Stevia rebaudiana Bertoni, Asteraceae, is an herbaceous perennial plant native to Paraguay. This species is considered since ancient times a medicinal plant with important bioactive compounds and pharmacologic and food properties, namely diterpenes glycosides. The high natural sweetener potential stevioside and rebaudioside A produced by S. rebaudiana plants are suitable sucrose substitutes, and their obtention is influenced by environmental, phytosociological, and genetic factors. The plants’ genetic profile and sweet potential depiction are needed for suitable plant selection for improvement and deployment. Thirty-one S. rebaudiana accessions grown in the same plot where leaves samples were collected in early 2019, were genotyped using six microsatellite markers, including two steviol glycosides biosynthesis functionally involved markers. Additionally, an aqueous extract of each sample was obtained in a water bath and purified by SPE for stevioside and rebaudioside A quantification by normal phase HPLC. Stevioside and rebaudioside A contents varied between 0.53–7.36% (w w−1) and 0.37–3.60% (w w−1), respectively. Two genotypes displayed interesting ratios of rebaudioside A/stevioside (number 3 and 33). The level of genetic similarity between genotypes was tested through a pairwise similarity coefficient, and two groups of individuals had the same fingerprinting. Strong relatedness was found within genotypes, possibly due to cloning, thus, influx of new germplasm ought to be made to prevent mating between relatives, and for further selection and genetic improvement.The online version contains supplementary material available at 10.1007/s11033-021-06308-x. Stevia consists of approximately 230 species [Stevia rebaudiana features the sweetest essence [Stevia genus is distributed worldwide, ranging from the southern parts of the USA to Argentina and Brazil. Nowadays, Stevia cultivation has spread worldwide, including Europe, propagated through seed and cuttings [S. rebaudiana Bertoni (2n = 22) is a perennial and branched shrub of the Asteraceae family, native to north-eastern Paraguay [Stevia sweetness potential is the biotransformation of stevioside into rebaudioside A. Adari et al. [Stevia leaves pre-treatment with cellulose. The steviol-glycosides are currently used as sweet-tasting non-caloric food additives, due to obesity and diabetes rates rise worldwide [The genus  species , 2, and Paraguay , 5. SincParaguay , found iParaguay . SteviosParaguay . In geneParaguay , 7. AccoParaguay , this raParaguay . Moreovei et al.  explaineorldwide , 7, 11. orldwide . The leaorldwide . Steviolorldwide . The exiorldwide ).S. rebaudiana and, recently, from freely available expressed sequence data (ESTs), and Cosson et al. [S. rebaudiana genotypes from Paraguay were studied with SSR and ISSR [Some genetic studies were reported in this species using markers with several limitations, such as RAPDs –18, AFLPn et al.  also devand ISSR , and anaThe use of microsatellite loci and the development of statistical tools made it possible inferring kin relationships from molecular data, in particular ‘relatedness’, meaning the estimation and assignment of pairs or groups of individuals to categories of relationship or a measure of the fraction of alleles shared identical by descent among individuals . Indeed,Stevia leaves steviol-glycosides were generally obtained subsequently to hot water application, followed sometimes by solid-phase extraction. However, extraction methods using other solvents, such as ethanol and methanol or even supercritical fluid extraction, were also described [S. rebaudiana leaves samples were reported in the literature [In addition to the genetic and environmental factors that can influence the steviol-glycosides content, the analytical methodology selection is important to obtain an efficient protocol for plant bioactive compounds present extraction, purification, and quantification. The escribed . These sescribed . Additioescribed . Severalterature . Matrix terature , 38. Finterature .S. rebaudiana established in the Pluridisciplinary Center for Chemical, Biological and Agricultural Research, State University of Campinas, São Paulo, Brazil (CPQBA/UNICAMP) were studied for breeding purposes. The aims of the present study were: (i) to genotype 31 individuals with microsatellite markers to compute the genetic diversity, the fingerprint, and the relatedness, and (ii) to identify the genotypes that produce the highest rebaudioside A/stevioside ratio.The genotypes collection of S. rebaudiana germplasm plot established in the campus of the CPQBA/UNICAMP . The S. rebaudiana seeds were previously collected in the species’ region of origin, but the exact location is unfortunately unknown. Four to five leaves sampled in each genotype from different parts of the plant were collected from January to February 2019. The genotypes were grown in the same plot with 1 × 0.5 m spacing, and 5 to 10 replications per genotype and the sampling was made just before flowering. Afterward, the leaves were lyophilized and frozen at – 80 °C.We have studied 31 individuals from the S. rebaudiana genotype, using the CTAB method as described by Doyle and Doyle [Stevia UniGene derived MicroSatellites) primers , 2.5 mM MgCl2, and 1.0 μM of each primer. The amplifications were performed on a UNO96 Gradient thermocycler . The PCR protocol consisted of an initial denaturation step of 4 min at 94 °C, followed by 35 amplification cycles composed of denaturation (1 min at 94 °C), annealing , 2.5 mM MgCl2, and 1.5 μM of each primer. The amplifications were performed in the same thermocycler, programmed with an initial denaturation step of 5 min at 95 °C, followed by 40 amplification cycles composed of denaturation (1 min at 95 °C), annealing , 2.5 mM MgCl2, and 2.0 μM of each primer . From each centrifuged extract, the supernatant was transferred to a 25 mL volumetric flask. The pellet was reused for two more extractions and the supernatants transferred to the same volumetric flask. Finally, the volumetric flask was filled to 25 mL with distilled water.Both steviol-glycosides were quantified according to the improved HPLC method described by Woelwer-Rieck et al. , with so−1. Both standard solutions and purified extracts were filtered with a 0.45 µm nylon filter  before loading in HPLC.Seven stevioside  and rebaudioside A  standard solutions were prepared in acetonitrile:Milli-Q water (75:25 v/v) in the concentration range of 5–800 µg mL−1, using six different concentrations, and all standard solutions were injected in two different days. Standards were loaded in triplicate and each purified extract in duplicate into HPLC , equipped with an NH2 column  under the following conditions: 20 µL of injection volume, 210 nm wave-length, mobile phase: HPLC grade acetonitrile:Milli-Q water (75:25 v/v), 1 mL min−1 constant flow rate, 20 min at 36 °C. The qualitative determination was achieved by comparing the stevioside and rebaudioside A standard solutions retention times with those of the samples. The quantification was possible by applying the computed calibration curves equations. The linear regression curves of stevioside and rebaudioside A are presented below were obtained (both adjusted the interception in 0):−1) and X is concentration (µg mL−1) (unpublished results). The stevioside and the rebaudioside A concentration were represented in % w w−1 (steviol glycoside/dry leave).The linearity was assessed by injecting standard solutions of stevioside and rebaudioside A from 10 to 800 µg mLEucalyptus globulus with known pedigree [The genetic diversity parameters Na , Ne , Ho (observed heterozygosity), He (unbiased expected heterozygosity), and F (fixation index) were obtained with the GenAIEx software version 6.502 . To testpedigree . The denpedigree . The genThe analysis of the genetic diversity parameters Table  revealedHowever, the loci with higher Na, in general, had higher Ne and Ho values (gi18465444 and gi18465673), which were found in putative non-coding regions, values in bold in Table The total observed heterozygosity (Ho) ranged from 0.45 to 0.87, with an average of 0.76. There was an excess of heterozygous in all loci since the observed is higher than that expected heterozygosity (Ho > He), while a lower difference is found in the SUGMS28 locus , individuals with the same genotype plotted together . According to the literature for a value of LR coefficient > 0.25 the genotypes may be considered half-sibs, resulting from cross-fertilization; for LR > 0.5 the genotypes may be full-sibs, sharing both mother and father, for LR > 0.67 they may be the result of self-pollination, and for LR = 1 for the same genotype or clones [The dendrogram (UPGMA method) obtained with the kinship matrix (LR) Fig.  reveals r clones . Indeed,−1 for both steviol glycosides standards and the linearity of the HPLC method was excellent (r2 > 0.99).The contents of stevioside and rebaudioside A in the different genotypes were determined using calibration curves obtained by authentic standards. The linearity corresponds to the method’s ability to provide results directly proportional to the analyzed compound concentration, within a given interval. The calibration curves were linear in the range of 10–800 µg mL−1 dry leaf) for each sample and the respective rebaudioside A/stevioside ratio. The rebaudioside A-concentrations were lower in all the genotypes compared to the stevioside ones. From the results, significant among sample differences existed in both steviol glycoside concentrations. Samples 10, 17, and 22 had the highest stevioside content (6.64–7.36%), while samples 19, 20, and 29 had the highest rebaudioside A value (2.53–3.60%). Samples 8, 11, and 28 showed a lower concentration of both steviol glycosides compared to the other samples. Several samples revealed undetectable rebaudioside A content , considering the HPLC conditions linearity method. The rebaudioside A/stevioside ratio should be as high as possible, given the rebaudioside A better sweetening properties than stevioside, as previously referred. The ratio ranged from 0.17 to 0.86, and the highest rebaudioside A/stevioside ratio was observed in samples 3 and 33, rating 0.81 and 0.86, respectively  were 5.28 ± 0.05 min and 6.91 ± 0.10 min (mean ± standard deviation) respectively, with the HPLC method. Figure t 6.64–7.6%, whileS. rebaudiana is undergoing domestication [S. rebaudiana is most likely due to allogamy in this species mating system [The tication , notwithtication  the authtication . Simultatication , was higtication , for theg system . MoreoveEstimators are useful to identify relatives and to minimize consanguinity in breeding populations in the absence of pedigree information . Differe−1) for stevioside, and 0.37–3.60% (w w−1) for rebaudioside A. In some samples, the rebaudioside A remained undetected, probably due to either steviol-glycoside production being lower than needed for quantification by the linearity HPLC conditions or to the genotype lack of production. In general, the steviol-glycosides concentrations observed in the thirty-one samples are following the values found in the literature, with stevioside content always higher than the rebaudioside A content [−1) and 4.93% (w w−1), respectively. Similar steviol-glycosides values were also obtained in another study, by water extraction, with stevioside values ranging between 3.84 and 9.36% (w w−1), and the rebaudioside A values ranging between 2.59 and 7.77% (w w−1) [The steviol-glycosides content and variability determination are important for the plant producers and industries to obtain performant cultivars through selection and breeding. Therefore, all individuals from the current study were grown in the same conditions and collected just before flowering, when steviol-glycosides content is expected to be highest. The steviol-glycosides concentrations in the current study varied between 0.53–7.36%  .−1) and 0.60–4.44% (w w−1) of stevioside and rebaudioside A, respectively [−1) and 1.62–7.27% (w w−1), respectively [Despite the glycosides great solubility in water, some studies reported extraction with other solvents . Methanectively . Analogoectively . Regardlectively , 38. ActComparing the genetics with the chemical results, it is worth noting that individuals 8, 11, and 28 revealed close genetic relatedness and similar chemical profile, with low steviol-glycosides concentration. Individuals 10 and 22 had displayed the same genetic fingerprint and the same chemical profile, both in stevioside concentration and absence or very low rebaudioside A synthesis. Individuals 23, 25, and 26 had the same fingerprint, and they also revealed a very similar chemical profile, being probably the same genotype/clone. Indeed, since the late 1980s, DNA fingerprinting has become an immensely important instrument for genotype identification in wild plant and cultivated species. Additionally, when the morphological characters are mostly quantitative, correspondence with DNA marker estimates is generally quite high as compared to qualitative characters, which are more likely to reflect only a small number of mutation events . But, duSupplementary file1 (DOCX 17 kb)Below is the link to the electronic supplementary material.


