to give a yellow oily product; yield 70%. 1H NMR  δ = 7.28 , 6.91 , 6.46 , 4.99 , 3.76–3.66 , 2.91 , 1.96–1.83 , 1.56 , 1.05  ppm. ES MS (m/z): [M-CO2NCO2]− 769.0831 . CHN analysis was obtained for potassium salt, which gives crystallizable powder suitable for analysis. Anal. calc. for C51H36EuK3N6O12·(H2O)6 (%): C, 47.04; H, 3.72; N, 6.45. Found: C, 47.37; H, 4.02; N 6.18.Synthesis of Eu2 Complex. The complex was synthesized according to a modified literature methodology + 1199.0510 . Anal. calc. for C43H32EuF9N8O6S3·CHCl3·C3H6O (%): C, 43.11; H, 2.80; N 9.07; S 7.79. Found: C, 43.10; H, 3.04; N 8.41; S 7.24.hodology . In a roSynthesis of Eu3 Complex. In a round-bottom culture tube, Eu(TTA)3·3H2O  and L3  were dissolved into acetone (4 mL); the mixture was stirred overnight. The precipitated powder was centrifuged and washed three times with diethyl ether to eliminate the ligand, which was then recrystallized using gas-phase diffusion of diethyl ether into dichloromethane solution of the product. Light-yellow powder, quantitative yield: 57%. 1H NMR  δ = 14.37 , 12.90 , 9.36 , 8.92 , 8.85 , 8.67 , 8.62 , 8.36 , 7.27 , 6.45 , 6.00 , 3.26  ppm. ES MS (m/z): [M + Na]+ 1256.0008 . Anal. calc. for C49H27EuF9N5O8S3·CH2Cl2 (%): C, 46.01; H, 2.11; N, 5.42. Found: C, 46.03; H, 2.53; N, 5.05.Synthesis of TPP-NHS ester. TPP-NHS ester was synthesized according to a slightly modified literature procedure (PF6)2 in water (rΦ = 0.042) as a standard [SΦ—the quantum yield of the sample, rΦ—the quantum yield of the reference, η—the refractive index of the solvent, sA, rA—the absorbance of the sample and the reference at the wavelength of excitation of emission, respectively, and sI, rI—the integrated area of emission band of the sample and the reference, respectively.standard . The refstandard :(1)ΦS=ΦrσTPE is the two-photon luminescence cross-section, σTPA is the two-photon absorption cross-section (cm4·s), Φ is the luminescence quantum yield, W is the is the number of emitted photons, C is the molar concentration of the solution, n is the refractive index of the solvent, and t is the exposure time in seconds. As the cross-section for two-photon absorption and luminescence, the Goeppert–Mayer value, 10−50 cm4·s = 1 GM, was used. Fluorescein in aqueous solution at pH = 11 was used as the standard, its quantum yield was considered to be 91% [σTPE = 43 GM [−4 M. The concentration of latex particles in the dispersions under study was ca. 10−4% in the units of dry residue mass fraction.The value of the two-photon absorption cross-section was calculated by finding the value of the two-photon luminescence cross-section :(2)σTPESo be 91% , σTPE =  = 43 GM . The conQuantum yield and lifetime measurements for latex nanoparticles. The quantum yields of latex nanoparticles in a water dispersion were measured using a Quanta-ϕ integrating sphere with a modular spectrofluorimeter Fluorolog-3 . A pulse laser TECH-263 Basic , an Ocean Optics monochromator , a FASTComTec (MCS6A1T4) multiple-event time digitizer, and a Hamamatsu (H10682-01) photon-counting head were used for lifetime measurements. Cyclic experiments for water dispersion of latex nanoparticles (~0.02 weight% of the dried residue) were performed in the temperature range of 20−40 °C using a temperature-controlled sample compartment qpod 2e .The lifetime vs. temperature calibration curves were obtained using the following protocol: (1) a sample of the probe aqueous dispersion was thermostated at 20 °C for 10 min (the optimized time that it made possible to stabilize the lifetime readings); (2) for the lifetime measurements, the resulting magnitude was the average of 5 times repetition; (3) the temperature was then increased by 5 °C, and the sample was thermostated for 10 min to make 5 independent measurements as above; (4) measurements were repeated in 5-degree increments until the temperature reached 40 °C. TS), relative sensitivity (rS), and reproducibility (R) are presented in The protocol for cyclic studies was similar to that used for building up the calibrations. The sensors’ characteristics were calculated in accordance with the literature guidelines . The calExperiments with CHO-K1 cell line. The Chinese hamster ovary CHO-K1 cells were cultured in DMEM/F12  medium supplemented with 10% FBS , 2 mM glutamine , and penicillin/streptomycin at a concentration of 100 U/mL . The cells were maintained in a humidified incubator at 37 °C with 5% CO2 and passaged using trypsin-EDTA . For living-cell confocal microscopy, the cells (1 × 105 CHO-K1 cells in 1 mL DMEM) were seeded in glass-bottomed 35 mm dishes  and incubated for 24 h. Latex nanoparticles NPs_Eu2 dispersed in water were dissolved in the growing media and added to the cells at a final concentration of 0.0195 wt.%. The dynamic of internalization was studied at different time points starting from 10 min. Before PLIM cell imaging, the cells were incubated with NPs_Eu2 for 24 h in the glass-bottom dishes and then the medium was replaced with a fresh one.MTT assay. The Chinese hamster ovary CHO-K1 cells (1 × 104 cells in 100 µL of culture medium/well) were seeded in 96-well plates  and incubated overnight. The particles were added to the cells at concentrations of 0.0012–0.039 wt.% for 24 h. The solid content was calculated gravimetrically per 100 mL of water. Then, the cells were treated with the MTT reagent 3-2,5-diphenyl-2H-tetrasole bromide  at a concentration of 0.5 mg/mL. Following further incubation at 37 °C under 5% of CO2 for 4 h, the formazan crystals were dissolved in DMSO, and the absorbance at 570 nm was measured using a SPECTROstar Nano microplate reader . The percentage of viable cells relative to the control was determined for each well as a ratio of the average absorbance value of the wells containing probes to that of the control wells. For each concentration of the particles, the experiment was repeated 12 times. Vital staining of organelles. LysoTracker Deep Red  was used for the vital staining of acidified compartments, lysosomes, and late endosomes. BioTracker 405 Blue Mitochondria Dye  was used for the vital staining of mitochondria. The CHO-K1 cells were incubated with NPs_Eu2 and NPs_Eu2_TPP . Then, the cells were rinsed with fresh media 3 × 1 mL, and incubated with a new portion of growing media for 15 min. LysoTracker Deep Red was added to the cells at a concentration of 50 nM and incubated 30 min prior to confocal imaging. BioTracker 405 was added to the cells at a concentration of 50 nM and incubated 15 min prior to confocal imaging.Confocal microscopy and PLIM. Living CHO-K1 cells were imaged by using a confocal-inverted Nikon Eclipse Ti2 microscope  with 60× oil immersion objective. The emission of europium was recorded in the range of 663–738 nm using single-photon excitation at 405 nm. In addition to luminescent microphotographs, differential interference contrast (DIC) images were also obtained. The images were processed and analyzed using ImageJ software . The emission of the particles was excited with a 405 nm laser and recorded in the 570–620 nm (red channel) range. The fluorescence of LysoTracker Deep Red was excited at 638 nm and recorded at 663–738 nm. The fluorescence of BioTracker 405 Blue Mitochondria Dye was excited at 405 nm and recorded at 425–475 nm.i2 confocal device. A picosecond laser (405 nm) was used as an excitation source. The phosphorescence of the probe was recorded using a 575 longpass filter and 630/75 nm bandpass filter, and a pinhole of 0.5. PLIM images were obtained using the following settings: frame time of 42.87 s, pixel dwell time of 10.46 ms, points number of 1024, time per point of 10.00 µs, time range of PLIM recording of 10.24 ms, total acquisition time of 87–131 s, and image size of 64 × 64 pixels. Oil immersion 