Coptidis Rhizoma extract and systematically investigated its effects and mechanisms on the pharmacokinetics of oral berberine hydrochloride (BBR), a co-existing bioactive constituent. First, three LC-MS/MS based methods were established and fully validated to determine the levels of 11 primary metabolites in Coptidis Rhizoma extract. According to the weight ratio of four major primary metabolites in the Coptidis Rhizoma extract, a stable “endogenous” NADES was prepared using the heating method by the addition of 350 μl of water to 1,307.8 mg of the mixture of malic acid (490.5 mg), glucose (280.6 mg), sucrose (517.7 mg), and choline chloride (19.0 mg). The prepared NADES showed significant acute toxicity in mice and cytotoxicity in MDCK-MDR1 cells. However, after being diluted 10 times or 100 times, the NADES had no significant acute toxicity or cytotoxicity, respectively. The dilutions of the NADES significantly increased the water solubility of BBR, reduced its efflux in gut sacs and MDCK-MDR1 cell monolayer, and improved its metabolic stability in intestinal S9. In addition, the NADES dilutions reversibly opened the tight junctions between the enterocytes in the gut sacs. Moreover, the NADES dilutions significantly improved the exposure levels of BBR in the portal vein and livers of mice that were administered oral BBR. Malic acid was identified as a major component in the NADES in terms of solubility, acute toxicity, cytotoxicity, and pharmacokinetic-improving effects on oral BBR. In conclusion, the primary metabolites of Coptidis Rhizoma extract could form “endogenous” NADES, and its dilutions improve the pharmacokinetics of oral BBR. This study demonstrates the synergistic interaction of the constituents of Coptidis Rhizoma extract and the potential use of the NADES dilutions in oral BBR delivery.Primary plant metabolites can be used for artificial preparation of natural deep eutectic solvents (NADESs), which have strong dissolving capacity, good biocompatibility, and biodegradability. In this study, for the first time, we verified that NADESs were present in  According to the Biopharmaceutics Classification System (BCS), water solubility is one of the fundamental parameters associated with the intestinal absorption of oral drugs . UnfortuCoptidis Rhizoma “(Coptis chinensis Franch.)”, a commonly used traditional Chinese medicine  has a wide range of pharmacological effects, and it is the main active constituent of medicine . Howevermedicine . Due to medicine . In addimedicine . In contpure BBR , suggestUsing a mixture of sugars, amino acids, organic acids, and other nitrogen-containing compounds, several NADESs have been artificially prepared to increase the solubility and intestinal absorption of BBR . InteresCoptidis Rhizoma extract. Subsequently, the influences and the related mechanisms of a prepared NADES on the pharmacokinetics of oral BBR in mice were investigated.Therefore, in this study, we aimed to verify if NADESs could be produced from several major small primary metabolites in Coptidis Rhizoma “(Coptis chinensis Franch.)” (No.180305) were purchased from Shanghai Kang Qiao Herbal Pieces Co., Ltd. . The herbal pieces were identified as the root of Coptis chinensis Franch. according to the Pharmacopeia of the People's Republic of China (2015 edition). The purity of all the reference compounds used in this study was greater than 98%. BBR, coptisine, epiberberine, palmatine, demethyleneberberine, carbamazepine, verapamil hydrochloride, tetraethylamine, caffeic acid, and mycophenolic acid were purchased from Shanghai Yuanye Biological Co., Ltd. . Dimethyl sulfoxide, acetonitrile, and fluorescein isothiocyanate-dextran (FD4) were purchased from Merck and Co., Inc. . Malic acid, sucrose, glucose, choline chloride, urea, fructose, proline, alanine, and glycine were obtained from China National Pharmaceutical Group Co., Ltd. . The BCA protein test kit was obtained from Shanghai Biyuntian Biotechnology Co., Ltd. . Pooled CD-1 mouse intestinal S9 fraction was obtained from SEKISUI Medical Co. Ltd. . Formic acid, ammonium formate, fetal bovine serum, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Thermo Fisher Scientific . Cell Counting Kit-8 (CCK-8) was purchased from Meilunbio® . Trypsinase and penicillin-streptomycin solution were obtained from Biosharp . The pure water used in the current study was prepared using a Millipore Milli-Q system .Herbal pieces of 2. Hank’s balanced salt solution (HBSS)  were provided and authenticated by Sandia Pharmaceutical Technology (Shanghai) Co., Ltd. . The cells were cultured at 37°C in DMEM supplemented with 10% FBS, penicillin-streptomycin solution, and HEPES (15 mM) in a humidified atmosphere of 5% COGrade II ICR mice  were purchased from Shanghai Slac Laboratory Animal Co., Ltd. . The mice were housed in an air-conditioned room at 22–24°C with a dark/light cycle of 12 h. Before the experiment, the mice were fasted for approximately 12 h but were allowed to drink freely. All animal experimental protocols were approved by (PZSHUTCM19011105) and all the experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine.Coptidis Rhizoma were extracted twice with 10 times volume of boiling water (1.5 h for the first and 1 h for the second extraction). The obtained aqueous extract was then filtered through eight layers of gauze and transferred for vacuum drying at 60°C. The alkaloids in the dry Coptidis Rhizoma extract were quantified for quality control using a liquid chromatography-mass spectrometry (LC-MS) method .The herbal pieces of Coptidis Rhizoma alkaloids present in relatively high concentrations (0.156–10 μg/ml). The LC-MS system was composed of a Shimadzu HPLC (LC-20AD) spectrometer  and a Thermo Scientific LCQ fleet mass spectrometer  and equipped with an electrospray ionization (ESI) source. An Eclipse XDB-C18  maintained at room temperature was used for chromatographic separation of the analytes. Water containing formic acid (0.0625%) and ammonium formate (4 mM) was used as mobile phase A, and methanol was used as mobile phase B. The following elution gradient was used at the flow rate of 0.3 ml/min: 0–7 min, 20–30% B; 7.01–10 min, 20%–20% B. Data acquisition was performed in the selected ion monitoring mode after the protonated (M + H)+ ions of berberine and epiberberine , coptisine (m/z 320.2), palmatine (m/z 352.1), and carbamazepine  were generated. The method was validated in terms of selectivity, sensitivity, linearity, intra-day and inter-day variation, and recovery (data not shown).A validated method based on a liquid chromatography-mass spectrometry (LC-MS) system was used for the quantitative analysis of A validated method based on a liquid chromatography tandem mass spectrometry (LC-MS/MS) system was used for the quantitative analysis of BBR with relatively low concentrations  in biological samples. Further details about the method can be found elsewhere .Coptidis Rhizoma extract. The LC-MS/MS system consisted of a Waters ACQUITY ultra performance liquid chromatography system  and an API 5,500 mass spectrometer , and it was equipped with an ESI source.Three LC-MS/MS methods were established and fully validated for the quantitative analysis of 15 primary metabolites in the μm) was used. Water with 0.1% formic acid was used as mobile phase A, while methanol was used as mobile phase B. Gradient elution was set as follows: 0–1 min: 5% B; 2–10 min: 5–40% B; 10–12 min: 40–90% B; 12–12.1 min: 90–5% B; 12.1–15 min: 5% B. The flow rate was 0.3 ml/min and the column temperature was maintained at 40°C. A 10-μL aliquot was injected into the LC-MS/MS system for analysis. Negative ion mode was used for the ESI source, and data acquisition was performed in the multiple reaction monitoring (MRM) mode: m/z 132.9→114.9 for malic acid, m/z 179.2→135.1 for caffeic acid, and m/z 319.0→191.0 for mycophenolic acid .For organic acids, an ACQUITY BEH C18 column  was used. Water containing 10 mM ammonium acetate was used as mobile phase A, while acetonitrile containing 10 mM ammonium acetate was used as mobile phase B. Gradient elution was set as follows: 0–6 min: 95%→60% B; 6–7 min: 60% B; 7–8 min: 60%→95% B; 8–10 min: 95% B. The flow rate was 0.3 ml/min, and the column temperature was maintained at 45°C. A 10-μL aliquot was injected for analysis. Both positive and negative ion modes were used for the ESI source and data acquisition was performed in the MRM mode: m/z 150.9→88.7 for xylitol, m/z 179.0→88.9 for glucose and fructose, m/z 341.1→160.9 for sucrose, m/z 115.8→70.1 for proline, m/z 90.3→44.1 for alanine, m/z 76.2→30.0 for glycine, m/z 319.0→191.0 for mycophenolic acid , and m/z 130.3→100.3 for tetraethylamine .For sugars and amino acids, an ACQUITY BEH amide column  was used. Water containing 0.1% formic acid was used as mobile phase A, while acetonitrile was used as mobile phase B. Gradient elution was set as 0–1 min: 95% B; 1–5 min: 95%→60% B; 5–6 min: 60%→95% B; 6–6.1 min: 60%→95% B; 6.1–8 min: 95% B. The flow rate was 0.3 ml/min, and the column temperature was maintained at 45°C. A 5-μL aliquot was injected for analysis. Positive ion mode was used for the ESI source and data acquisition was performed in the MRM mode: m/z 61.0→44.0 for urea, m/z 104.2→60.1 for choline chloride, and m/z 130.3→100.3 for tetraethylamine .For urea and choline chloride, an ACQUITY BEH amide column  was prepared and then treated with ultrasound for 60 min. After centrifugation at 20,000 g for 10 min, the supernatant was collected. After certain dilution, the supernatant was injected into the LC-MS/MS system for analysis.A methanol-water  solution of the Coptidis Rhizoma extract were mixed together according to different combinations. That is, two, three or four compounds of 490.5 mg malic acid, 280.6 mg glucose, 517.7 mg sucrose and 19.0 mg choline chloride were mixed together according to their contents in the extract . The mixtures were then, respectively, transferred to a glass bottle with lid. After 350 μL water was, respectively, added, the mixtures were stirred and heated in a water bath at 50°C for 60 min to determine whether a transparent liquid, i.e., a NADES, was formed. The stability of the prepared NADESs was evaluated after placing them for 48 h at 20°C by observing whether the solutions were still transparent. In order to investigate the effect of water addition on the formation and stability of the prepared NADESs, 200, 250, 300, 350 or 400 μL pure water was added into the mixture of the four metabolites  to prepare four-component NADESs and evaluate their stability.NADESs were prepared by using the heating method . In brieμL water, the same below) or its 30 and 10% water dilutions, respectively. The symptoms of the mice were then observed, and the death time and the number of dead mice within 3 days were recorded.Sixty ICR mice were randomly divided into three groups according to gender and body weight, with 20 mice in each group. After fasting for 12 h, the mice were orally administered with 0.2 ml/10 g body weight the NADES .BBR was added to the prepared NADES or its 3, 10, or 30% water dilutions to produce a final concentration of 20 mg/ml. BBR was dissolved in pure water as a control. The solutions were ultrasonically treated for 1 h, and after full vortexing, the solutions were placed at room temperature for 1 h. Next, the solutions were centrifuged at 20,000 g for 10 min at 20°C. The obtained supernatant was filtered through a 0.22 The above method was also used to evaluate the influences of solutions of malic acid, glucose, sucrose, and choline chloride on the solubility of BBR. The concentrations of the primary metabolites were equal to their concentrations in the NADES, i.e., 490.5 mg/ml, 280.6 mg/ml, 517.7 mg/ml, and 19.0 mg/ml, respectively. In addition, the influences of different concentrations  of malic acid on the solubility of BBR were also evaluated.