60× objective with zoom 7.11 provided a scan area of ca. 0.04 mm × 0.04 mm. Phosphorescence lifetime images were processed using SPCImage 8.1 software . All PLIM measurements were performed in a humidified Stage Top Incubator Tokai HIT  at 25, 30, or 40 °C and 5% CO2. The phosphorescence decay curves were fitted in monoexponential mode. The average number of photons per curve was not less than 10,000 at binning 5–6.Phosphorescence lifetime imaging microscopy (PLIM) of CHO-K1 cells was carried out using the time-correlated single-photon counting (TCSPC). A DCS-120 module  integrated with the Nikon Eclipse TNPs_Eu1, NPs_Eu2, and NPs_Eu3) using nanoemulsion polymerization. Additionally, the surface of the NPs was modified with amino functions that provide a potential opportunity for their vectorization aimed at probe-targeted delivery. The photophysical properties of the europium chromophores remain nearly unchanged in the NPs, including lifetime sensitivity to temperature, with the highest one of 1.3%/K found for NPs_Eu2. For this probe, the investigation of cytotoxicity, internalization dynamics, and localization in CHO-K1 cells showed that it is possible to apply it safely in cell experiments. We also examined the NPs_Eu2 lifetime dependence on temperature under different conditions: in aqueous solution, in phosphate buffer, and in a mixture of the growth media (DMEM) and fetal bovine serum (FBS). The obtained data allow building up the calibration curve for temperature estimation in biological samples. PLIM experiments on the CHO-K1 cell line showed that this probe and obtained calibration curve can be used for the estimation of temperature in cells as well as for detection of the cell response to chemical shock, expressed as an increase in intracellular temperature. Unfortunately, vectorization of the probe surface with a phosphonium cation did not give its preferential localization in mitochondria that implies further studies to search for effective ways to deliver the probe to target cell compartments.The obtained novel europium complexes display desirable photophysical properties, namely, excitation with the bio-friendly visible (405 nm) and NIR  radiation and temperature dependence of emission characteristics (intensity and lifetime) in a physiologically relevant interval (20–40 °C). To avoid europium emission quenching in aqueous media, the complexes were successfully embedded into biocompatible latex nanoparticles (


Passiflora edulis fruits (PESE), a major by-product of the juice industry, and piceatannol (PIC), one of the main phytochemicals of PESE. PESE, PIC, and acarbose (ACB) exhibited IC50 for alpha-amylase, 32.1 ± 2.7, 85.4 ± 0.7, and 0.4 ± 0.1 µg/mL, respectively, and IC50 for alpha-glucosidase, 76.2 ± 1.9, 20.4 ± 7.6, and 252 ± 4.5 µg/mL, respectively. The IC50 of PESE, PIC, and sitagliptin (STG) for dipeptidyl-peptidase-4 (DPP-4) was 71.1 ± 2.6, 1137 ± 120, and 0.005 ± 0.001 µg/mL, respectively. PESE and PIC inhibited the formation of advanced glycation end-products (AGE) with IC50 of 366 ± 1.9 and 360 ± 9.1 µg/mL for the initial stage and 51.5 ± 1.4 and 67.4 ± 4.6 µg/mL for the intermediate stage of glycation, respectively. Additionally, PESE and PIC inhibited the formation of β-amyloid fibrils in vitro up to 100%. IC50 values for 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) scavenging activity of PESE and PIC were 20.4 ± 2.1, and 6.3 ± 1.3 µg/mL, respectively. IC50 values for scavenging hypochlorous acid (HOCl) were similar in PESE, PIC, and quercetin (QCT) with values of 1.7 ± 0.3, 1.2 ± 0.5, and 1.9 ± 0.3 µg/mL, respectively. PESE had no cytotoxicity to the human normal bronchial epithelial (BEAS-2B), and alpha mouse liver (AML-12) cells up to 100 and 50 µg/mL, respectively. However, 10 µg/mL of the extract was cytotoxic to non-malignant breast epithelial cells (MCF-10A). PESE and PIC were found to be capable of protecting cultured human cells from the oxidative stress caused by the carcinogen NNKOAc at 100 µM. The in vitro evidence of the inhibition of alpha-amylase, alpha-glucosidase, and DPP-4 enzymes as well as antioxidant and antiglycation activities, warrants further investigation of the antidiabetic potential of P. edulis seeds and PIC.The objective of this work was to investigate the antidiabetic, antiglycation, and antioxidant potentials of ethanolic extract of seeds of Brazilian  Passiflora (Passifloraceae family) genus has more than 500 species; however, Passiflora edulis Sims f. flavicarpa O. Degenerer (yellow or sour form passion fruit) and P. alata (sweet form) are the two main economically important species  × 100, where A0 is the absorbance of the positive control subtracted from the blank, Abs 1 is the absorbance in the presence of the extract subtracted from sample control. The IC50  was calculated graphically, using an analytical curve by plotting the concentration versus the inhibition percentage (I%).Plant extract screening for alpha-amylase inhibition was conducted using the previously described method , with so® 200 PRO, TECAN, Männedorf, Switzerland) to quantify the amount of p-nitrophenol (PNP) released. ACB, an antidiabetic drug used as an alpha-glucosidase inhibitor for T2DM, was used for comparison purposes. The positive control was the mixture of enzyme and substrate without inhibitors. The sample control consisted of the mixture of sample and buffer. The buffer was used as a blank. The percentage of the alpha-glucosidase inhibition and IC50 was calculated as described in The alpha-glucosidase inhibition assay was performed using a previously described method  with min® 200 PRO, TECAN, Männedorf, Switzerland). STG, a standard DPP-4 inhibitor, was used to compare the effectiveness of PESE and PIC. The percentage of the DPP-4 inhibition and IC50 was calculated as described in The DPP-4 inhibition assay was performed according to an established method . Briefly3 (0.02% w/v). The plant extract was prepared in a buffer containing 60% ethanol. The mixture was incubated in the dark at 37 °C for 7 days with constant stirring. After the incubation, 200 µL of the reaction mixture was transferred to a 96-well black plate, and fluorescent AGEs were measured using a microplate reader  at λex = 360 and λem = 440 nm. X. Aminoguanidine (AMG), a known AGE formation inhibitor, was used as a standard. The reaction mixture without inhibitors was used as a positive control. Color interference from the sample was eliminated by a sample control (buffer and sample). The fluorescence readings for the experimental treatment were blanked against extract blanks to eliminate the baseline fluorescence of the sample. The percentage of the AGE inhibition and IC50 was calculated as described in The formation of AGEs was measured after incubation of a system containing protein and carbohydrates. This assay was based on previous methods . The rea3 (0.02% w/v). Then, 300 μL of MGO was incubated with samples at different concentrations (dissolved in a buffer containing 60% ethanol) for an hour at 37 °C, in the dark, with constant stirring. After BSA (3 mg/mL) was added to the reaction mixture and incubation time was extended to 48 h at 37 °C in darkness. The rest of the procedure was the same as that for the BSA-glucose/fructose model that is described above. The percentage of the AGE inhibition and IC50 was calculated as described in The BSA-MGO assay was based on those described before , with so® 200 PRO, TECAN, Männedorf, Switzerland). Fresh BSA solution (1 mg/mL) and PBS were used as controls.The fibrillar state of modified BSA was determined with ThT as previously described . ThT is v/v, solvent A) and methanol (solvent B) were used as mobile phases, the flow rate of 1.0 mL/min, and the injection volume was 20 μL. The linear gradient for elution was: starting at 5% of solvent B, 0–3 min, 5 to 50% B; 3–16 min, isocratic in 50% B; 16–17 min, 50–90% B; 17–19 min, isocratic in 90% B and 19–19.5 min, 90–5% B. The derivatization product, 2-methylquinoxyline (2-MQ), was detected at 315 nm, with a retention time of 10 min. The percentage of trapping efficiency of MGO was calculated using the following equation: % trapping MGO = [100 − (peak area after adding sample/peak area without adding sample) × 100].MGO scavenging was tested using a described procedure , with slw/v) were added to 120 μL of diluted samples. After incubation in a water bath at 40 °C in the dark for 20 min, the absorbance of the resulting mixture was measured at 760 nm using a UV–Vis spectrophotometer (Agilent 8453). The results were expressed as milligrams of gallic acid equivalents (mg GAE) per gram of dry extract.The TPC was estimated using the Folin–Ciocalteu (FC) method, as described , with so• method [• solution (40 μg/mL in methanol). After incubation in the dark for 30 min, the absorbance was read at 516 nm, using a UV–Vis spectrophotometer . Results were expressed as the half-maximal inhibitory concentration (IC50) in μg/mL.Antioxidant activity of PESE was determined using the DPPH• method . Briefly2SO4, was diluted to 30 µM, using 100 mM phosphate buffer pH 7.4. For the analysis, in a 96-well plate, the following reagents were added at the indicated final concentrations: 150 μL of buffer solution , 50 μL of PESE , 50 µL of DHR 123 (5 µM) and 50 µL of HOCl (5 µM). QCT was used as means of comparison. Fluorescence assays were performed in a microplate reader , at 37 °C, at wavelengths of 505 ± 10 nm and 530 ± 10 nm, for excitation and emission, respectively. The results were expressed as IC50 (μg/mL) of extract solution.HOCl scavenging activity of PESE and PIC was determined . Briefly® 200 PRO, TECAN, Männedorf, Switzerland), and the absorbance was measured at 560 nm. The results were expressed as IC50 of extract solution in μg/mL.The potential of the samples to scavenger superoxide anion radicals was determined , with mi® 200 PRO, TECAN, Männedorf, Switzerland).The generation of intracellular ROS in BEAS-2B, AML-12, and MCF-10A cells after treatments was measured as described previously . Briefly2. Culture flasks (polystyrene T75) were pre-coated with a mixture of 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I, and 0.01 mg/mL bovine serum albumin were dissolved in LHC-9 medium overnight. MCF10-A cells were cultured in mammary epithelial basal medium (MEBM) culture medium supplemented with the Mammary Epithelial Cell Growth Medium Kit (Lonza), and AML-12 cells in DMEM-F12 medium (ATCC) were supplemented with insulin-transferrin-selenium , 10% fetal bovine serum, and 1% antibiotic  at 37 °C in a 5% CO2 incubator. Cells were grown to around 70% confluence on the culture flask, and passages (<10) were employed for all experimental conditions.BEAS-2B, MCF10-A, and AML-12, cells were purchased from the American Tissue Type Culture Collection  and cultured with a specific medium. BEAS-2B cells were cultured with LHC-9 media at 37 °C in an incubator with 5% CO4 cells were plated on a 96-well plate with a growth medium of 100 μL/well. After 24 h, cells were treated with PESE or PIC for an additional 24 h. Fifteen microliters of MTS reagent (with PMS) was then added to each well and incubated for 3 h in the dark. Absorbance was recorded at 490 nm using a microplate reader . For each experiment, cells with DMSO media served as control, and cells with only culture medium and MTS reagent served as the blank.MTS Cell Titer 96™ aqueous cell proliferation assay was used to determine cell viability . BEAS-2Bn = 3) with three independent studies and using Graph-Pad Prism software . Data were presented as the mean ± standard deviation (SD), and analyses of variance, one-way ANOVA, followed by Tukey test and p ≤ 0.05 were considered significant between experimental groups.All analyses were performed in triplicate (Passion fruit seeds, by-products of the juice industry, have the potential for use as a low-cost antioxidant and bioactive source for developing nutraceuticals and dietary supplements, for managing blood glucose levels, and consequently, reducing the progression of complications of T2DM. In this work, PESE showed a potent antidiabetic, antiglycant, and antioxidant potential without being toxic to BEAS-2B cells at bioactive concentrations. Furthermore, it was observed that PESE could protect some of the tested cultured cell lines from the oxidative stress caused by the presence of the chemical carcinogen, NNKOAc. Although it is still early to suggest the use of ethanolic extract from passion fruit seeds in the treatment of T2DM, this work demonstrates its potential dietary use to manage T2DM and glycation-associated complications. Further investigations on the efficacy and safety of PESE, using pre-clinical experimental animal models are encouraged and are underway.


Before training, ambulatory systolic BP (ASBP) and diastolic ABP (ADBP) decreased by 3.2 ± 2.1 mmHg and 2.5 ± 1.5 mmHg, respectively, from baseline after the GEST versus CONTROL (p < 0.05). After training, ASBP tended to decrease by 3.5 ± 2.2 mmHg (p = 0.055) and ADBP decreased by 1.7 ± 2.5 mmHg (p = 0.001) from baseline after the GEST versus CONTROL. Before training, HRV high frequency (HFms2) (β = −0.441), age (β = 0.568), and resting SBP (β = 0.504) accounted for 66.8% of the ASBP response (p = 0.001), whereas the low frequency (LF)/HF ratio (β = 0.516) and resting DBP (β = 0.277) accounted for 35.7% of the ADBP response (p = 0.037). After training, the standard deviation of NN intervals (SDNN) (β = −0.556), age (β = 0.506), and resting SBP (β = 0.259) accounted for 60.7% of the ASBP response (p = 0.004), whereas SDNN (β = −0.236) and resting DBP (β = 0.785) accounted for 58.5% of the ADBP response (p = 0.001). Our preliminary findings show that adults with hypertension and parasympathetic suppression  may elicit PEH to the greatest degree independent of training status versus adults with parasympathetic predominance, suggesting that resting HRV may be an important determinant of PEH.Because data are scarce, we examined the relationship between postexercise hypotension (PEH) and heart rate variability (HRV) before and after aerobic exercise training among adults with hypertension. Participants completed a 12 w aerobic training program. Before and after training, they performed a peak graded exercise stress test (GEST) and nonexercise control (CONTROL) and were left attached to an ambulatory BP monitor. Prior to CONTROL, HRV was measured supine for 5 min using a 12-lead electrocardiogram (ECG). The participants ( Hypertension is the most prevalent cardiovascular disease (CVD) risk factor, affecting ~105 million Americans ~47%) . Partici7% . PartNo consensus exists on the precise mechanisms by which PEH occurs. However, it has been hypothesized that most agree that PEH occurs owing to the lasting reductions in total peripheral resistance without adjacent increases in cardiac output among otherwise healthy individuals with hypertension ,8,14. Su. Enhanced endothelial function and vascular distensibility  following exercise training may also account for decreases in sympathetic activity and BP  . To assen = 18) were overweight to obese, mostly white, middle-aged men and women with normal HR and stage 1 hypertension. Half of the sample was taking antihypertensive medications that included angiotensin II receptor antagonists, angiotensin-converting-enzyme inhibitors, and diuretics (2peak was “fair” according to the ACSM reference standards for men and women of this age [2peak (2.7 ± 6.7 mL/kg/min) was not significant following 12 w of aerobic exercise training (p = 