μg/ml BBR in the absence or presence of 1, 3, or 10% NADES dilutions. For transportation from the serosal side to the mucosal side, the gut sac was everted and filled on the inner serosal side with 1 ml Krebs-Ringer buffer containing 200 μg/ml BBR in the absence or presence of 1, 3, or 10% NADES dilutions. After incubation, aliquots of buffer (100 μL) were, respectively, taken from the serosal side or mucosal side every 15–60 min and replaced with an equal volume of blank Krebs-Ringer buffer. The LC-MS system  was used for the quantitative analysis of BBR.The influence of NADES dilutions on the transportation of BBR was determined as previously described . For train vitro transportation of FD4 (3 mg/ml) across the mouse gut sac was determined. The concentrations of FD4 were determined using a fluorescence microplate reader , with excitation and emission at 485 and 528 nm, respectively, (Fluorescein isothiocyanate-labeled dextran (FD4) with a molecular weight of 4,400 g/moL, is usually used to test the integrity of intestinal paracellular tight junctions . The infctively, . In addiThe above method was also used to evaluate the influences of different concentrations of malic acid on the transportation of BBR and FD4 in gut sacs. The concentrations of malic acid were 4.9, 14.7 and 49 mg/ml, which were respectively equal to the concentrations of malic acid in the 1, 3 or 10% water dilutions of the NADES.5 cells/mL. The cells were cultured until the tight junctions were formed . Before the experiment, the media on both sides of the chamber were replaced with a warmed HBSS solution. The cells were then incubated for 20 min at 37°C. For the transportation test from the apical (AP) to the basolateral (BL) sides, 0.2 ml of HBSS solution containing BBR (10 μg/ml) in the absence or presence of 0.3% or 1% of NADES was added to the AP side, and 0.7 ml blank HBSS solution was added to the BL side. For the transportation test from the BL side to the AP side, 0.2 ml blank HBSS solution was added to the AP side and 0.7 ml of HBSS solution containing BBR in the absence or presence of 0.3% or 1% of NADES was added to the BL side. After incubation for 2 or 4 h, the solutions on both sides were aspirated, and the concentration of BBR was determined using the LC-MS/MS method .MDCK-MDR1 cells were seeded on a Merck Transwell polycarbonate membrane  at a density of 2 × 10μg/ml) in the absence or presence of 0.3% or 1% of NADES was mixed with intestinal S9 (2 mg/ml) in 100 μL Tris-HCl  buffer solution and then pre-incubated at 37°C for 5 min. Next, reduced nicotinamide adenine dinucleotide phosphate  was added to initiate the reaction. After 0.5 or 1 h, the incubation was terminated with the same amount of chilled methanol, which contained carbamazepine as the internal standard. After centrifugation at 20,000 g for 6 min, the concentration of demethylberberine, a representative metabolite of BBR, in the obtained supernatant was determined using the LC-MS/MS method . The metabolic stability of BBR was characterized by using the concentration of demethylberberine.BBR .Mice were randomly divided into five groups, and they were orally administered with BBR that were dissolved in water, water dilutions of the NADES (1 and 10%), or solutions of malic acid (98 or 980 mg/kg). The dosages of malic acid were, respectively, equal to the dosages of malic acid in the 1 and 10% water dilutions of the NADES. The dosage of BBR in each group was 200 mg/kg, which was consistent with the dosage (0.1–0.3 g/kg) reported in previous studies on mice . Six micPapp) of BBR in the MDCK-MDR1 experiment was calculated using the equation: Papp = ΔQ/(Δt × A × C0), where ΔQ is the drug amount transported within Δt time, A is the surface area (0.33 cm2 in this experiment), and C0 is the initial concentration of BBR on the donor side. The unit of Papp is cm/sec.The permeability (Papp(BL-AP)/Papp (AP-BL).The efflux rate (ER) of BBR in the MDCK-MDR1 experiment was calculated using the following equation: ER = A non-compartmental analysis using WinNonlin® software  was performed to obtain the pharmacokinetic parameters of berberine in mice. It should be noted that the pharmacokinetic parameters were calculated based on the average drug concentration at each time point because the mice were not continuously sampled.p value < 0.05 indicated statistical significance and a p value < 0.01 indicated high statistical significance.The results are expressed as Mean ± SD, and statistical significance was determined with one-way or two-way analysis of variance (ANOVA) for multiple comparisons. A Coptidis Rhizoma extract. The calibration curves exhibited good linearity, and the corresponding typical regression equations are shown in Coptidis Rhizoma extract sample at three concentration levels. The recoveries of the analytes were between 88.41 and 110.14%, with RSD % between 0.03 and 5.16%. Taken together, these results suggest that the developed methods were specific, sensitive, reliable, and could be applied to quantitatively analyze the primary metabolites of Coptidis Rhizoma extract.The three methods were fully validated in terms of selectivity, sensitivity, linearity, intra-day, and inter-day variation, reproducibility, stability, and recovery. Representative MRM chromatograms of the analytes are shown in Coptidis Rhizoma extract. The levels of the primary metabolites are shown in Coptidis Rhizoma extract, malic acid, glucose, sucrose, and choline chloride were the major primary metabolites, which had contents of 28.14, 16.10, 29.70, and 1.09 mg/g, respectively.Four primary metabolites, including malic acid, sucrose, glucose, and choline, were detected in nine batches of Coptidis Rhizoma extract, therefore it was used in subsequent studies.Several transparent and viscous NADESs  were preμL of water was added to 1,307.8 mg of the mixture malic acid (490.5 mg), glucose (280.6 mg), sucrose (517.7 mg), and choline chloride (19.0 mg), the prepared NADESs were stable at room temperature for at least 48 h (The level of water added to the formulations positively correlated with the stability of the formed NADESs. When more than 350 ast 48 h . Therefop < 0.01). The dissolving capacity of the NADES decreased sharply after dilution with water (p < 0.01) than that in water ; the viability of the cells incubated with 1% dilution of the NADES was not influenced after incubation for 4 h but significantly decreased after incubation for 24 h (p < 0.01); while 0.3 and 0.1% dilutions of the NADES had no significant cytotoxicity after incubation for 4 and 24 h (p > 0.05). Therefore, 1% (v/v) was the highest concentration of the NADES in subsequent transportation experiment in cells and metabolic experiment in intestinal S9.The results  showed tin vitro transportation experiment with mouse gut sacs, NADES dilutions (1–10%) increased (p < 0.01) the transportation of BBR from the mucosal side to the serosal side in a concentration-dependent manner (p < 0.01) the transportation of BBR from the serosal side to the mucosal side when its concentration was higher than 10%  by the NADES dilutions when its concentration was higher than 1%  the absorption of FD4 across mouse gut sacs (p < 0.01)  , which smax and increased Cmax and AUC0–12 h of BBR in the portal vein indicated that NADES dilutions promoted the intestinal absorption of BBR. In the portal vein, the Cmax of BBR in the 1 and 10% dilutions of NADES treated group was 3.04 and 4.57 times of that in the control group, while the AUC0–12 h was 2.45 and 3.22 times, respectively. Furthermore, the pharmacokinetic parameters of BBR were improved in the liver, one of the major target organs. In the liver, the Cmax of BBR in the 1 and 10% dilutions of NADES treated group was 1.75 and 2.08 times of that in the control group, while the AUC0–12 h was 1.12and 1.65 times, respectively.As shown in p < 0.01) (p < 0.01) (p < 0.01), but no significant cytotoxicity was observed after incubation with 1.47 or 0.49 mg/ml malic acid for 4 and 24 h (p > 0.05) (p < 0.01)  . Interes < 0.01) . The res < 0.01) . In addi > 0.05) . In gut  > 0.05) ; it also > 0.05) . Malic a < 0.01) , but the < 0.01) . FinallyCoptidis Rhizoma extract. These metabolites were selected because they were usually used for artificial preparation of NADESs. The methods were simple, reliable, and suitable for quantitative analysis. The results showed that sucrose, malic acid, glucose, and choline are the four major primary metabolites in Coptidis rhizoma extract.In this study, three LC-MS/MS-based methods were first established and fully validated to determine the levels of 11 primary metabolites of Coptidis Rhizoma extract. In other words, NADESs would be “endogenously” present in the Coptidis Rhizoma extract. The most stable stoichiometric proportion of HBA and HBD is usually 1:1 , especially CYP3A, play a major role in the phase I metabolism of BBR . There ain vivo in a concentration-dependent manner, which was manifested by a significant increase in BBR exposure levels (AUC0–t and Cmax) in the portal vein of the mice. The level of BBR distributed in the liver increased mainly due to increased intestinal absorption. The influence of the NADES dilutions on the pharmacokinetics of oral BBR in systemic circulation was not investigated due to the poor dose-exposure relationship of BBR in systemic circulation (in vivo exposure levels are undoubtedly beneficial for improving its systemic effects. However, it should be noted that BBR itself has acute toxicity (in vivo exposure level of BBR should be controlled within a certain range. Researches on the relationship between dose-exposure level-toxicity are expected to achieve a balance of benefits and risks. In this experiment, no toxic reaction was found after oral administration of BBR that was dissolved in the NADES dilutions.The extremely low oral bioavailability limits the further research and development of BBR . Therefoculation . The inctoxicity , with a toxicity . TherefoCoptidis Rhizoma extract and pure BBR (Coptidis Rhizoma extract. In addition, it was found that BBR in Coptidis Rhizoma extract had a non-linear dose exposure relationship after oral administration (Coptidis Rhizoma extract reached 3 g/kg, the in vivo exposure level of BBR was much higher than the exposure level that was predicated according to dose proportion relationship (Coptidis Rhizoma extract is orally administered at a dosage of 3 g/kg and a volume of 20 ml/kg. The results suggested that malic acid and the malic acid based NADES may contribute to the non-linear dose exposure relationship of BBR in oral Coptidis Rhizoma extract.It has been reported that there are large pharmacokinetic differences between BBR in pure BBR . The resstration . That istionship . It is wIt is undeniable that the NADES has shortcomings as a CPE for oral BBR. Above all, the NADES itself showed acute toxicity to mice. It has been reported that NADESs may block and halt blood flow due to high intrinsic viscosity . For thiAfter being diluted, the NADES can no longer be regarded as a NADES. However, the NADES dilutions cannot be regarded as an aqueous solution of the four primary metabolites, because they may retain weak intermolecular interactions . The resmax of BBR was, respectively, 3.04 and 4.57 times and the AUC0–12 h was, respectively, 2.45 and 3.22 times of that in the control group. In contrast, in the 98 mg/kg and 980 mg/kg malic acid treated groups, the Cmax was, respectively, 3.03 and 3.91 times while the AUC0–12 h was, respectively, 2.00 and 2.74 times of that in the control group.Malic acid was identified as a major component in the NADES in terms of solubility, acute toxicity, cytotoxicity, and influences on the intestinal absorption and pharmacokinetics of oral BBR. The results are consistent with the literature reports, that is, the physicochemical and biological properties of NADESs, such as solubility , cytotoxCoptidis Rhizoma extract. The dilutions of the NADES promoted the intestinal absorption of oral BBR by increasing solubility, reducing intestinal efflux, improving intestinal metabolic stability, and reversibly opening the tight junctions between enterocytes. This study showed for the first time that “endogenous” NADESs could be prepared from herbal extracts and could improve the pharmacokinetic properties of coexisting active constituents. This study demonstrated the pharmacokinetic synergy of the constituents of Coptidis Rhizoma extract. In addition, this study was the first to systematically show the mechanisms through which NADESs improve the pharmacokinetics of another compound, which may help to promote the use of NADESs in oral drug delivery.In summary, a stable NADES was prepared according to the content proportion of the four primary metabolites in