0.111). However, the ACSM age-and-sex-specific normative VO2peak percentiles increased from “fair” to “good” following training [p = 0.034) decreased following training. The markers of body composition, BMI  and WC , were not different before versus after training.All data used in the results are shared in the iuretics . Pre-exethis age  Table 1n = 18 wemax; 61.2 ± 8.2% HRR; 64.3 ± 7.4% of VO2peak; and 12.1 ± 1.1 of RPE) treadmill-based aerobic exercise 2.6 ± 0.4 d/w for 12 w [Training sessions were performed in consistent environmental conditions with an average room temperature and humidity of 71.4 ± 2.6 °F and 19.6 ± 14.4% . The parfor 12 w . There wfor 12 w .p = 0.016) from baseline over awake, 1.4 ± 3.4 mmHg (p = 0.043) over sleep, and 2.3 ± 2.4 mmHg (p = 0.010) over 19 h (p = 0.015) from baseline over awake and 2.7 ± 1.5 mmHg (p = 0.029) over 19 h (p = 0.206).After versus before the GEST versus CONTROL, ambulatory systolic blood pressure (ASBP) decreased by 3.2 ± 2.1 mmHg (ver 19 h . After vver 19 h . Howeverp = 0.028) from baseline over 19 h and tended to decrease by 3.5 ± 2.2 mmHg (p = 0.055) over awake and 7.4 ± 3.0 mmHg (p = 0.111) over sleep (p < 0.001) from baseline over awake, 3.4 ± 2.7 mmHg (p = 0.015) over sleep, and 2.5 ± 2.4 mmHg (p = 0.001) over 19 h  (ps < 0.05), while the decrease in ADBP sleep was not different (p = 0.081)  . ADBP aw= 0.081) .p = 0.358), sleep , and 19 h  following exercise training were not different. Similarly, the changes in ADBP awake  and 19 h  following exercise training were not different. However, ASBP sleep  increased after exercise training.The changes in ASBP awake  and 5.6 ± 9.4 mmHg (p = 0.023), respectively, after 12 w of exercise training.Resting SBP and DBP measured in the laboratory by auscultation decreased by 7.9 ± 10.2 mmHg (p < 0.001), RMSSD , and pNN50 ; the frequency domain measures of absolute LF , HF , and the LF/HF ratio ; and the nonlinear measure of SampEn  indicated a shift towards parasympathetic dominance after versus before aerobic exercise training in the laboratory  decreased and age (β = 0.568) and resting SBP (β = 0.504) increased, ASBP over awake increased following the GEST versus CONTROL. The regression model combining HFms2, age, and resting SBP explained 66.8% of the variance in the ASBP response over the same time period (p = 0.001). As the resting SBP (β = 0.596) increased, ASBP over sleep increased following the GEST versus CONTROL. The regression model containing LF/HF (β = −0.178) and the resting SBP and BMI (β = 0.371) accounted for 41.2% of the variance in the ASBP response over the same time period, but this model did not achieve statistical significance (p = 0.053). As resting SBP (β = 0.578) and age (β = 0.437) increased, ASBP over 19 h increased following the GEST versus CONTROL. The regression model including SampEn  and resting SBP and age accounted for 54.1% of the variation in the ASBP response over the same time period (p = 0.010) (p = ns) explained 35.7% of the variance in the ADBP response over awake (p = 0.037). As LF/HF (β = 0.473) increased, ADBP over 19 h increased following the GEST versus CONTROL, accounting for 22.3% of the variation in the ADBP response over the same time period (p = 0.048). There were no statistically significant models that emerged explaining a clinically important amount of the ADBP response over sleep  . As LF/Her sleep .p = ns) explained 60.7% of the variance in the ASBP response over the same time period (p = 0.004). There were no statistically significant models that emerged explaining a clinically important amount of the ASBP response over sleep and 19 h (p = ns) and resting DBP explained 58.5% of the variance in the ADBP response over the same time period (p = 0.001). As the resting DBP (β = 0.689) increased, ADBP over 19 h increased following the GEST versus CONTROL. The regression model combining LF/HF  and resting DBP accounted for 48.2% of the variance in the ADBP response over the same time period (p = 0.007). There were no statistically significant models that emerged explaining any meaningful amount of the ADBP response over sleep  decreased and age (β = 0.506) increased, ASBP over awake increased following the GEST versus CONTROL. The regression model combining SDNN, age, and resting SBP , ∆LFnu, and resting SBP  explained 43.2% of the ∆SBP (p = 0.042) (p = ns), LF/HF, and resting DBP  accounted for 44.0% of the variation in the ∆DBP (p = 0.039)  decreased, the resting SBP change (∆SBP) decreased after versus before training. The regression model combining the pretraining ASBP awake response to the GEST versus CONTROL  . In addi= 0.039) .2 and SDNN, before and after training. Before training, resting HFms2 age and resting SBP accounted for 67% of the variation in the magnitude of PEH. After training, resting SDNN age and resting SBP explained 61% of the variation in the magnitude of PEH. For ADBP, the magnitude of PEH over the awake hours became greater as resting parasympathetic predominance decreased and/or sympathetic predominance increased, assessed by higher LF/HF, before training only. Before training, resting LF/HF and resting DBP accounted for 36% of the variation in PEH. Similarly, the magnitude of PEH over sleep and 19 h increased as resting parasympathetic predominance decreased before and after training.We examined the relationship between PEH over 19 h and resting autonomic activity as measured by HRV among a small group of adults with hypertension before and after aerobic exercise training. For ASBP, we found that the magnitude of PEH over the awake hours became greater as resting parasympathetic predominance decreased, assessed by lower HFmsIn addition to statistical significance, the magnitude of the PEH response observed in our study was also clinically meaningful as reductions in ASBP and ADBP of ≥2 mm reduce the incidence of CVD in both individuals with hypertension and normal BP ,24. SimiHypertension is considered the most important global risk factor for CVD morbidity and mortality . Hence, PEH may occur due to reduced total peripheral resistance without a concomitant increase in cardiac output in otherwise healthy individuals with hypertension . The supAnother novel finding is the magnitude of PEH was not different before and after training under ambulatory conditions in the same participants over 19 h. Thus, PEH does not appear to depend on training status, suggesting that sedentary and trained individuals may benefit from its antihypertensive effects. Surprisingly, in contrast to reports in the literature ,13, we fThe present study is subject to limitations. Additional factors associated with PEH, such as the properties of the vascular system , may have contributed to PEH response due to their relationships with ANS. In addition, the small sample size and the lack of sex and racial/ethnic diversity limit the extrapolation of our findings to the general population of adults with hypertension. Further, our correlational findings should be interpreted with caution as they do not imply causality. Nevertheless, the current study is the first rigorously designed substudy of a larger randomized control trial  to measuIn accordance with our hypothesis, we found that with hypertension, the magnitude of PEH became greater as resting parasympathetic predominance decreased and resting BP increased among a small group of middle-aged adults regardless of training status obtained following 12 w  of moderate-intensity  aerobic exercise training. Furthermore, PEH was of similar magnitude before and after training under ambulatory conditions in the same participants over 19 h. Further studies are needed to more definitively determine the characteristics of patients that can maximally benefit from the antihypertensive effects of PEH.


Radiotherapy using the deep inspiration breath-hold (DIBH) technique compared with free breathing (FB) can achieve substantial reduction of heart and lung doses in left-sided breast cancer cases. The anatomical organ movement in deep inspiration also cause unintended exposure of locoregional lymph nodes to the irradiation field.From 2017–2020, 148 patients with left-sided breast cancer underwent breast conserving surgery (BCS) or mastectomy (ME) with axillary lymph node staging, followed by adjuvant irradiation in DIBH technique. Neoadjuvant or adjuvant systemic therapy was administered depending on hormone receptor and HER2-status.CT scans in FB and DIBH position with individual coaching and determination of the breathing amplitude during the radiation planning CT were performed for all patients. Intrafractional 3D position monitoring of the patient surface in deep inspiration and gating was performed using Sentinel and Catalyst HD 3D surface scanning systems . Three-dimensional treatment planning was performed using standard tangential treatment portals (6 or 18 MV). The delineation of ipsilateral locoregional lymph nodes was done on the FB and the DIBH CT-scan according to the RTOG recommendations.mean) in axillary lymph node (AL) level I, II and III in DIBH were 32.28 Gy (range 2.87–51.7), 20.1 Gy (range 0.44–53.84) and 3.84 Gy (range 0.25–39.23) vs. 34.93 Gy (range 10.52–50.40), 16.40 Gy (range 0.38–52.40) and 3.06 Gy (range 0.21–40.48) in FB (p < 0.0001). Accordingly, in DIBH the Dmean for AL level I were reduced by 7.59%, whereas for AL level II and III increased by 22.56% and 25.49%, respectively.The mean doses (Dmean for the supraclavicular lymph nodes (SC) in DIBH was 0.82 Gy (range 0.23–4.11), as compared to 0.84 Gy (range 0.22–10.80) with FB (p = 0.002). This results in a mean dose reduction of 2.38% in DIBH.The Dmean for internal mammary lymph nodes (IM) was 12.77 Gy (range 1.45–39.09) in DIBH vs. 11.17 Gy (range 1.34–44.24) in FB (p = 0.005). This yields a mean dose increase of 14.32% in DIBH.The DThe DIBH technique may result in changes in the incidental dose exposure of regional lymph node areas. Incidental irradiation of cardiac structures in left-sided breast cancer increases the risk of subsequent ischaemic cardiac events . NotablyCurrently, deep inspiration breath hold technique (DIBH) in the supine position allows reproducible cardiac shift from the irradiation field. Therefore, it is a widely used protective heart approach . DIBH caTangential 3DRT of the mammary gland tissue often unintentionally includes the locoregional lymph nodes in the radiation fields with therapeutic dose. This dose may be sufficient for the eradication of microscopic tumor residues. This hypothesis is supported by the results of the ACOSOG Z0011 study, where patients with 1–2 involved sentinel lymph nodes upon sentinel lymph node dissection had similar axillary recurrence rates whether they received secondary axillary lymph node dissection or not . HoweverWe propose that DIBH may lead to differences in the incidental radiation dose of the axillary, supraclavicular and internal mammary lymph nodes due to changes in their position , 15 in rIn the absence of published data in this regard from randomized trials of DIBH vs free breathing (FB) RT in the supine position, reporting institutional experiences is necessary. The goal of this single-institutional retrospective study was to investigate dosimetric differences of incidental locoregional lymph nodes irradiation between DIBH and FB for left-sided breast cancer patients.From December 2017 to July 2020, 148 out of 247 patients with left-sided or bilateral breast cancer which were screened for irradiation in DIBH technique were included in this analysis. The majority of patients  received 3DRT in DIBH. The remaining 17 (11.5%) patients received RT in FB either due to suboptimal compliance or lack of dosimetric benefits of DIBH.BCS or mastectomy with axillary lymph node staging was performed according to institutional protocols. Systemic therapy was administered according to current guidelines  and indiAll patients received coaching for DIBH in the CT room using a Surface Image Guided RT (SGRT) system . The patients were asked to take a deep breath and hold it for a duration of 20 s. The width of the gating window was set to 5 mm. All patients received two CT scans with a slice thickness of 2 mm  in FB and DIBH. Treatment planning ) was carried out using standard tangential treatment portals .Adjuvant WBI or thoracic wall RT was delivered using either moderate hypofractionation (40.05 Gy in 15 fractions) or conventional fractionation (50.00–50.40 Gy in 25–28 fractions). Boost irradiation was delivered sequentially (10–16 Gy in 5–8 fractions), with simultaneous integrated boost (58.80–61.60 Gy in 25–28 fractions) or IORT (single dose of 20 Gy with 50-kV photons ).During follow up, all patients were examined every three to six months for the first two years in the radiation oncology department, followed by annual visits thereafter. Breast ultrasound was performed every 6 months for the first three years. Mammograms were obtained six months after WBI, and yearly after the first mammography. Suspected recurrences were biopsy confirmed.mean, D50%, Dmax, Dmin, V30, V40).For the planned dosimetric evaluation, lymph nodes levels of interest  and internal mammary (IM) lymph nodes) as well as the contralateral breast were retrospectively delineated Fig. . The delP-values < 0.05 were considered statistically significant. Effect size was assessed according to Cohen (1988) . n = 100) and N0 stage  with positive estrogen and progesterone receptor status. Poor differentiation (G3) and Ki-67 > 20% were present in 25.7% and 41.9 of patients, respectively. A minority of patients required re-resection to achieve clear margins (12.8%). Only four patients received mastectomy (2.7%). About 23% of patients received hypofractionated radiotherapy.Altogether, 148 patients with 296 CT scans were analyzed. Baseline characteristics are shown in Table The DVH parameters for levels I-III, SC and IM are summarized in Table mean) in level I, II and III in DIBH were 32.28 Gy (range 2.87–51.7), 20.10 Gy (range 0.44–53.84) and 3.84 Gy (range 0.25–39.23) vs. 34.93 Gy (range 10.52–50.40), 16.40 Gy (range 0.38–52.40) and 3.06 Gy (range 0.21–40.48) in the FB group   and increased for level II and III by 22.56% (r = 0.45), and 25.49% (r = 0.47).The mean dose  (Table r = 0.39), whereas it was increased for level II and III D50% by 28.52% (r = 0.42), and 35.71% (r = 0.46) , 19.65 Gy (range 0.44–52.32) and 3.23 Gy (range 0.25–44.97) vs. 39.02 Gy (range 2.75–52.05), 15.29 Gy (range 0.38–52.37) and 2.38 Gy (range 0.21–47.19) in the FB group , respectively. There was an increase in the mean values of V30, and V40 for level II in DIBH 27.88%  and 29.31%  respectively. There was a non-significant increase in the mean values of V30Gy and V40Gy for level III with DIBH  but not for level II by 1.28% (p = 0.062).The mean volumes of the lymph node levels were assessed in FB and DIBH. There were small, but significant volume decreases in DIBH for level III by 3.83% (mean for the SC in DIBH was 0.82 Gy (range 0.23–4.11), as compared to 0.84 Gy (range 0.22–10.80) with FB (p = 0.002). This results in a Dmean reduction of 2.38% (r = 0.26) in DIBH  vs. DIBH: 26 cm3 (range 13–37.3). This resulted with moderate volume decrease in DIBH for SC by 2.4% (p < 0.0001).The Drange 0.2–4.11, asmean for IM was 12.77 Gy (range 1.45–39.09) in DIBH vs. 11.17 Gy (range 1.34–44.24) in FB (p = 0.005). This yields a Dmean increase of 14.32% (r = 0.23) in DIBH  and 33.54%  (Table 3 (range 3.80–9.5) compared to 7.5 cm3 (range 3.7–9.5) in DIBH. This corresponded to a small, but significant decrease in volume in DIBH for IM by 1.3% (p < 0.0001).The D range 1.5–39.09 i3 (range 128–2762.7) for FB and 801.2 cm3 (range 121.80–2759.1) for DIBH. The mean relative difference between DIBH and FB was small (relative increase of 0.72%), but statistically significant (p = 0.023).Mean values for right  breast were 795.5 cmIn this retrospective single-center analysis, we could demonstrate that the use of DIBH leads to significant changes in dose-volume parameters for patients with left-sided breast cancer treated with tangential 3DRT. For level I, DIBH lead to a decrease in incidental dose whereas DIBH was associated with increased doses for level II-III, supraclavicular and internal mammary lymph nodes.Since publication of the ACOSOG Z0011-trial, there has been a controversial discussion regarding the radiation dose required for control of subclinical disease in the axilla . Althoug2 had significantly higher median doses in level I-III. Most patients received 3DRT (76.1%), while DIBH was only used in 1 patient [A recent systematic review identified 13 retrospective studies with a total of 475 patients which analyzed dosimetric parameters of axillary lymph node levels for patients planned for adjuvant whole breast radiotherapy . There w patient .DIBH is a standard technique for patients with left sided breast cancer due to a significant dose reduction of cardiac structures –26. In 2Borm et al. contoured axillary lymph node levels according to the RTOG atlas for 32 patients and compared incidental doses during DIBH and FB . They dePazos et al. analyzed the influence of DIBH on doses to level I-III, supraclavicular and internal mammary nodes for 35 patients planned for radiotherapy of the breast or chest wall and the regional lymph nodes. Three-dimensional movement was between 0.79 cm for level I and 1.44 cm for internal mammary nodes. DIBH led to a significant decrease in the dose to level I and II compared to FB, however there were no significant differences for the other regions of interest .40Gy and V30Gy. While only small amounts of level II were exposed to these doses, DIBH led to an absolute decrease in the V40Gy and V30Gy of 6.3% and 5.9% for Level I, respectively. Level III as well as the supraclavicular and the internal mammary nodes were only minimally exposed to incidental irradiation. The observed changes, though in some cases statistically significant, may not be clinically relevant due to relatively small absolute differences. Compared to the analysis by Borm et al. [mean and V40Gy and V30Gy are somewhat smaller in magnitude. Contrary to their findings, we saw an increase in all dosimetric parameters for level II as well as Dmean to level III, but nor for V40Gy and V30Gy for level III. We observed considerably lower doses to level II and III, for example for Dmean of level II: 16.40 Gy (FB) and 20.10 Gy (DIBH) in our analysis compared to 23.7 Gy (FB) and 24.1 Gy (DIBH) for Borm et al. [In our analysis, the largest absolute changes were observed for level I with a decrease of 2.65 Gy (from 34.93 Gy to 32.28 Gy) and for level II with an increase of 3.7 Gy (from 16.40 Gy to 20.10 Gy). However, the clinical relevance may be more adequately addressed by comparing exposure to higher doses, such as Vm et al. , the cham et al. . This mam et al. . No detaOur analysis represents the largest cohort both in terms of dosimetric analysis in FB and in DIBH. We included intraindividual comparisons based on CT-scans in FB and DIBH for each patient. To exclude confounding by different radiotherapy techniques, only tangential 3DRT-plans were analyzed. It has been previously shown that IMRT or VMAT can also significantly impact incidental dose to the axilla –29. ThusIn conclusion, we could demonstrate a significant variability in incidental dose exposure to regional lymph node areas in patients with left-sided breast cancer by the use of DIBH. Further studies are needed to determine the clinical significance of these findings and to establish predictors of dosimetric changes based on patient-related factors.


Belamcanda chinensis is a common garden herb. The extraction technology of B. chinensis seed oil (BSO) was optimized by ultrasonic-assisted extraction (UAE) method, the composition, relative content of main fatty acids and physicochemical properties of BSO were determined, and the isolation, identification and determination of chemical constituent in BSO residue (BSOR) were also investigated. The optimum process conditions of BSO by UAE were optimized as ultrasound time 14 min, extraction temperature 42℃, the ultrasound power 413 W and the liquid–solid ratio 27:1 mL/g. Under this condition, the extraction yield was 22.32 % with the high contents of linoleic acid and oleic acid in BSO. Ten compounds were isolated and identified from BSOR, and belamcandaoid P (9) was a new compound. The contents of the determined compounds were all at high level in B. chinensis seed. The study provided a certain scientific reference for the comprehensive development and utilization of B. chinensis seeds. The original plants and samples were identified by the pharmacist Hai-chen Liu of Luoyang Bencaotang Pharmaceutical Co., ltd. 50 g of voucher specimen each were deposited in School of Chemistry and Chemical Engineering, Henan University of Science and Technology (access number: 2022–015). The seeds of B. chinensis were crushed, sieved through 60 mesh sieve and kept at −4℃ for further use.The seeds of n-hexane  were purchased from Tianjin Jindong Tianzheng Fine Chemical Reagent Factory .The ethyl acetate, acetone, petroleum ether (PE) (60-90℃) and 2.2B. chinensis seeds for the advantages of simple operation, short time and high efficiency Ultrasonic-assisted extraction (UAE) was employed to extract the oil from 2.3After esterification by the method described in GB/T 5009.168–2016  with NaOH-methanol, the composition and relative content of the FAs in BSO were determined by GC–MS according the conditions detailed described in literature 2.4The acid value (AV) , iodine value (IV) , peroxide value (POV) , saponification value (SV)  and unsaponifiable matter (USM)  were determined based on the International Organization for standardization (ISO).2.5The dried powder of BSOR (10 Kg) was extracted with 40 L 90 % ethanol using reflux extraction method for 4 h for two times. The mixture was filtered to get the extraction solution, and extraction solution was concentrated by decompress concentration to obtain ethanol extract.n-butanol in the order of increasing polarity to obtain different fractions. Then, silica gel column chromatography (CC), LH-20 CC, semi-preparative HPLC and other chromatographic methods were employed to isolate the compounds from each fraction of PE, EtOAc and n-butanol.The ethanol extract of BSOR obtained above was suspended with water, and then extracted with PE, ethyl acetate (EtOAc) and The structures of the isolated compounds were identified by NMR, UV and MS.2.6B. chinensis seeds by HPLC method.According to the results of the chemical composition, the representative compounds, irisquinone E, belamcandaphenol P, belamcandaphenol B, iridal, isoiridogermanal and iridogelamal A were selected as the reference substance to determine the content of these main constituent in 2.6.1Waters e2695 HPLC system equipped with UV detector was used to determine the contents of the selected six compounds. The HPLC conditions were Thermo BDS HYPERSIL C18  column, column temperature 25℃, detection wavelength 254 nm, injection volume 10 μL, and the mobile phase of methanol (A)-0.1 % phosphoric acid (B) with gradient elution.2.6.2The precisely weighed reference substance of irisquinone E (7.30 mg), belamcandaphenol P (9.60 mg), belamcandaphenol B (8.65 mg), iridal (6.5 mg), isoiridogermanal (5.53 mg) and iridobelamal A (5.9 mg) was dissolved in methanol to prepare the reference solutions with mass concentrations of 11.62 mg/mL, 2.40 mg/mL, 1.922 mg/mL, 1.625 mg/mL, 1.383 mg/mL and 1.475 mg/mL, respectively. The mixed reference stock solution containing irisquinone E (0.1296 mg/mL), belamcandaphenol P (0.1440 mg/mL), belamcandaphenol B (0.2306 mg/mL), iridal (0.1300 mg/mL), isoiridogermanal (0.1106 mg/mL) and iridobelamal A (0.1180 mg/mL) was prepared by added appropriate amount of the above reference solution into a 5 mL-volumetric flask and fixed the volume with methanol. The mixed reference stock solution with the volume of 0.167, 0.250, 0.334, 0.500, 1.000, 1.670 and 5.000 mL were added into 5 mL-volumetric flasks, respectively, and fixed the volume with chromatographic grade methanol to obtain standard solution with different concentration gradient.2.6.3The powder of BSOR (5 g) was extracted with methanol in a Soxhlet reflux device for 4 h. After extraction, the mixture was cooled, and then filtered. The filtrate was transferred into a 100 mL-volumetric flask and fixed the volume with methanol to get the sample solution. After filtration with an organic filter membrane (0.22 μm), the sample was determined.33.1B. chinensis seeds powder with the same quality were extracted by Soxhlet extraction method with acetone, n-hexane, PE and EtOAc as solvents, respectively. The results revealed that the extraction yields of BSO with four solvents were as follows: acetone > n-hexane > PE > EtOAc. Compared with those using other solvents, the yield of BSO using acetone (26.01 %) was the highest. However, acetone is more toxic and thus less used in industry to extract oil. The yield of BSO extracted by EtOAc (20.63 %) was the lowest, and the color of the oil extracted by EtOAc was the deepest, which would increase the complexity of subsequent decolorization and other processes. The BSO yield extracted by PE (24.92 %) and n-hexane (25.42 %) was close, and with insignificant difference (P > 0.05). In addition, the color of BSO extracted by PE was better than that of the other three solvents, and the price of PE was lower than that of n-hexane. Therefore, PE was employed as the solvent in the follow-up oil extraction experiment considering the cost, toxicity and easy to obtain.Four samples of 3.23.2.1Four factors such as extraction temperature, ultrasound power, ultrasound time and liquid–solid ratio were selected to optimize the effect of different factor on BSO yield. The results were shown in 3.2.2Y) of BSO and ultrasound time (1X), ultrasonic extraction temperature (2X), liquid–solid ratio (3X) and ultrasound power (4X) was as follows:According to the single factor experiments results, four factors and three levels were selected for RSM experiments . The BoxP-value and F-value. The P-value of < 0.01, between 0.01 and 0.05 and > 0.05 means extremely significant, significant and no-significant, respectively F-value can reflect the significance of the fitting degree of the experimental model equation. High F-value imply the good fitting degree of the model equation and high significance of the model P-value (<0.0001) of liquid–solid ratio (3X) revealed that liquid–solid ratio had a significant effect on the yield of BSO. The influence order of each factor on the yield of BSO was liquid–solid ratio > extraction temperature > ultrasound time > ultrasound power (3X > 2X > 1X > 4X). The P-values (≤0.0001) of interactive and quadratic terms 1X3X and 32X, far<0.01, implied that the liquid–solid ratio and ultrasound time had a significant effect on BSO yield. At the same time, the P-value (<0.0001) of the RSM regression model and the F-value (14.89) revealed that the model was extremely significant. And the P-value (0.1174) of the lack of fit was far>0.05, indicating that the equation of the RSM regression model was good and could be employed to optimize the data in the experiment. The regression coefficient (2R = 0.9520) of this equation was close to the correction coefficient  could be directly reflected from the diagram. The curvature of the three-dimensional surface plots could reveal the influence of the interaction of the factors on BSO yield. At the same time, the shape of the contour line corresponding to the three-dimensional surface plots could also reveal the strength of the interaction between the factors. When the contour line shape was close to elliptical, the interaction of factors was strong and the effect on the yield of BSO was more significant. By comparing the response surface plots and contour map of the interaction of various factors (A), (B) and (C) in X3) and ultrasound time (X1) on BSO yield was the largest (X3) and ultrasound power (X4) on BSO yield was the smallest  was 0.08 %, indicating that the regression equation of the model could truly reflect the influence of various factors on the yield of BSO. The model was feasible for the optimization of UAE process of BSO.2) according to the methods described in literatures 2 (9.45 %) was lower than that extracted by UAE (22.32 %). The extraction time of SC-CO2 (180 min), SE (360 min) and IE (480 min) was much longer than that of UAE (14 min). SC-CO2 is a high-pressure extraction equipment, which requires high operating conditions and is difficult for industrial production 2 were much lower than that of UAE, and UAE was suitable for the extraction of BSO.The BSO was also extracted by Soxhlet extraction (SE) and impregnation extraction (IE) with PE as extraction solvent, and supercritical carbon dioxide (SC-CO2 (91.24 %), and the extraction time (48 min) was much shorter than that by other extraction methods (130–360 min) Prinsepia utilis (32.5 %) was higher than that of SC-CO2 (31.9 %) and pressing method (28.2 %) Nitraria sibirica Pall by UAE (7.05 %) was higher than that by traditional extraction (5.24 %) and SE (5.73 %), and the extraction time (40 min) was much shorter than that of traditional extraction (360 min) and SE (480 min) 2 (180 min), and the oil yield by UAE (10.07 %) was higher than that by SC-CO2 (9.04 %) and slight lower than that by SE (10.56 %) Ultrasonic-assisted extraction method has been widely used in the extraction of oil, showing a good extraction effect 3.32 were determined by GC–MS  and the total content of the four major FAs (97.88 %) were slightly higher than those of other three solvents. The content of linoleic acid extracted by n-hexane (47.40 %) was the highest, while by EtOAc (44.81 %) was the lowest. The content of oleic acid extracted by PE (37.81 %) was the highest, while by EtOAc (33.63 %) was the lowest. The total content of linoleic acid and oleic acid extracted by PE (83.49 %) and n-hexane (84.49 %) were higher than those by acetone (80.28 %) and EtOAc (78.44 %). The above results implied that PE and n-hexane were the suitable solvents for extracting BSO. Considering the factors such as oil yield and solvent cost, PE is an ideal solvent for extracting BSO.The composition and relative content results of the FAs of BSO extracted by four solvents suggested that the compositions of FAs in BSO extracted by 2 (43.31 %) and IE (42.75 %), and the content of oleic acid by SE (37.81 %) and SC-CO2 (37.81 %) was higher than that by UAE (37.53 %) and IE (33.41 %). Which revealed that the total content of linoleic acid and oleic acid extracted by UAE (85.75 %) was higher than that by SE (83.49 %), SC-CO2 (81.12 %) and IE (76.16 %). And the content of UFAs in the seeds oil of Prinsepia utilis obtained by UAE was higher than that by SC-CO2The content of linoleic acid extracted by UAE (47.57 %) was higher than that by SE (45.68 %), SC-CO3.42/100 g), POV (7.805 mmol/kg), SV (147.378 mg KOH/g) and USM (3.91 %) were determined by ISO methods. The physicochemical properties of common edible vegetable oils in China were shown in 2/100 g) was higher than that of olive oil (86–87 g I2/100 g), peanut oil (86–107 I2/100 g), oil-tea camellia seed oil (83–89 I2/100 g) and palm oil (50–55 I2/100 g), but was lower than that of maize oil (107–135 I2/100 g), flaxseed oil (165–208 I2/100 g), sesame seed oil (104–120 I2/100 g), grapeseed oil (118–141 I2/100 g), sunflower seed oil (118–141 I2/100 g), walnut oil (140–174 I2/100 g), soya been oil (124–139 I2/100 g) and safflower seed oil (136–148 I2/100 g). The results also implied that BSO had a high degree of unsaturation, the conclusion was consistent with the research results of high content of UFAs. The AV (33.79 mg KOH/g) was at high level, which was higher than that of most common vegetable oils (The AV (33.79 mg KOH/g), IV , EtOAc-fraction (15.5 g) and n-butanol-fraction (14.5 g).The extraction and separation process of BSOR after oil extraction was shown in 1–7. Using the same methods, n-butanol-fraction (14.5 g) was separated to obtain compound 8–10. The isolation process of the compounds was shown in The PE-fraction (100 g) was separated by silica gel CC, LH-20 CC and semi-preparative HPLC (semi-prep-HPLC) to obtain compounds 1) 2) 3) 4), iridal (5) 6) 7) 8) 9) and β-sitosterol (10) 9 was a novel one, compounds 5–7 were firstly isolated from B. chinensis seeds. The chemical structures of the ten compounds were shown in The isolated compounds was identified as belamcandaphenol P , which revealed seven degrees of unsaturation of compound 9. It could be seen from 1H NMR  that compound 9 had two terminal olefinic proton signals  and 7 methyl protons, including 5 methyl singlet signals , 0.83 , 0.96 , 0.99 , 1.78 ) and 2 methyl double peak signals , 0.91). The chemical shift of methyl ), combined with the molecular formula of compound 9, revealed that the methyl group ) was connected to the double bond, which could be further confirmed through the HMBC cross-peaks of H-24 ) with C-23(δC: 113.5), C-4(δC: 147.5) and C-5(δC: 46.4), and H-23  with C-4 (δC: 147.5), C-5 (δC: 46.4) and C-24 (δC: 23.3). The H—H COSY cross-peaks of H-30 ) with H-22 ), and of H-29 ) with H-22 ), along with the HMBC cross-peaks of H-30 ) with C-21 (δC: 59.8), C-22 (δC: 30.8) and C-29 (δC: 23.0), and of H-29 ) with C-21 (δC: 59.8), C-22 (δC: 30.8) and C-30 (δC: 22.1) implied that there was an isopropyl structural fragment in the structure of compound 9.Compound 13C NMR  of compound 9 revealed that there were 30 carbon signals, including a carboxyl carbon signal (δC: 178.1), two double bond carbon signals , and seven methyl carbon signals in high field . Through the analysis of carbon spectrum, hydrogen spectrum and two-dimensional NMR data of compound 9, the spectral characteristics of compound 9 were consistent with those of the 3,4-sec-triterpenoids with fernane type, and were very similar to those of belamcandaoid B 9 was of fernane type triterpenoids with 3,4-seco-structure.The 9 were shown in 9. In addition, the position of the double bond was also significantly different from that of belamcandaoid B. According to DEPT 13C NMR, the double bond carbon signals  of compound 9 were quaternary carbon atom. The HMBC correlation from H-28 to C-8, H-25 to C-9, H-5 to C-8 and H-11 to C-8 confirmed that the double bond of compound 9 was at C8-C9 position. Unlike belamcandaoid B, the chemical shift of the carbonyl carbon signal in compound 9 obviously moved to the low field (δC:178.1), indicating that the carbonyl group existed in compound 9 in the form of carboxylic acid rather than in the form of ester in belamcandaoid B. Which could be further confirmed by the high-resolution mass spectrometry of compound 9 and the reduced methoxy group in the signals. The spectral data of other positions of compound 9 could be further confirmed by two-dimensional spectrum , H-25(δH 0.96), H-26(δH 0.83) and H-21(δH 1.01) were β-configuration, and H-27(δH 0.99) and H-28(δH 0.80) were α-configuration. The configuration at the corresponding positions  were also confirmed. It was found that compound 9 had not been reported in the literature, so it was determined that this compound was a new triterpene, and named belamcandaoid P (9).The configurations of spatial structure of compound 1H NMR δH: 4.9, 4.7, 2.44 , 2.20, 2.18, 2.16, 2.12, 2.03, 1.96, 1.95, 1.83, 1.78, 1.70, 1.68, 1.64, 1.58, 1.54, 1.43, 1.38, 1.29, 1.01, 0.99, 0.96, 0.91, 0.85, 0.83, 0.80; 13C NMR δC: 178.1 (C-3), 147.5(C-4), 138.5(C-8), 129.9(C-9), 113.5(C-23), 59.7(C-21), 52.7(C-18), 46.4 (C-5), 42.9(C-17), 41.8(C-15), 40.8(C-10), 36.5(C-13), 35.9(C-12), 32.1(C-1), 30.8(C-22), 30.5(C-16), 29.5(C-2), 29.3(C-20), 28.2(C-7), 26.9(C-16), 24.6(C-6), 23.3(C-25), 23.3 (C-24), 23.0(C-29), 22.1(C-30), 21.8(C-27), 21.6(C-11), 20.3(C-19), 16.7(C-26), 14.7 (C-28).3.6B. chinensis seed.Compounds irisquinone E, belamcandaphenol B, belamcandaphenol P, iridal, isoiridogermanal and iridobelamal A with typical structural characteristics and high purity (>98 %) isolated from BSOR were selected as reference substance to determine the content of these compounds in The maximum absorption peaks of these compounds were recorded by wavelength scanning of UV, and the results revealed that the maximum absorption wavelength of all the compounds were at 230–270 nm. Based on the maximum absorption wavelength and the results in the literatures B. chinensis seed.The reference solution was analyzed with the above chromatographic conditions. The regression equation, precision, stability, repeatability, sample recovery, LOD (limit of detection)) and LOQ (limit of quantification) of the determination method were investigated according to the method literatures B. chinensis seed were 0.6139 ± 0.042, 1.0655 ± 0.032, 0.7058 ± 0.02, 9.8677 ± 0.048, 6.3042 ± 0.076 and 6.9585 ± 0.144 mg/g, respectively. Compound irisquinone E is benzoquinones, compounds belamcandaphenol B and belamcandaphenol P belong to phenols, and iridal, isoiridogermanal and iridobelamal A are iridal-type triterpenoids. Benzoquinones and phenols are common chemical components in B. chinensis seeds B. chinensis seed. Iridal-type triterpenoids, a kind of special compounds which come from the metabolism of squalene, are the representative components in the rhizoma of B. chinensis and Iridaceae plants with various and vital biological activities B. chinensis seeds was firstly established. At present, a variety of bioactive compounds had been isolated and identified from B. chinensis, flavonoids were mainly isolated in the roots and leaves of B. chinensis, and phenols, benzoquinones and volatile oil were mainly found in the seeds of B. chinensisB. chinensis seed were all at high level.The sample solution was determined according to the optimized HPLC chromatographic conditions , EmpowerB. chinensis seeds as a new resource food, and provided a scientific basis for the quality standard of B. chinensis seeds.This study provided a foundation for the comprehensive development and utilization of 4Overall, the extraction technology of BSO was optimized, the composition and relative content of main FAs and physicochemical properties of BSO were determined, and the isolation, identification and determination of chemical constituent in BSOR were also investigated in this study. The results revealed that UAE with PE as the extract solvent was the proper method for the extraction of BSO with the high yield (22.32 %) and good quality of the oil. The high content of oleic acid and linoleic acid makes BSO has an important potential application value as an edible oil. After further refining, BSO could be developed into edible oil, lubricating oil, daily chemicals, biodiesel and other industries.9) was a new compound, and iridal, isoiridogermanal and iridobelamal A were firstly isolated from B. chinensis seed. The bioactive evaluation and the underlying pharmacological mechanisms may be further studied to promote the potential application of those compounds. The content of isoiridogermanal, iridobelamal A, iridal, irisquinone E, belamcandaphenol B and belamcandaphenol P were all at high level in B. chinensis seed. The results implied that in addition to rhizoma of B. chinensis, B. chinensis seed might also become a main source of iridal-type triterpenoids. The high oil yield, UFAs content, along with the abundant secondary metabolites make B. chinensis seed possible to become a new food resource. The further study is suggested to establish the standard of quality control, and evaluate the safety and security of potential products of B. chinensis seed. The study provided a certain scientific reference for promoting the comprehensive development and utilization of B. chinensis seed as a new resource food, and also provided a new possible source for the development of natural food, medicine, beauty and daily chemicals from the seed of B. chinensis.Ten compounds were isolated and identified from BSOR, belamcandaoid P (Pu Liu: Methodology, Supervision, Visualization. Shuang Kang: Investigation, Writing – original draft. Yu-yang Huang: Investigation, Writing – original draft. Tian-peng Song: Investigation, Software. Zi-yue Wu: Investigation, Software. Zong-yuan Lu: Resources. Rui-xue Deng: Conceptualization, Supervision, Funding acquisition, Writing – original draft, Project administration.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.