Dear Editor,Rheumatoid arthritis (RA) is a common chronic autoimmune disease that causes progressive joint destruction and involves severe damage to physical function and life quality.Neuron navigator 2 (NAV2) belongs to the neuron navigator family which mainly regulates growth, migration, and regeneration of neurons in the nervous system and is highly expressed in the brain, kidney, liver, and also existed in the skeleton, heart, placenta, as well as abundantly elevated in colon cancers.We first examined NAV2 protein level in blood samples and found a significant increase of NAV2 expression in RA patients compared with osteoarthritis (OA) patients or healthy samples Figure . The cliNAV2 mRNA level reached a peak at 3 h and then declined, NAV2 protein level showed a continuous increase from 0 to 24 h and decreased slightly at 48 h  for 0, 3, 6, 12, 24 and 48 h to evaluate an inflammatory response Figure . Interesh Figure . After th Figure . Intriguh Figure . Besidesh Figure  and G. An Figure . More imn Figure  and J. In Figure . As Wnt/n Figure , the pron Figure . Furtherd Figure .E2F1 also resulted in decreased E2F1 and NAV2 significantly (Figure We next investigated the underlying mechanisms by which NAV2 promoted inflammatory response in RA. We found that NAV2 could be regulated by transcription factor E2F1. First, we found E2F1 increased in RA FLS and AIA rat synovial tissues Figure  and B, ay Figure . Furthery Figure . Then thy Figure  and S.In conclusion, the present study revealed an important novel finding that NAV2 plays a critical role in RA, and provided a new understanding of the molecular mechanism of RA which is unique and previously unreported. We also speculate targeting NAV2 might not just affect inflammation in RA but could also interfere with a major cell‐cell interaction involved in sensitization of joint‐innervating neurons that drive pain in arthritis.R. Wang, J. Mao, and Y. Zhu designed research, analyzed data, and wrote the manuscript; and R. Wang, M. Li, W. Wu, Y. Qiu, W. Hu, Z. Li, Z. Wang, Y. Yu, J. Liao, W. Su performed research. All authors read and approved the manuscript.All the experimental processes were conducted within the approved guidelines of the Ethics Review Committee for Animal Research of Macau University of Science & Technology. Appropriate measures were taken to minimize the use of animals as well as their suffering.The authors declare that they have no conflict of interest.Supporting InformationClick here for additional data file.


Rubus chingii var. suavissimus (S. K. Lee) L. T. Lu (RS)—a sweet plant also known as Tiancha distributed in the south of China where it is used as a beverage—recently gained extensive attention as adjuvant therapy of diabetes and hypertension. Although pharmacological studies indicate that RS has beneficial effects in regulating lipid metabolism disorder characteristics, the active chemicals responsible for this effect remains unclear. The present study aims to predict the effective substances of RS on regulating lipid metabolism disorder through the analysis of the chemical profile of RS, the absorbed prototype components in rat plasma, and network pharmacology. Also, a UPLC method able to quantify the screened potential effective chemicals of RS products was established. First, a total of 69 components—including diterpene, triterpenoids, flavonoids, polyphenols, and lignans—were systematically characterized in RS. Of those, 50 compounds were detected in the plasma of rats administered with RS extract. Through network pharmacology, 9 potential effective components, 71 target genes, and 20 pathways were predicted to be involved in RS-mediated regulation of lipid metabolism disorder. The quantitative analysis suggested that the contents of potential effective components varied among samples from different marketplaces. In conclusion, the presented results provide a chemical basis for further research of Rubus chingii var. suavissimus. Lipid metabolism disorder is an important pathogenic factor of diseases characterized by an abnormal lipid metabolism, such as atherosclerotic vascular disease, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD) . While sRubus chingii var. suavissimus (S. K. Lee) L. T. Lu (Rubus suavissimus S. Lee (RS) (Siraitia grosvenorii (Swingle) C. Jeffrey ex Lu et Z. Y. Zhang and Stevia rebaudiana Bertoni (L. T. Lu  or RubusLee (RS)  also knoLee (RS) . As an i Bertoni  and is m Bertoni . Rubusos Bertoni . Our pre Bertoni  and rubu Bertoni  could al Bertoni . Moreove Bertoni . HoweverAnalysis of compounds migrating into the blood proved to be a very useful approach for the identification of bioavailable components of traditional Chinese medicine (TCM) . Moreovein vivo absorbed components from RS extract-administrated rats. Based on this, a network pharmacology approach was developed to systematically predict the main active components and possible molecular action mechanisms. In addition, the main chemicals predicted to have the potential activity to regulate lipid metabolism disorder were quantified by a validated UPLC method. These results provide a reference for further research and exploration of pharmacodynamics material basis and mechanisms of RS.Therefore, this study aims to predict the active components and molecular mechanisms underlying the protective action of RS on regulating lipid metabolism disorder. First, an ultra-perfomance liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) method was established to characterize the chemical profile of RS as well as the Rubus chingii var. suavissimus (S. K. Lee) L. T. Lu by Professor Yi Cai, Guangxi University of Chinese Medicine. The voucher specimen is deposited in the herbarium of School of Pharmacy, Guangxi University of Chinese Medicine.Thirteen batches of plant materials from markets were collected  and authGallic acid, caffeic acid, ellagic acid, rutin, hyperoside, isoquercitin, quercitrin, quercetin, kaempferol, rubusoside, steviol, isosteviol, ursolic acid, and oleanolic acid (purity ≥ 98%) were provided by Chengdu PFID Co., Ltd. Formic acid and acetonitrile of LC-MS grade were purchased from Thermo Fisher Scientific. Other chemicals were of analytical grade.ad libitum. Before experiments, they were fasted for 24 h but with free access to water.Male Sprague Dawley (SD) rats (200 ± 20 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd. . Animal care and procedures were approved by and conducted according to the standards of the Guangxi University of Chinese Medicine  (Certificate DW20200713-003). The rats were housed in an animal room  on a 12 h dark/12 h light cycle with food and water Accurately weighted references of caffeic acid, rutin, hyperoside, isoquercitin, quercitrin, quercetin, kaempferol, rubusoside, steviol, isosteviol, ursolic acid, and oleanolic acid were dissolved in methanol, except for gallic acid (in water) and ellagic acid (in DMSO) to get the stock solutions of each reference compound. All reference stock solutions were stored at 4°C.0.5 g weighed sample powders (RS08) were extracted with 10 mL 60% ethanol at room temperature for 60 min by ultrasonication, then complemented for the weight loss, and centrifuged at 12,000 rpm for 10 min, after which the supernatant fluid was diluted 10 times with 60% ethanol and filtered through a 0.22 μm filter before analysis.Each reference stock solution was mixed and diluted with methanol to a suitable concentration and then filtered through a 0.22 μm filter before analysis.200.0 g RS powder (RS08) was extracted three times with 1,600 mL 95% ethanol (60 min per time) by ultrasonication, filtered, and recycled the solvent under reduced pressure to obtain the dried extract. It was diluted by 0.5% sodium carboxymethyl cellulose (CMC-Na) to prepare the suspension for animal experiments.Ten male SD rats were randomly divided into control group (two) and RS group (eight). Rats in the RS group were gave at a fourfold human equivalent dosage (4 × 2.06 g/kg), and the blood was collected from the orbital veins in heparinized centrifuge tube at 0.17, 0.33, 0.5, 1, 1.5, 2, 4, 6, 8, and 12 h. Rats in the control group received an equal volume of 0.5% CMC-Na orally after which the blood was collected by the abdominal aortic method. The blood samples were centrifuged at 4,000 r/min for 10 min at 4°C to obtain the plasma which was stored at −80°C.50 μL plasma samples of the above time points were combined (or 500 μL control plasma samples) and loaded onto a pre-equilibrated solid-phase extraction (SPE) column and successively eluted by 3 ml of water and 3 ml of methanol. The methanol eluent was collected and evaporated to dryness under nitrogen at 40°C. The residue was reconstituted in 100 μL methanol and centrifuged at 12,000 rpm for 10 min to obtain the supernatant fluid for analysis.Dionex UltiMate3000 coupled with a hybrid quadrupole orthogonal time-of-flight (Q-TOF) tandem mass spectrometer . The chromatographic separation was carried out on an InertSustain ODS-3 LC column . The mobile phase consisted of  0.1% formic solution (A) and acetonitrile (B) using the following gradient program: 0–1 min, 5% B; 1–6 min, 5–15% B; 6–20 min, 15–24% B; 20–25 min, 24–33% B; 25–30 min, 33–54% B; 30–35 min, 54–80% B; 35–38 min, 80% B; 38–40 min, 80–95% B; 40–43 min, 95% B; 43–45 min, 95–5% B; and 45–50 min, 5% B with a sample injection volume of 5 μL. The solvent flow rate was 0.5 ml/min and the column temperature was set at 40°C.The UPLC-Q/TOF-MS analysis was performed on a –), with a drying gas flow and temperature of 8 L/min at 200°C, a nebulizer pressure of 2.0 bar (=29 psi), a collision RF of 500 Vpp, a transfer time of 60.0 µs, and a prepulse storage of 5 µs. Automatic MS/MS experiments were performed adjusting the collision energy values as follows: m/z 50, 10 eV; m/z 500, 25 eV; and m/z 1,000, 40 eV. Argon was used as collision gas for CID in the MSE mode. The ion scan ranged from m/z 50 to 1,000. Sodium formate solution was used as a calibration solution. The MS data were processed through DataAnalysis 4.3 software .The ESI source was operated at an optimized capillary voltage of 3.5 V (ESIhttp://www.lilab-ecust.cn/pharmmapper/), STITCH (http://stitch.embl.de/) and SwissTargetPrediction (http://www.swisstargetprediction.ch/). Beyond this, some known molecular targets of identified compounds were collected for comprehensive analysis through literature mining. All targets were combined and the duplicates were removed.According to the results of the chemical profiling and components absorbed in plasma of RS, the absorbed prototypes in rat plasma were selected as candidate compounds. The molecular targets of RS were identified by PharmMapper (https://www.genecards.org/). Next, the overlapping genes between RS and lipid metabolism disorder were matched by the Venn diagram.The related targets of lipid metabolism disorder were collected from GeneCards (https://string-db.org/). For the analysis, the study organism was limited to Homo sapiens, while all other settings were kept as default values.To gain insight into the molecular mechanisms underlying the action of RS on lipid metabolism disorder, a bioactive compound–therapeutic target network was constructed with Cytoscape 3.7.1 to depict the interactions between target genes and bioactive compounds. Next, the degree value was calculated by using the NetworkAnalyzer plug-in. Compounds with a high degree value were deliberated as main bioactive RS-derived compounds potentially involved in the RS-mediated alleviation of lipid metabolism disorder. The protein–protein interaction (PPI) network was supported by STRING (https://david.ncifcrf.gov/); terms with p < 0.05 were considered significant and captured.To decipher the possible signaling pathway involved in the intervention of RS on lipid metabolism disorder, the KEGG pathway enrichment analysis was performed with DAVID 6.8 . The mobile phase consisted of 0.1% phosphoric acid (A) and acetonitrile (B) using a gradient program of 0–6 min, 5–15% B; 6–20 min, 15–24% B; 20–37 min, 24–80% B; 37–38 min, 80–95% B; 38–38.5 min, 95–5% B; and 38.5–40 min, 5% B. The solvent flow rate was 0.35 ml/min and the column temperature was set at 40°C. The detection wavelengths were the following: 205 nm for 0–6 min; 254 nm for 6–23 min; and 205 nm for 23–40 min. An aliquot of 1.0 μL was injected for analysis.UPLC analysis was performed on an Agilent 1290 I system equipped with a 1290 VWD detector, a 1,290 vial sampler, and a 1290 high-speed pump. The column configuration was an InertSustain CStock reference solutions of gallic acid, caffeic acid, rutin, ellagic acid, rubusoside, and kaempferol were prepared and diluted to six appropriate concentrations using the corresponding solvent to get the reference working solutions. Six calibration curves were constructed by plotting the content (Y) vs. the peak areas (X) of each analyte. The limit of quantitation (LOD) and limit of detection (LOQ) were determined at a signal-to-noise ratio (S/N) of about 3 and 10, respectively. The intraday precision was examined by six repetitive injections within one day, and the interday precision was carried out for three consecutive days. Repeatability was acquired by analyzing six replicates of the same sample solution (RS08). To determine the stability, the same sample solution (RS08) was injected at 0, 1, 2, 4, 6, 8, 12, and 24 h, respectively. The accuracy of the method was expressed by the recovery. Approximately 100% amount of each reference standard equivalent to those in 0.25 g extracts were added to the same RS sample (RS08), after which the sample solutions were prepared in six replicates. Recoveries were calculated by the following equation: recovery (%) = 100 × /spiked amount.All data were expressed as the mean ± standard deviation (SD). T-test was used to compare the two groups. The multigroup comparisons were analyzed by one-way analysis of variance (ANOVA) with SPSS 24.0 .n fragments data were compared with reference compounds, a self-built chemical database, and reported literature. In addition, a ClogP value was calculated by ChemDraw 11.0  to estimate the retention time of the isomers.A UPLC-QTOF/MS method was established to characterize the chemical profile. As MS and fragmentations were most informative in the negative ion mode, all analyses were conducted in the negative ion mode. The base peak intensity (BPI) chromatogram of RS in the negative ion mode is shown in In total, 69 compounds were identified or tentatively characterized, including 20 diterpenes, 13 triterpenes, 19 flavonoids, 15 polyphenols, and 2 lignans. Compounds 1, 9, 18, 19, 20, 21, 26, 32, 44, 50, 64, 66, 68, and 69 were identified as gallic acid, caffeic acid, ellagic acid, rutin, hyperoside, isoquercitrin, quercitrin, quercetin, kaempferol, rubusoside, steviol, isosteviol, ursolic acid, and oleanolic acid through the comparison of the retention time and quasimolecular ions with reference compounds, respectively. Compounds 4 (corilagin), 19 (rutin), and 50 (rubusoside) were taken as examples for explaining the fragmentation process .2O, –OCH2, –CO2, and –C6H9O5 (glucose unit). The main characteristic fragmentation behaviors of RS-flavonoids were the loss of glycosyl, elimination of small neutral fragments such as H2O and CO, and retro Diels–Alder (RDA) cleavage. RS-derived polyphenols are mainly galloyl-oxygen-diphenyl-type ellagitannins and gallotannins of which the fragmentation patterns could mainly be summarized as successive elimination of the galloyl group and lactonization , cyanidin-3,5-diglucoside (16), 5-hydroxy-7-methoxydihydroflavone (17), cyanidin 3-O-rutinoside (22), rubuside J (57), and euscaphic acid (63) in Rubus chingii var. suavissimus.Noteworthy, based on the reported chemicals of r plants , the curConsidering that the time to being absorbed into the blood is different for each RS extract-derived component, plasma samples were first collected at 10 different time points (0.17 ∼ 12 h) after the administration of RS extract and then finally combined. An SPE column was used for protein precipitation and separation.in vivo absorbed prototype component profile of the plasma of RS extract-administrated rats was obtained and analyzed.Based on reference compounds and the chemical profile of RS and by comparing pre- and postdose rat plasma, an n fragments data, is listed in – ion at m/z 641 which yielded characteristic fragment ions at m/z 479 [M–H–glucose]–, 317 [M–H–2glucose]–, and 273 [M–H–2glucose–CO2]–, it could be identified as rubusoside through comparison of the MS behavior to the reference.As shown in Most diterpenoids and triterpenoids existed as unchanged prototypes in rat plasma and displayed high peak intensity in the BPI chromatogram. Compared to flavonoid aglycones, more flavone glycosides were detected. The fact that RS-polyphenols displayed low peak intensity or were even hard to detect might be due to their bioavailability and the liver first-pass effect .in vivo absorbed components profile of RS. Then, a total of 1,882 molecular targets were obtained from PharmMapper, STITCH, and SwissTargetPrediction. Next, the name of the identified targets was converted into an official gene symbol by UniProt (https://www.uniprot.org/). Finally, a target library with 521 target genes was established after integrating the results and removing the duplicates.First, 50 candidate compounds were selected based on the 1,112 targets (relevance score>22.01) related to lipid metabolism disorder were gathered from GeneCards. Through Venn diagram matching, 213 overlapping target genes between RS and lipid metabolism disorder-related targets were identified. Of note, a target with a higher relevance score as calculated by GeneCards indicated a higher pertinence to the disease. As such, the top 71 targets  selectedAs shown in This network suggested the involvement of the 36 compounds and 71 genes in the beneficial effect of RS on lipid metabolism disorder. In network analysis, a high degree value generally indicates the node is closely connected with other target genes. Based on the calculated results from NetworkAnalyzer, compounds with a degree value ≥ 11 were considered as potential bioactive ingredients. As such, nine compounds including ursolic acid (21.0), oleanolic acid (21.0), quercetin (18.0), kaempferol (16.0), gallic acid (14.0), ellagic acid (14.0), caffeic acid (11.0), rubusoside (14.0), and rutin (16.0) might be the main effective RS-derived compounds involved in the beneficial effect of RS on lipid metabolism disorder.p < 0.01) are presented in p = 7.02 E−08, count = 11), the PPAR signaling pathway , the AMPK signaling pathway , and the HIF-1 signaling pathway .The top 20 KEGG pathways , 71 target genes, and 20 pathways are directly related to lipid metabolism disorder.Rubus leaf. Also, caffeic acid ameliorates hepatic steatosis and reduces endoplasmic reticulum stress in obese mice by regulating autophagy . Meanwhile, IR could promote the transcription of fatty acid synthetase (FAS) , lipoprotein lipase (LPL), and fatty acid binding protein 2 (aP2). PPARα and AMPK play important roles in regulating the processes of FFA transport and lipid oxidative metabolism (α (HIF-1α) mainly improves abnormal lipid metabolism by regulating oxidative stress  are deposited primarily in the liver and se (FAS) . On the tabolism . Hypoxiae stress . Interesα and PPARγ, resulting in the activation of downstream adipocyte genes aP2, Glut4, and LPL .First, several variables including solvent, extraction methods, extraction time, and number of cycles were optimized to obtain the maximal extraction efficiency. Maximal extraction was achieved through ultrasonication with 20 volumes of 60% ethanol for 60 min once or eight volumes of 95% ethanol for three times in case of intragastric administration to rats.18 LC column at 40°C using a linear gradient of 0.1% phosphoric acid and acetonitrile within 40 min. Considering that the maximum absorption wavelength of rubusoside situates at 205 nm while that of the other references situates at 254 nm, a wavelength conversion program was used to obtain a better resolution.Next, the conditions for chromatographic analysis including the type of column, mobile phase gradients, and detection wavelength were optimized. The results showed that optimal separation was obtained by eluting RS samples on an InertSustain Cr2 ≥ 0.9993) within the test ranges while the overall LODs and LOQs were ranged from 0.09 to 1.00 ng and 0.30 to 3.00 ng, respectively. The RSDs of precision, repeatability, stability, and recovery are shown in The regression equation, linearity range, correlation coefficients, and LODs and LOQs of gallic acid, caffeic acid, rutin, ellagic acid, rubusoside, and kaempferol are summarized in The obtained peaks in the chromatograms were identified through the comparison of the retention times and on-line UV spectra with those of the standard constituents . RetentiThe results showed that the contents of the determined compounds varied in the RS samples obtained from different markets. For example, high levels of ellagic acid and rubusoside were present in all batches. However, even though the samples were purchased from the same origin, the contents could show significant differences. For example, while samples RS06∼ RS08 were all purchased from Laibin of Guangxi, the content of rubusoside and ellagic acid in these samples ranged from 40.15 to 67.92 mg/g and 0.85 to 2.67 mg/g, respectively. Similarly, the content of rubusoside and ellagic acid in samples RS01∼ RS05—obtained from Guilin—ranged from 36.85 to 68.38 mg/g and 0.38 to 1.15 mg/g, respectively. Importantly, the effectiveness of herbal medicine is highly related to the quality of the raw materials which on itself is affected by a complex set of factors such as the place of origin, growing conditions, medicinal parts, and processing methods. Previously, a higher content of rubusoside in tender tissues of RS  which waIn order to strengthen the quality control of RS, a strict control of the quality of raw materials from the origin and processing procedure is required. Thus, simultaneous determination methods to identify effective bioactive components could be of importance not only to analyze underlying mechanisms but also to establish future quality control of RS.Rubus chingii var. suavissimus by UPLC-Q/TOF-MS. Among them, 50 absorbed prototype components were detected in rat plasma. Next, bioactive compounds–therapeutic target network analysis predicted nine active components , 71 target genes, and 20 pathways to be involved in RS-mediated alleviation of lipid metabolism disorder. Finally, UPLC analysis could show significant variability in the contents of six selected potential active compounds in RS samples obtained from different marketplaces. Taken together, the study provided a basis for further exploration of the mechanism on the regulation of lipid metabolism disorder and the development of Rubus chingii var. suavissimus–derived therapeutic drugs and healthcare products.In this study, a total of 69 compounds—including diterpenes, triterpenes, flavonoids, polyphenols, and lignans—were identified or tentatively characterized in


This study was aimed to evaluate the value of DNA index(DI) among pediatric acute lymphoblastic leukemia (ALL) treated on Children’s Oncology Group (COG) protocols between 2000 and 2015.Retrospective study were analysis among pediatric ALL patients from the TARGET dataset.HR = 0.6, 95% CI 0.4–0.8, P = 0.003) and OS  compared to patients with no ETV6/RUNX1. On the contrary, BM NR on Day+ 29 showed a significant decrease in EFS  and OS .Totally, 1668 eligible pediatric patients were enrolled in this study. Of them, 993 are male and 675 are female with a median age of 7.6 years old. The median follow-up for those patients was 7.7 years (range 0.1–15.7 years). The probability of 15-year EFS and OS were reported to be 67.5 ± 3.1% and 78.3 ± 2.5%, respectively. BCR/ABL1 fusion gene affected the early treatment response and the survival of childhood ALL. Moreover, those patients with ETV6/RUNX1 fusion gene were also significantly associated with better EFS  for DI < 1.1 while 8.8  for DI ≥ 1.2 and 0.0  for 1.1 ≤ DI < 1.2. Generalized additive models revealed that the lowest rates of the adverse outcomes estimated to occur among DI between 1.1 and 1.2.Multivariate analysis showed that DI was significantly associated with better EFS and OS. The threshold effect of DI on poor outcome was significant after adjusting for potential confounders. The adjusted regression coefficient (Log RR) was 0.7 (95%For those childhood ALL treated on COG protocols between 2000 and 2015, ETV6/RUNX1 and BM NR were closely related to the prognosis. Moreover, the DI between 1.1 and 1.2 can serve as a significant cut-point discriminating the risk group, which indicated a favourable prognostic factor. Acute lymphoblastic leukemia (ALL) is the most common hematological malignant tumor in children, accounting for 80% of childhood leukemia . ALL is n = 17; Down syndrome, n = 33), 272 were not assessable (inadequate information on diagnosis or treatment), and 1668 childhood ALL were enrolled in our study finally.The results published here are in whole based upon data generated by the TARGET (https://ocg.cancer.gov/programs/target) initiative, phs000218. The data used for this analysis are available at https://portal.gdc.cancer.gov/projects. The trial was approved by COG, and informed written consent was obtained from all study participants. All methods were performed in accordance with the relevant guidelines and regulations such as French-American-British (FAB) criteria in the manuscript. Chemotherapy protocols for childhood ALL included 9906, AALL0232, AALL0331 and AALL0434, the application of different treatment protocols were related to the update time of the protocols and the choice of researchers in different centers.The clinical and laboratory data about pediatric ALL aged≤18 years old was downloaded from the Therapeutically Applicable Research to Generate Effective Treatments dataset . Between May 2000 and August 2015, 1990 patients were registered in the TARGET database: 50 were not eligible  samples were transported overnight and processed at the blood analysis laboratory within 24 h. Diagnosis of ALL was based on French-American-British (FAB) criteria: less than 3% blasts positive for myeloperoxidase or Sudan black, and negative for nonspecific esterase . ImmunopComplete remission (CR) was defined as non-leukemic signs, no leukemic cells detected in blood smears, active hematopoiesis in bone marrow, less than 5% of leukemic primordial cells, and normal cerebrospinal fluid (CSF). Bone marrow aspiration was examined on Day+ 29 at the end of phase IA. According to the dataset, the year of diagnosis is from 2000 to 2015. The last follow-up year was from 2000 to 2017.http://www.empowerstats.cn/). A 2-tailed p < 0.05 was considered to be statistically significant in all analyses.Baseline characteristics were presented as mean ± SD for continuous variables and as frequency (%) for categorical variables. Comparisons between groups were made using the chi-square test for categorical variables and analysis of variance or the Kruskal-Wallis test for continuous variables. To assess outcome, the following parameters were used: CR rate, event-free survival , overall survival . EFS and OS were estimated by the Kaplan–Meier method and compared using the log-rank test. The univariate and multivariate logistic regression analyses were performed to identify risk factors of early treatment response and prognostic factors. A generalized additive model (GAM) with a spline smoothing function was applied to examine the relationship between DI and adverse clinical outcomes . Adjusted OR with 95% CIs were estimated to evaluate the association of DI and adverse clinical outcomes, which was with adjustments for gender, age, race, chemotherapy protocol, white blood cell(WBC); central nervous system(CNS) status, fusion gene, prednisone response, BM blasts on Day+ 29, immunophenotype and karyotype. All statistical analyses were performed using the IBM SPSS Statistics version 22.0, and EmpowerStats (9/L (range 0.4–1306.0 × 109/L), and the median DI was 1.0 (range 0.0–1.9).Totally, 1668 eligible pediatric patients were enrolled in this study. The baseline characteristics of the eligible patients were presented in Table n = 1625) achieved CR at the end of remission induction chemotherapy (Day + 29).As a result, 222 patients accept 9906 protocol, 789 patients accept AALL0232, 415 patients received AALL0331 protocol and the remaining 242 patients received AALL0434 protocol. As for the early treatment response, 1154 patients responded well to prednisone on Day+ 8, while the remaining 514 patients did not respond well to prednisone. Most of the pediatric patients (The median follow-up for those patients was 7.7 years (range 0.1–15.7 years). Finally, 309 patients subsequently developed hematological relapse (presence of leukemic blasts > 25% in BM), 113 patients developed CNS relapse and testes site of relapse occurred in 11 children. The probability of 10-year EFS and OS were reported to be 67.5 ± 3.1% and 78.3 ± 2.5%, respectively , B Cell ALL, B precursor , hyperdiploid, BCR/ABL1 fusion gene, poor prednisone response , AALL0232 protocol  and AALL0434  protocol were the independent risk factors  and OS . Those patients with ETV6/RUNX1 fusion gene were also significantly associated with better EFS  and OS  compared to patients with no.When we included DI with other risk factors in the Cox model, including age, gender, race, WBC, fusion gene, prednisone response, BM blasts Day+ 29, immunophenotype and karyotype as co-variables, we identified DI as an independent factor for both EFS and OS in pediatric patients with ALL Table . DI was HR = 3.1, 95%CI 2.1–4.5, P < 0.001) and OS .ETV6/RUNX1. On the contrary, BM NR on Day+ 29 showed a significant decrease in EFS  for DI < 1.1 while 8.8  for DI ≥ 1.2 and 0.0  for 1.1 ≤ DI < 1.2 , but no significant difference was found. However, the OS of pediatric ALL with a DI between 1.1–1.2 were significantly higher than those with DI of < 1.1 or ≥ 1.2 . As a result, the EFS of pediatric ALL with a DI between 1.1–1.2 were higher than those with DI of < 1.1 or ≥ 1.2  investigators from different institutions. After the inclusion and exclusion criteria, 1668 pediatric ALL were eventually enrolled in our study, which was the largest-scale study to evaluate the value of the DI among childhood ALL treated with recent chemotherapy protocols since 2000s.First of all, we investigated the value of DI in early treatment response of childhood ALL. In the existing evaluation system, the main factors affecting the early treatment response of childhood ALL include age, initial WBC, specific gene fusions and so on. According our univariate and multivariate analysis, which showed that age ≥ 10 years, BCR/ABL1 fusion gene and poor prednisone response had a significant negative impact on early treatment response, while hyperdiploid and an improved chemotherapy regimen had a significant positive impact, and these results were basically consistent with those reported in previous literature –20. It hETV6/RUNX1 positive ALL is considered to occur in prenatal period, which may precede the preleukemic stage . FurtherMoreover, we showed an independent favorable outcome for the patients on DI in terms of 10-year EFS and OS.We further revealed a threshold effect based on the DI and provide clear evidence of a nonlinear association between DI and adverse clinical outcomes. To the best of our knowledge, this is the first study to describe a U-shaped relationship between DI and adverse clinical outcomes. Interestingly, using these data, we were able to identify DI ranging from 1.1 to 1.2 in which the rates of an adverse clinical outcome of induction failure, relapse, progression or death of any cause were lowest in our population. Next, we explored the survival as just three groups having different DI levels among pediatric patients with ALL. Finally we found that DI between 1.1 to 1.2 conferred favorable prognostic impact on survival in patients with DI of <1.1 or ≥ 1.2. As for the EFS was not significantly different in the 1.1–1.2 group, but the OS was much different and statistically significantly. We supplied that there were many factors that affect EFS, but the only factor that affects OS was whether the patient was death.Inconsistent with previous studies –9, DI ofThe study has several limitations. Most importantly, it was a retrospective study, so bias in this study was inevitably. Secondly, the lack of some data may lead to the incompleteness of the results. In spite of these limitations, this study is a multicenter, large-scale retrospective study to re-evaluate the value of DI, which provide strong evidence for those pediatric ALL treated with recent chemotherapy protocols. In conclusion, the DI between 1.1 and 1.2 can serve as a significant cut-point discriminating the risk group as ever, which remained an independent prognostic factor.


To establish reference data on required competition age regarding performance levels for both sexes, all swimming strokes, and race distances and to determine the effect of competition age on swimming performance in the context of other common age metrics. In total, 36,687,573 race times of 588,938 swimmers (age 14.2 ± 6.3 years) were analyzed. FINA  points were calculated to compare race times between swimming strokes and race distances. The sum of all years of race participation determined competition age.β = 0.19 to 0.33). Increased race distance from 50 to 1500 m, decreased effects of chronological age (β = 0.48 to − 0.13) and increased relative age effects (β = 0.02 to 0.11). Reference data from the present study should be used to establish guidelines and set realistic goals for years of competition participation required to reach certain performance levels. Future studies need to analyze effects of transitions between various swimming strokes and race distances on peak performance.Across all events, swimmers reach top-elite level, i.e. > 900 FINA points, after approximately 8 years of competition participation. Multiple-linear regression analysis explained up to 40% of variance in the performance level and competition age showed a stable effect on all race distances for both sexes (The online version contains supplementary material available at 10.1186/s13104-022-05969-6. At international competitions, i.e. European championships, chronological age was related to success , possiblWhile the concept of deliberate practice , 3 is haAdditionally, the relative age, i.e. age-related difference between athletes born early and late in the year, affects talent selection and progression towards top-elite performance , 11. By In total, 36,687,573 race times of 588,938 swimmers  from the years 2000 until 2019 were included in the present study. Data were provided from the official database [To compare race times between swimming strokes and race distances, FINA  points for each race time were calculated . FINA poAnalysis step 01 From the 2019 race data, each individual swimmer’s best race, i.e. most FINA points, was used to establish the ranking for each race category. As race categories were defined: (a) all events including all swimming strokes and all race distances from 50 to 1500 m; (b) each swimming stroke, i.e. BU, backstroke (BA), BR, freestyle (FR), individual medley (IM), using pooled data of its 50, 100, and 200 m races; (c) each race distance using the 50, 100, 200, 400, 800, and 1500 m FR events. The rankings were established based on long-course races, due to their superior recognition, i.e. at the Olympics, compared to short-course events (25 m pool length).Analysis step 02 Within each race category, each swimmer was tracked retrospectively, and the races extracted for every year from 2000 to 2019. Total number of years with race times of the particular race category determined competition age. As short-course races contribute to the training and development process, short- and long-course races were included for determination of competition age . Table P < 0.001). Regarding race distances, there was a stable effect of competition age from 50 to 1500 m in males  and females (β = 0.39 to − 0.13). The effect of relative age was fairly small yet increased with race distance from 50 to 1500 m in males (β = 0.02 to 0.11) and females (β = 0.03 to 0.08).Regression analysis explained up to 40% of variance in swimming performance, i.e. FINA points, and showed a significant effect of competition age, chronological age, and relative age (Swimmers accumulated approximately 8 years of competition practice to reach top-elite level, i.e. > 900 FINA points, regardless of event. The present descriptive data show years of competition practice needed to reach various performance levels for each swimming stroke and race distance. While the regression model explained up to 40% of variance in swimming performance depending on the event, the effect of competition age remained stable across all race distances. The effect of chronological age continuously decreased, and the effect of relative age continuously increased the longer the race distance, i.e. from 50 to 1500 m.Previous studies showed the largest relative age effect at early junior age that decreased the older the swimmers  and evenThe present study showed a stable effect of competition age for all swimming events, which supports previous findings that top-elite performance needs to be developed over time and requires accumulated years of practice , 3. HowePrevious studies show the value of deliberate play which is the unspecific and unstructured involvement in the same or other sports , 26. ComThe stable effect of competition age across all swimming events shown in the present study supports the findings that accumulated practice contributes to elite age success , 3, in pThe present study determined competition age, assuming that regular competition participation is part of a well-structured training process. However, future studies should use questionnaires and training diaries to determine training age. FINA point ranking was established based on each swimmer’s best race within the race category. As swimmers specialize in a particular swimming stroke rather than race distance , the swiAdditional file 1: Figure S1. Flow chart of the data analysis procedure.


Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). Results show the nanosystems have a quasi-spherical shape of 12–18 nm in size for CDs_P and 15–20 nm in size for CDs_C. UV–Vis characterization indicates a broad absorption band with two main peaks at about 270 nm and 300 nm, respectively, attributed to the π-π* and n-π* transitions of the CDs, respectively. Both samples show photoluminescence (PL) spectra excitation-dependent with a maximum at λem = 420 nm (λexc = 300 nm) for CDs_P and a red-shifted at λem = 445 nm (λexc = 300 nm) for CDs_C. Band gaps values of ≈ 1.48 eV for CDs_P and ≈ 1.53 eV for CDs_C are in agreement with semiconductor behaviour. ζ potential measures show very negative values for CDs_C compared to CDs_P . The evaluation of the antibacterial properties highlights that both CDs have higher antibacterial activity towards Gram-positive than to Gram-negative bacteria. In addition, CDs_C exhibit bactericidal behaviour at concentrations of 360, 240, and 120 µg/mL, while lesser activity was found for CDs_P . This finding was correlated to the higher surface charge of CDs_C compared to CDs_P. Further investigations are in progress to confirm this hypothesis and to gain insight on the antibacterial mechanism of both cultivars.Carbon nanomaterials have shown great potential in several fields, including biosensing, bioimaging, drug delivery, energy, catalysis, diagnostics, and nanomedicine. Recently, a new class of carbon nanomaterials, carbon dots (CDs), have attracted much attention due to their easy and inexpensive synthesis from a wide range of precursors and fascinating physical, chemical, and biological properties. In this work we have developed CDs derived from olive solid wastes of two Mediterranean regions, Puglia (CDs_P) and Calabria (CDs_C) and evaluated them in terms of their physicochemical properties and antibacterial activity against  Since their serendipitous discovery as byproducts of the arc-discharged synthesis of single-walled carbon nanotubes , carbon 2 core, surrounded by sp3 imperfections and high oxygen content on their surface  for both CDs.The bactericidal activity of both CD dispersions was evaluated against both S. aureus viability reduction of about 99.9% (MIC99) at 360 µg/mL and 90% bacterial inhibition (MIC90) at about 120 µg/mL, while no bacterial inhibition was observed against P. aeruginosa. On the contrary, no bacterial inhibition was observed for CDs_P at all using concentrations (ranging from 60 to 360 µg/mL) against either bacterial strain.Our results showed that CDs_C exhibit an To further evaluate the antibacterial properties of both CD types, we also performed an MTS cell viability assay .S. aureus viability at the higher concentrations , while at the lowest concentration (60 µg/mL), it is reduced by about 50% compared to the control (untreated). On the contrary, MTS data of CDs_P indicated a bacterial cell reduction of only 30% at the highest concentration (360 µg/mL), showing very poor antibacterial activity. In more details, the bacterial viability of CDs_C was found of 47.5 ± 2.37% at 60 µg/mL, 4.75MTS data showed that CDs_C dispersion reduces almost completely P. aeruginosa showed that only CDs_C at the higher concentration (360 µg/mL) exhibited a bacterial reduction of about 20%. In more detail, the bacterial viability of CDs_C was found to be 98.4 ± 1.17% at 60 µg/mL, 95.75On the other hand, MTS data obtained against S. aureus than in P. aeruginosa probably due to the different cell wall compositions. Several action mechanisms have been suggested to explain the antibacterial activity of CDs closely related to their physicochemical properties, including their dimensionalities, lateral size, shape, number of layers, surface charges, the presence and nature of surface functional groups, and doping [In addition to the above considerations, we also observed a different level of antibacterial activity between CDs_P and CDs_C, more evident in d doping ,36,37,38E. coli (Gram negative), such as DNA fragmentation, extracellular exposure of phosphatidylserine, condensation of the chromosome, and loss of structural integrity, but the same effects did not occur with uncharged glucose carbon dots (GC-dots). Based on these considerations, our results could be compared with negatively charged candle-soot C-dots (CC-dots). Although similar results were obtained against P. aeruginosa, we found that S. aureus viability was almost totally and slightly reduced when treated with CDs_C and CDs_P, respectively. Since our systems, CDs_C and CDs_P, exhibit similar sizes of nanoparticles, and no surface functionalization was carried out, neither factor is relevant to the findings, indicating that CDs_C show higher antibacterial activity compared with to CDs_P. These results suggest, rather, different antibacterial mechanisms for the two cultivars, probably attributable to the different surface charges and ζ potential. Although both CDs from the two cultivars exhibited negative surface charges, CDs_C have much more of a negative charge than do CDs_P (about −38 mV for CDs_C and −18 mV for CDs_P at pH = 7). Actually, during the bacterial growth, the medium pH decreased over time (0–8 h), reaching more acidic values (from 7.2 to about 5.5)  See . By consS. aureus. The UV–Vis characterization shows a typical broad absorption band with two main peaks at about 270 nm and 300 nm, attributed to the π–π* and n–-π* transitions of CDs, respectively. The PL spectra are excitation-dependent, and CDs_P shows emission maximum at λem = 420 nm (λexc = 300 nm), while CDs_C has an emission maximum red-shifted at λem = 445 nm (λexc = 300 nm). Band gaps values of ≈ 1.48 eV for CDs_P and ≈ 1.53 eV for CDs_C, respectively, were found, in agreement with semi-conductor behaviour. ζ potential values are negative for both cultivars. However, CDs_C feature a more negative charge, in fact three times more negative than that of CDs_P (−38 mV vs. −18 mV at pH = 7). The TEM morphological inspection shows quasi-spherical nanoparticles 12–18 nm in size for CDs_P and 15–20 nm in size for CDs_C. The evaluation of antibacterial properties highlights that both CDs have higher antibacterial activity towards Gram-positive bacteria than to Gram-negative bacteria. In addition, the evaluation of the antibacterial properties towards S. aureus of both CD types highlights that CDs_C exhibit antibacterial properties at concentrations of 360, 240, and 120 µg/mL, while CDs_P shows a bacterial cell reduction of 30% at the highest concentration of 360 µg/mL. This finding was correlated to the highest surface charge of CDs_C compared to CDs_P, which is still very negative during bacterial growth, reaching an acidic pH of 5.5. Further investigations are in progress to confirm this hypothesis and gain insights on the antibacterial mechanism of both cultivars.In this paper, we report on the development of CDs derived from olive solid wastes of two Mediterranean regions, Puglia (CDs_P) and Calabria (CDs_C) and their evaluation in terms of their physicochemical properties and antibacterial activity against


Objective: To evaluate the efficacy and safety of different doses of sildenafil for persistent pulmonary hypertension of the newborn (PPHN) with Bayesian random effects network meta-analysis.Methods: We searched Chinese and English databases for randomized controlled trials (RCTs) concerning sildenafil in newborns with persistent pulmonary hypertension from 1998 to December 2020.Results: Twenty-two RCTs including over 2131 patients were included. Sildenafil was administered by nasal feeding at 0.3–2 mg/kg every 4–6 h. The network meta-analysis revealed that 1.5 mg/kg of sildenafil led to a significant decrease in pulmonary artery systolic pressure (PASP) compared with 0.3 and 0.6 mg/kg (p < 0.05); 1.5 mg/kg was better than 0.3, 0.5, and 1.0 mg/kg at increasing the partial pressure of oxygen (PaO2) (p < 0.05); 1.5 mg/kg was better than 0.5, 0.6 and 1.0 mg/kg at reducing the partial pressure of carbon dioxide (PaCO2) (p < 0.05); and 1.2 mg/kg was better than 0.3, 0.5 and 1.0 mg/kg at increasing the arterial oxygen saturation (SaO2) (p < 0.05). The surface under the cumulative ranking analysis (SUCRA) results showed that 1.5 mg/kg had the best effect in reducing PASP  and PaCO2 (91.1%) and increasing PaO2 , 2.0 mg/kg had the best effect in increasing SaO2  and total effective rate ). No severe adverse effects were observed with the different doses of sildenafil.Conclusion: Different doses of sildenafil can significantly improve PPHN, and 1.5 mg/kg of sildenafil has better clinical efficacy and does not increase the probability of adverse reactions. Persistent pulmonary hypertension of the newborn (PPHN) is one of the most serious fatal diseases in the neonatal period; this condition mostly occurs during the transition from fetal to adult circulation. It is characterized by elevated pulmonary vascular resistance (PVR) that leads to labile hypoxia in the immediate postnatal period with or without respiratory distress . The incPPHN remains a major cause of morbidity and mortality in neonatal centers across the globe. It is therefore essential to take the available treatment modalities to ensure better outcomes. Phosphodiesterase (PDE) inhibitors have recently been studied as therapeutic agents for PPHN. Sildenafil is the most well-researched PDE5 inhibitor for PPHN. It was approved for the treatment of pulmonary hypertension (PH) in adults in 2005. Notably, sildenafil is not approved for use in PPHN. To date, most case reports and prospective or retrospective studies  have shoUnfortunately, pharmacokinetic data on the optimal dose regime in neonates are limited. Sildenafil dosing data vary widely between sources for PPHN, and the indications have been derived from data from adults and older children. To date, there is still considerable controversy about the dosage of sildenafil for PPHN, and an optimal dose has not yet been recommended. The European Medicines Agency (EMA) recommends its use at “low doses.” NeoFax, a well-known neonatal drug manual published in 2006, states that a dose of 0.3–1 mg/kg can be given every 6 h for newborns with PPHN. In 2011, the recommended dose of sildenafil was 0.5–2 mg/kg every 6 h with a maximum dose of 3 mg/kg. The British National Formulary suggested including incremental doses ranging from 0.5 to 2 mg/kg every 6 h . The AmeNewborns from 0 to 28 days postpartum who met the diagnostic criteria for PPHN of pulmonary artery systolic pressure (PASP) > 35 mmHg were included. PPHN can be formed by special anatomical and physiological characteristics in the neonatal period, and can also be secondary to parenchymal lung diseases, meconium aspiration syndrome (MAs), respiratory distress syndrome (RDs), pneumonia or sepsis with or without asphyxia . All inf1) newborns with a confirmed diagnosis of PPHN, 2) randomized controlled clinical trials, 3) studies that involved various different doses for PPNH, and 4) outcome indices including pulmonary artery pressure. Studies were excluded for the following reasons: texts that were not in English or Chinese, duplicate publications, case reports or reviews and texts with no observation indicators.The inclusion criteria were as follows: We searched the Wanfang, China National Knowledge Infrastructure (CNKI), China Biology Medicine disc (CBMdisc), PubMed, EMBASE, Web of Science and Cochrane Central Register of Controlled Trials databases for RCTs that examined the efficacy of sildenafil for PPHN from 1998 to December 2020 by using “sildenafil,” “PPHN,” “persistent pulmonary hypertension of the newborn,” “pulmonary hypertension,” “Viagra,” “persistent fetal circulation syndrome,” “persistent fetal circulation,” and “newborn.”Two independent investigators reviewed the titles, abstracts and full articles that satisfied the inclusion criteria and independently extracted data into a predetermined database. The following information was extracted from each trial: the first author’s name, year of publication, demographic characteristics of participant, sample size, study design, interventions and treatment duration, and observation indicators. We assessed the risk of bias using the Cochrane risk of bias tool version 2  as low, high, and unclear risk of bias, and any disagreements were resolved by consensus.baseline and SDfinal represent the SD at baseline and follow-up, respectively, and Corr represents a correlation coefficient that describes the similarity between baseline and follow-up measurements (If the mean and standard deviation (SD) of the change in scores were not directly extracted from the literature, they were calculated by using the following formula recommended by the Cochrane Handbook for Systematic Reviews of Interventions, where SDurements . Here, wA 10% decrease from baseline in PASP or an PASP <35 mmHg was considered effective after the newborn is treated with sildenafil. The calculation method of total effective rate was the proportion of neonates with PASP decreased by 10% or PASP<35 mmHg after sildenafil treatment.p value of <0.05 was considered to be significant. A value of I2 = 0–50% was considered as low heterogeneity; 50–75% as moderate heterogeneity; and 75–90% as high heterogeneity. If the difference was not statistically significant, it indicates that the direct and indirect comparison results were consistent, and the consistency model was used for analysis. If there were differences, we used inconsistent model analysis. Surface under the cumulative ranking analysis (SUCRA) was used to rank the effects of the treatment regimens. The SUCRA results are expressed as percentages to compare each intervention to an imaginary intervention, which was always the best intervention without uncertainty. Throughout the meta-analysis process, we had followed the Preferred Reporting Project (PRISMA) guidelines for systematic reviews and meta-analysis.We conducted to pool the results of direct and indirect comparisons using a Bayesian approach. We will fit our model using STATA 14 statistical software  and Aggregate Data Drug Information System 1.16.8 software . The odds ratio (OR) with a 95% confidence interval (CI) was applied to evaluate the end points. For the NMA, ORs with 95% credible intervals (CrIs) were used. The NMA under the Bayesian framework, using Markov chain Monte Carlo (MCMC) random effects model. A consistency model based on the MCMC simulation method was applied by using 50,000 simulation iterations for each four chains with a burn-in period of the first 20,000 iterations. Node-splitting analysis and inconsistency standard deviation (ISD) were then performed to evaluate the consistency of the data. A two-tailed We will also assess the quality of evidence contributing to network estimates of the main outcomes with the GRADE framework, which characterises the quality of a body of evidence on the basis of the study limitations, imprecision, inconsistency, indirectness and publication bias.The search strategy identified 1754 studies. Finally, 22 RCTs with over 2,131 patients were included in the analysis . The floThe majority of RCTs had a low risk of bias . The risp = 0.001), H value 2.124 [95% CI ], I2 = 65.589%. However, after stratification of factors that may affect heterogeneity such as dosing frequency, duration, study year, and trial design, the heterogeneity test was performed, Q test (p > 0.05), and I2 = 24.245–49.479% indicated that there was no heterogeneity between studies within each layer. The results showed the comparison of each split node, direct comparison and indirect comparison was not statistically significant (p > 0.05), suggesting that there was no evidence that the network model was inconsistent.The clinical heterogeneity and methodological heterogeneity of the basic characteristics of the included studies were tested. The results showed that there was significant heterogeneity between studies, including Q test (p > 0.05), except between 0.3 and 0.6 mg/kg (p = 0.046). The network meta-analysis was performed using an inconsistency model. The NMA revealed that, compared with 0.3, 0.6 and 1.5 mg/kg showed a significant decrease; compared with 0.6, 1.0 and 1.5 mg/kg showed a significant decrease in PASP . The network meta-analysis was performed using a consistency model. The NMA revealed that there was not a significant difference in reducing SBP , except between 0.3 and 1.0 mg/kg, 0.5 and 1.0 mg/kg, and 0.5 and 2 mg/kg (p < 0.05). The network meta-analysis was performed using an inconsistency model. The NMA revealed that 0.6 and 1.2 mg/kg were better than 0.3, and 1.5 mg/kg was better than 0.5, 0.6 and 1.0 mg/kg in reducing the partial pressure of carbon dioxide . The NMA was performed using a consistency model. The NMA revealed that 0.5, 1.0, 1.5, and 2 mg/kg were better than 0.3 mg/kg; 1.5 and 2.0 mg/kg were better than 0.5, 1.0, and 1.5 mg/kg; 2 mg/kg was better than 0.6 mg/kg; and 1.5 mg/kg was better than 1.0 mg/kg at increasing the partial pressure of oxygen. The differences were statistically significant . The network meta-analysis was performed using an inconsistency model. The NMA revealed that 0.5, 1.2 and 1.5 mg/kg were better than 0.3 mg/kg; and 1.2 mg/kg was better than 0.5 and 1.0 mg/kg at increasing the arterial oxygen saturation . The network meta-analysis was performed using a consistency model. The NMA revealed that 0.5, 1.0, 1.5, and 2.0 mg/kg had higher clinical effectiveness than 0.3 mg/kg; and 1.0, 1.5, and 2.0 mg/kg had higher clinical effectiveness than 0.6 mg/kg , urticaria (three cases), tachycardia (two cases), and gastrointestinal bleeding (one case). Among them, the 1 mg/kg group and 2.0 mg/kg group experienced four types of adverse reactions. Hypotension did not occur at different doses of sildenafil among the newborns.The influence of different direct comparisons on the results of NMA showed that the direct comparison of 1.2 and 2.0 mg/kg had the greatest influence on the results of the whole results (10.2%), followed by 0.5 and 1.0 mg/kg group (9.7%).PPHN is an important cause of neonatal death and is considered a complex condition with a more or less unknown pathophysiology. PPHN can be primary (such as severe pulmonary hypoplasia) or can occur secondary to pulmonary parenchymal disease . In addition, polycythemia, hypoglycemia, sepsis, maternal diabetes mellitus, cesarean section delivery, high maternal body mass index, and maternal use of aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs) and serotonin reuptake inhibitors are among the factors associated with an increased risk of PPHN . The degThe purpose of PPHN treatment is to reduce pulmonary vascular resistance (PVR), maintain systemic blood pressure, correct right-to-left shunting and improve oxygenation . TreatmeConsequently, in terms of reducing PASP, the effect of reducing PASP was more obvious with increasing doses in the range of 1.5 mg/kg, while 2.0 mg/kg showed no difference, indicating that increasing the dose could not further reduce PASP. Similarly, 1.5 mg/kg had the most significant effect in increasing the PaO2 and reducing the PaCO2. Our found that some infants have increased or decreased SBP after treatment, but it is not statistically significant compared to before sildenafil treatment. But the cause of the increase in SBP is unclear. All the RCTs in this study used different doses of sildenafil to compare the clinical efficacy within 3 days, indicating that short-term high-dose sildenafil is more effective than low-dose sildenafil, but the long-term efficacy still needs further research and observation. Barst RJ et al. (Sildenafil is a selective phosphodiesterase 5 (PDE-5) inhibitor and a vasodilator specifically for the pulmonary vasculature that increases the intracellular cAMP and cGMP levels by inhibiting PDE, leading to vascular smooth muscle relaxation in the pulmonary vascular bed . Most ofJ et al.  have shoDespite the fact that we have limited knowledge of sildenafil safety in PPHN, previous studies have reported that the most frequently reported adverse reactions (AEs) were priapism, facial flushing, headaches, nasal stuffiness, irritability, diarrhea, vomiting, cough, abnormal vision and upper respiratory tract infection . The AEs1) This study only included literature published in Chinese; other studies in English were not found; 2) the 22 studies included were all RCTs, but the quality of the literature was not high; one study did not describe the method of random sequence generation and allocation concealment; 3) the number of included studies was small, and the results of the publication bias analysis may not be reliable; 4) the follow-up time of the included studies was 1–3 days, which is short, suggesting that follow-up studies need to increase the follow-up time to obtain safety data; and 5) in the study, infants were treated the brand name drug and generic drugs. The brand name drug and generic drug of sildenafil have bioequivalence, but the clinical equivalence of the brand name drug and generic drugs needs further study. Therefore, large RCTs are needed before advocating the clinical use of this agent in neonates with PPHN. Furthermore, the dose route and time intervals of sildenafil administration still need to be further elucidated.In conclusion, sildenafil is effective and safe for decreasing the PASP in newborns with PPHN. Our research proves that 1.5 mg/kg has the best effect in reducing pulmonary arterial pressure and the partial pressure of carbon dioxide and increasing the arterial blood oxygen pressure without severe adverse effects. However, the limitations of this study were as follows: