N-phenacyl derivatives of 2-mercaptobenzoxazole, including analogues of 5-bromo- and 5,7-dibromobenzoxazole, were screened against Candida strains and the action mechanism was evaluated. 2--1-(4-bromophenyl)ethanone (5d), 2--1-ethanone (5i), 2--1-ethanone (5k) and 2-[sulfanyl]-1-phenylethanone (6a) showed anti-C. albicans SC5314 activity, where 5d displayed MICT = 16 µg/mL (%R = 100) and a weak anti-proliferative activity against the clinical strains: C. albicans resistant to azoles (Itr and Flu) and C. glabrata. Derivatives 5k and 6a displayed MICP = 16 µg/mL and %R = 64.2 ± 10.6, %R = 88.0 ± 9.7, respectively, against the C. albicans isolate. Derivative 5i was the most active against C. glabrata (%R = 53.0 ± 3.5 at 16 µg/mL). Benzoxazoles displayed no MIC against C. glabrata. Benzoxazoles showed a pleiotropic action mode: (1) the total sterols content was perturbed; (2) 2--1-ethanol and 2--1-ethanol (8h–i) at the lowest fungistatic conc. inhibited the efflux of the Rho123 tracker during the membrane transport process; (3) mitochondrial respiration was affected/inhibited by the benzoxazoles: 2--1-(4-chlorophenyl)ethanol and 2--1-(4-bromophenyl)ethanol 8c–d and 8i. Benzoxazoles showed comparable activity to commercially available azoles due to (1) the interaction with exogenous ergosterol, (2) endogenous ergosterol synthesis blocking as well as (3) membrane permeabilizing properties typical of AmB. Benzoxazoles display a broad spectrum of anti-Candida activity and action mode towards the membrane without cross-resistance with AmB; furthermore, they are safe to mammals.A newly synthetized series of  Candida’s potential of new benzoxazoles. The benzoxazole ring is an important pharmacophore, that has for years been used to synthesize new biologically active compounds, including antifungal ones [Candida spp. [5–8, the confirmed activity of the 2-mercaptobenzoxazole derivatives against Candida spp. is particularly important [S-functionalized with the phenacyl or halogen-phenacyl group also exhibit antifungal activity [Fungal diseases represent a critical worldwide health problem and they are one of the main causes of morbidity and mortality. Moreover, only a few classes of antifungals are currently available to treat patients with fungal infections due to their high toxicity . Therefogal ones ,5,6,7,8.ida spp. ,10,11,12mportant ,14. On tactivity ,18,19,20N-phenacyl derivatives of 2-mercaptobenzoxazole, including analogues of 5-bromo- and 5,7-dibromobenzoxazoles. The ketones were reduced to respective alcohols. In the study, we evaluated the toxicity of various benzoxazoles towards the mammalian cell lines as well as the fungistatic and fungicidal effect against the reference and clinical isolates resistant to azoles. The exogenous ergosterol affinity of the most active and randomly selected benzoxazoles was tested. Benzoxazoles inhibitory activity towards ergosterol synthesis was studied using the biochemical (high-performance liquid chromatography HPLC) and spectrophotometric method (SPE). Benzoxazole permeability/lytic activity against the fungal membrane was assessed via flow cytometry. Benzoxazoles exhibit the candidacidal mechanism via accidental cell death (ACD).This prompted us to synthesize the 5–8 is outlined in 2 and 5,7-dibromo-2-mercaptobenzoxazole 3 were obtained according to procedures reported earlier [4 in the presence of activated carbon in the THF-H2O mixture [5–7 were synthesized from 2-mercaptobenzoxazoles 1–3 and phenacyl bromides or chlorides 4 by S-alkylation carried out in the K2CO2/MeCN system . Additionally, attempts to obtain respective alcohols, derivatives of bromobenzoxazoles 9 and 10, by the reduction in ketones 6 and 7 were rather unsuccessful, because of the formation of complicated mixtures of products. All alcohols 9 and 10, isolated in low yields and moderate purity, were unstable so they were not fully characterized and biologically tested.Reduction in ketones oxazoles . Decreasd yields . All alc1H NMR, 13C NMR, IR and HRMS analyses.All new stable compounds were fully characterized by 5d, 5i, 5k and 6a showed anti-C. albicans SC5314 ATCC activity  and MIC were also determined . Derivatives: activity , where 5termined  against isolates . Among tIn the study, the primary cell lines suitable and routinely employed for the compound cytotoxic examination were tested ,30. Thes5d, 6a and 7a tested against the Vero cells, a moderate cytotoxic effect (≤63.8% ± 8.7) was observed at 256 µg/mL  against MRC-5  for the ref. strain vs. the untreated control. The 6a- or 7a-treated clinical isolate displayed the mean decrease app. by 1-fold (6a and 7a) at conc. of 16 µg/mL and by 1.5-fold at 4 µg/mL, compared to the untreated control. Using the SPE analyses, we showed that the differences in the ergosterol content between the benzoxazole-treated vs. untreated control were statistically significant (p = 0.003).The results of the spectrophotometric (SPE) analyses showed a conc.-dependent decrease in the ergosterol content when the mL of 5d . In comp5d at 16 µg/mL (MICT), the treatment favoured the increase in the ergosterol content app. by 0.9-fold vs. the untreated control, while 4 µg/mL inhibited the ergosterol content app. by 1.3-fold vs. the control , 2-fold (6a) or 1.4 (7a). Using HPLC, we showed the statistically significant differences in the ergosterol content between the benzoxazole-treated and the untreated control (p = 0.007).Moreover, the ergosterol content was measured using HPLC . Interes control . Contrarp = 0.12 indicated a moderate correlation between these two methods. The SPE method produced slightly more accruable results due to the smaller p-value. In spite of this, there was p < 0.05 for these two methods; thus, both were worth applying.Based on the method comparison studies (Regression and Pearson correlation coefficient), it was shown that there was a large bias in these two methods used to assess the ergosterol content. The Pearson correlation coefficient (r = 0.5) and C. albicans, the blastoconidial cells were treated with compounds at 16 µg/mL  in 16 µg/mL  or 4 µg/16 µg/mL  and % of5j and 8h at 0.125 µg/mL as well as 5a and 5e at 16 µg/mL increased the efflux vs. the untreated control (OD600nm = 3901). Generally, the fungistatic compounds tested at lower conc. induced the cell efflux activity. In details, when all compounds at two different conc. were tested, there were differences in the efflux as follows: 5j (1-fold increase in the efflux at the lowest conc. tested vs. the highest one); 8d (1.2-fold); 5a and 8c (1.6-fold); 5e (1.7-fold). On the contrary, 8i caused a decrease in the efflux (4.6-fold) at the lowest conc. tested vs. the highest one. Although all benzoxazoles exhibited a comparable fungistatic effect, they showed a diversity in the cell efflux inhibition. Derivatives: 8c, 8i and 8d inhibited the blastoconidial efflux. The Rho123 efflux in the benzoxazole-treated SC5314 was stimulated by the glucose addition, but since it gradually decreased along with the increasing concentration of 8d, this derivative was taken as an illustrative example.The least active fungistatic compounds were tested for the Rho123 efflux at the highest (160 or 16 µg/mL) or the lowest (1.25 or 0.125 µg/mL) antifungal conc. tested . The SPEC. albicans, blastoconidia at log phase were harvested and then treated with benzoxazoles at various concentrations in a medium containing ergosterol at 20 mM  in the in vitro checkboard interactions against C. glabrata. The FIC index interpretations for the activities of 5d with AmB against C. glabrata recorded an indifference for all combinations. Synergism or antagonism were not recorded for any combinations.As it was shown in Candida activity  were tested against the C. albicans clinical isolates (resistant to Flu and Itr) and C. glabrata displaying an intrinsically decreased susceptibility to azoles [5k) or 5-bromobenzoxazole (6a) derivatives showed modest activity (spectrophotometric SPE end-point of %R = 64.2 ± 10.6 and %R = 88.0 ± 9.7 at 16 µg/mL) against the C. albicans clinical strain, respectively. The 2,3,4-trichlorophenacyl derivative (5i) exerted the most potent anti-C. glabrata action (%R = 53.0 ± 3.5 at 16 µg/mL). In spite of the total MICT = 16 µg/mL (%R = 100.3 ± 3.2) against the reference strain, 5d functionalized with the 4-bromophenacyl group displaying low activity against both azole-resistant isolates . The above was consistent with the cell proliferation inhibition data  for the ref. strain vs. the untreated control. The 6a- or 7a-treated clinical isolate displayed a mean decrease of one-fold (6a or 7a) compared to the untreated control (5d = MICT against the SC5314 ref and the cell growth reduction (%R = 100) were noted. Additionally, a close agreement between the sterol quantification and broth microdilution results was seen previously by Arthington-Skaggs et al. [5d at 16 µg/mL corresponded with Arthington-Skaggs’s et al. [5d-treated Candida still has to be elucidated, we clearly demonstrated that the trailing growth phenomenon classified 5d as active against the blastoconidial cells. Interestingly, the outcomes of our work point to the plasma membrane as a key player in the benzoxazoles’ anti-Candida action  and MICP (6a and 7a) were subjected to the negative feedback regulation of the ergosterol biosynthesis [C. albicans strains. Thus, when the benzoxazole (5j or 8h) treatment was conducted, the lipophilic molecule, such as the Rho123 tracker, was reduced in the uptake and/or retention in blastoconidials. It was noted that the total conc. of Rho123 in the loading medium was higher (from 1- to 3.3-fold) than within the cells when a lower conc. of 5a, 5e, 5j and 8h was tested. Our observation demonstrated that mitochondrial respiration was affected/inhibited by the benzoxazoles 8c–d and 8i. Although, all benzoxazoles exhibited a comparably weak fungistatic effect, they showed diversity in the cell efflux inhibition. The disparity was due to the sequestration of Rho123, leading to the heterogenous sub-cellular localization [8c–d and 8i at a higher conc., showing a stronger sequestration of ~3.3-fold vs. the lower conc. Therefore, the derivatives substituted with Cl or Br at C-4 or trisubstituted with Cl at C-2,3,4 blocked the fluorophore dye diffuse out of the sequestrating body in the blastoconidial cells. Thus, benzoxazoles could be the uptake transporter of ABCD1′s substrates [5j appeared as the most effective compound inducing an efflux (Cefflux = 16 μg/mL or 1.25 μg/mL). This could have be expected, when CDR1 was overexpressed in Candida [8h at Cefflux = 1.25 μg/mL was also the most potent inducer of the Rho123 efflux in C. albicans, although this value was app. by 4.6-fold higher than at 0.125 μg/mL.Exposure to  control . Our SPE control , where 5s et al. . Our HPLs et al.  studies s et al.  for the a action . In compynthesis . We showlization . The degbstrates . Thus, ibstrates , we show Candida . Derivat8d or 8f with ergosterol, which led to the inhibition of the cell growth  induced a leakage in ergosterol even at a very high concentration  as well as hydrogen bounds. The activity of 5a, 5j, 8i and 8c  encoded by the CDR transporters and the major facilitator (MF) encoded by MDR1. Benzoxazoles based on their ergosterol biosynthesis-inhibiting capacity, membrane permeabilization activity and increased activity against the azole-resistant isolates, could be an interesting approach to address the problem of resistance. To conclude, the antifungal action of benzoxazoles and the lack of cytotoxicity as well as physico-chemical properties, e.g., good stability, water solubility, rendered 5d a potential candidate for the development of new antifungal drugs.In general, benzoxazoles can be considered as effective, safe and stable and could be used alone or in a combination therapy with AmB to treat candidiasis (including azole-resistant strains). Since the azoles’ efficiency is limited by the development of 254  (0.2 mm thickness film). The column chromatography was performed using Silica gel 60  of 40–63 μm.Commercially available reagents from Sigma-Aldrich, Fluka and Avantor were used as supplied. Thin-layer chromatography was carried out on TLC aluminium plates with silica gel Kieselgel 60 FThe melting points were measured with a commercial apparatus Thomas Hoover “UNI-MELT”  on samples placed in glass capillary tubes and were not corrected. Infrared (IR) spectra were taken on a Carl Zeiss SPECORD M80 instrument .1H and 13C NMR spectra were measured with a Varian 500 spectrometer  operating at 500 MHz for 1H and 125 MHz for 13C nuclei. Chemical shifts (δ) were given in parts per million (ppm) relative to the residual solvent signal ; signal multiplicity assignment: s, singlet; br s, broad singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublets of doublets; m, multiplet. Coupling constant (J) was given in hertz (Hz). All these measurements were conducted in Warsaw University of Technology.The High-resolution mass spectrometry (HRMS) was carried out on Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer, ESI (electrospray)  with spray voltage 4.00 kV at IBB PAS Warsaw. The most intensive signals were reported. Ketones 5–7 were synthesized according to procedures reported previously ,22.2O (30 mL) and THF (15 mL), activated carbon (9.2 g) was added. The mixture was heated to 50–60 °C, NaBH4  was added carefully in portions keeping the temperature within the range of 50–70 °C (ca 2–3 h). The reaction was carried out in 60–70 °C  and monitored by TLC (hexane/acetate 9/1 v/v). After full conversion of bromonitrophenol, the mixture was cooled, EtOAc (100 mL) was added, stirring was continued overnight, filtered through a pad of celite, washed with EtOAc , phases were separated, the organic phase was dried (MgSO4) and evaporated to dryness. 4-Bromo-2-aminophenol was obtained as orange solid  and used without purification for synthesis of 5-bromo-2-mercaptobenzoxazole 2.To a stirred suspension of 4-bromo-2-nitrophenol  in H2O (20 mL), THF (10 mL) and activated carbon (8 g) was reduced with NaBH4 . 4,6-Dibromo-2-aminophenol was obtained as orange solid  and used without purification for synthesis of 5,7-dibromo-2-mercaptobenzoxazole 3.According to procedure reported above, 4,6-dibromo-2-nitrophenol  in suspension with H1–3 (10 mmol) in MeCN (40 mL) K2CO3 (15 mmol), appropriate amount of phenacyl chloride or bromide (10 mmol) was added. The reaction was carried out at room temperature (20–22 °C) and monitored by TLC (hexane/acetate 9/1 v/v). After full conversion of substrates , the mixture was poured on ice (120 g), the crude product was filtered, washed with H2O and dried on air. Ketones 5 were purified by crystallization, 6 and 7 by column chromatography. After full conversion of substrates , the mixture was poured on ice (120 g), the crude product was filtered, washed with water and dried on air. Crude ketones 5 were used for synthesis of alcohols 8 and purified by crystallization for biological research. Ketones 6 and 7 were purified by column chromatography .To a stirred solution of 2-mercaptobenzoxazole 4 (12 mmol) was added in portions keeping the temperature below 2–3 °C (ca 0.5 h). The reaction was carried out at 0–2 °C and monitored by TLC (hexane/acetate 5/1 v/v). After full conversion of ketone 5 , the mixture was poured on ice (120 g). Solid products  were filtered and washed with H2O. Oily products were extracted with EtOAc (3 × 50 mL), washed with H2O (5 × 50 mL), dried (MgSO4), filtered and evaporated. Alcohols 8 were purified by crystallization (hexane/EtOAc 10/1 v/v) or by column chromatography .To a stirred suspension of ketone 5 (8 mmol) in MeOH (40 mL) cooled to 0–2 °C, NaBHCandida albicans SC5314 strain from American Type Culture Collection (ATCC). Additionally, two clinical isolates were used in biological activity testing: C. albicans (fluconazole- and itraconazole-resistant) and C. glabrata. The strains were stored at −80 °C in Microbank system . Prior to biological examinations, yeasts were cultured overnight in YEPD (Yeast Extract Peptone Dextrose) or in RPMI 1640  at 30 °C with shaking at 100 rpm. Then, cells were harvested at 3,000 rpm for 5 min at 4 °C and washed twice with sterile water. The final inoculum ranged from 1.9 × 107 to 4.0 × 108 CFU/mL.Antifungal activity of benzoxazole derivatives was tested against the reference 5-fold diluted in sterile water and then 1:20 in a medium. Sets of compound test wells (CTW) and sterility control wells (SCW) were prepared in 96-well plates in triplicate for each conc. containing the tested compounds and inoculum (CTW) or sterile water instead of inoculum (SCW) in YEPD or RPMI 1640 medium. Growth control wells (GCW) with inoculum and DMSO (the same amount as in the CTW/SCW wells) in suitable medium were prepared. Plates were incubated for 18 h at 30 °C and evaluated using Synergy H4 Hybrid Reader  or with Spark Control M10  at 405 nm. Percentage of cell growth inhibition was calculated as follows: % of inhibition = 100 × (1 − (ODCTW − ODSCW)/(ODGCW − ODSCW). MIC (Minimum Inhibitory Concentration) was determined as the lowest concentration in which a prominent decrease in growth was visible according to the standard criteria from CLSI [Antifungal activity was assessed using broth microdilution assay according to the reference CLSI  M27-A3 method ,28,46. S5d, 6a and 7a was assessed against mammal Vero cell line  using MTS test (3-(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTS, Promega, USA) and for 5a, 5e, 5j, 8c, 8d, 8h, and 8i against mammal MRC-5 cell line (ATCC CCL-171) by MTT assay (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide, MTT, Thermo Fisher Scientific, USA) [2. Cells were incubated in 96-well microtiter plates for 24 h. Vero and MRC-5 cell monolayers were treated with different conc. of the benzoxazoles. Following 10 μL of MTS or MTT, reagents were added to each well and then each plate was incubated for 3 h in darkness.Cytotoxicity of ic, USA) ,48. Vero490/495 − A660. The cytotoxicity was calculated using the following formula: % cytotoxicity = (SA Positive control − SA Test)/(SA Positive control − SA Blank) × 100. Positive control means Vero or MCR-5 monolayer in EMEM and Blank means EMEM with tested conc. of compounds [The optical densities were measured at 490 nm (for MTS assay) and 495 nm (for MTT assay) with 660 nm as the reference wavelength using Synergy H4 Hybrid Reader  or Spark Control M10 . Specific absorbance (SA) was calculated using the following formula: SA = Aompounds .282 × 5/290)/pellet weight (g); %DHE = (A230 × 5/518)/pellet weight (g); %ergosterol = (%ergosterol + %DHE) − %DHE.Extraction of ergosterol was performed as previously described in literature with slight modifications . Samplesv/v as the mobile phase.For quantification of ergosterol by HPLC, organic solvent was evaporated. Sterols were re-dissolved in methanol (HPLC grade) and stored at 4 °C. HPLC analysis were performed using 200 Series Nelson NCI 900 system with UV/VIS detection at 282 nm  and COSMOSIL C18-MS-II column . Gradient elusion was performed starting with acetonitrile: methanol 97:3 C. albicans SC5314 were prepared following the method described in previous article [5d, 6a and 7a at conc. of 16 or 4 μg/mL with shaking at 200 rpm at 30 °C for 24 h and cells were treated with the following derivatives: 5e, 5a, 5j, 8h, 8c, 8i, 8d and 8f at 16 μg/mL for 24 h. After centrifugation at 3000 rpm at 4 °C for 5 min, the cells were washed and resuspended in sterile water. Staining was preformed according to the annexin and propidium iodide staining kit’s procedure  [5d, 6a and 7a, and with FACSCanto II  for 5e, 5a, 5j, 8h, 8c, 8i, 8d and 8f. Data were analysed with FACSuite Software 1.4 RUO .Protoplasts of  article . Cells arlandia) . SuspensC. albicans SC5314 cells were added to YEPD medium and incubated at 28 °C for 20 h with shaking at 150 rpm. Cells were then washed with water and PBS/NaOH. Two microliters of 2-deoxy-D-glucose and Rhodamine123 (Rho123) both at 20 mM were added to 1 mL of the cell suspended in PBS for each tested compound . After 18 h incubation at 28 °C and 150 rpm, suspensions were centrifuged and cells were washed with water and PBS. Tested compounds were added to final conc. Of 0.125 or 1.25 μg/mL  and 16 or 160 μg/mL . Then, 200 μL of 20 mM glucose were then added to cells and after 18 h incubation at 28 °C with shaking at 150 rpm, 100 μL of supernatant was placed into 96-well plate. Fluorescence of Rho123 was examined with excitation at 521 nm and emission at 600 nm using plate reader Spark Control M10 .Procedure was based on the protocol in Gbelska’s article ,54. C. a5a, 5e, 5j, 8c, 8d, 8f, 8h, 8i, exogenous ergosterol in YPD or RPMI 1640 medium were prepared in triplicates. Sterility control wells (SCW) contained tested compounds and ergosterol in medium. The growth control wells (GCW) contained inoculum, DMSO and ergosterol suspended in medium. Final concentration of exogenous ergosterol in each well was 400 μg/mL. Additionally, for examination antifungal activity of 5a, 5e, 5j, 8c, 8d, 8f, 8h, 8i, wells containing inoculum, ergosterol and itraconazole ITR  in medium (instead of tested derivatives) were prepared. Plates were incubated at 30 °C for 18–96 h and analysed using Synergy H4 Hybrid Reader  or with Spark Control M10  at 400 or 405 nm.To test compounds’ affinity to ergosterol, their antifungal activities in the presence of exogenous ergosterol were examined. Ergosterol  was dissolved in 96% DMSO and Triton X-100 or Tween 20  . Emulsio5d and AmB, a modification of checkerboard microdilution method was performed analogically to the CLSI M27-A3 method described above [5d (16–0.25 μg/mL) or combination of these two compounds. GCW and SCW wells were prepared as described above in CLSI [5d)/MIC(5d) + C(AmB)/MIC(AmB), where MIC of 5d or AmB and C stands for conc. of 5d or AmB in well containing combination of two compounds resulting in at least 50% growth inhibition in a given well in the 96-well microtiter plate in spectrophotometric (SPE) method [To determine Fractional Inhibitory Concentration (FIC) index between ed above . Compoun in CLSI . Samples) method . Interac) method  by the fp values ≤0.05) between the control and test values were determined by means of the Wilcoxon-signed rank-matched pair test using SPSS 16.0 software (Spss 16.0 free download). Moreover, the method comparison studies were determined by the means of regression and Pearson correlation coefficient.All experiments were performed in triplicate. Data were represented as mean ± SD. Statistically significant differences (


Angiography headache (AH) is common but not negligible, and the criteria for AH have been based on only a few studies. The purpose of this study was to investigate the incidence, risk factors and possible mechanism of AH and reappraise the diagnostic criteria for AH in the International Classification of Headache Disorders 3 (ICHD-3).Two hundred and seventy-nine patients completed this prospective, non-randomized study, including 107 patients who underwent cerebral angiography, 101 patients who underwent coronary intervention and 71 patients who underwent extremities arterial intervention. Patients were followed up with questionnaires immediately after the procedure and 24 h, 72 h, 1 week and 2 weeks after the procedure.p = 0.003 in cerebral angiography group, p = 0.006 in coronary intervention group, and p = 0.016 in extremities arterial intervention group). In addition, female (p = 0.008) was a risk factor for cerebral angiography group. Headache characteristics were described in detail.The incidence of headache was 22.4% (24/107) in cerebral angiography group, 23.8% (24/101) in coronary intervention group, and 16.9% (12/71) in extremities arterial intervention group. Headache still occurred in 12.1% (13/107), 14.9% (15/101) and 11.3% (8/71) of patients 24 h after the procedure in the three groups, respectively. Two types of headache were observed in cerebral angiography group and coronary intervention group, one during and one after the procedure, while only postoperative headache was observed in extremities arterial intervention group. Previous headache history was a risk factor for headache in the three groups (6.7.2 angiography headache in ICHD-3 may miss a number of cerebral AH with onset later than 24 h after the procedure. Therefore, it is recommended to revise it according to the literature and further studies. The incidence of headache was high during and after angiography and interventional procedure. It was suggested that the definition of headache due to coronary intervention and headache due to extremities arterial intervention should be added in ICHD.The diagnostic criteria for  Previous studies have reported that the incidence of headache in cerebral angiography ranges from 6.9–55.6% –6. The pBased on the above points, in order to further discuss the characteristics and potential mechanisms of cerebral AH and improve the applicability of cerebral AH diagnostic criteria in ICHD-3, we designed this prospective study.This project was approved by the Research Ethics Committee of the Chinese People’s Liberation Army (PLA) General Hospital. All of the patients provided written informed consent.The study included three groups from the First Medical Center of PLA General Hospital, respectively patients admitted to the Department of Neurology and the Department of Cardiology from May 2020 to January 2021, patients admitted to the Department of Vascular and Endovascular Surgery from October 2020 to January 2021. The inclusion criteria were: 1. Older than 18 years of age; and 2. Cerebral angiography or coronary intervention or extremities arterial intervention under local anesthesia. The exclusion criteria were as follows: 1. Patients with subarachnoid hemorrhage, patients who presented with secondary headaches due to the reason for DSA or had a headache occurring within 24 h prior to the procedure; 2. Renal failure requiring dialysis; 3. Severe organ failure, unconsciousness or endotracheal intubation; and 4. Patients who underwent intervention under sedative or general anesthesia; 5. Refusal to complete the questionnaire or follow up regarding the questionnaire.All patients underwent preoperative serum biochemical tests, blood coagulation, electrocardiogram, chest radiographs or chest CT, and fasting for 8 h and drinking abstaining for 4 h before the procedure. All procedures were performed by senior interventional specialist with the assistance of residents. Cerebral angiography used one or two of Iodoxanol, Ultravist, loversol, lohexol and lopamidol as contrast agents. Coronary intervention includes coronary angiography (CAG) and percutaneous coronary intervention (PCI). Iodixanol or Ultravist was used as contrast agent, and a small amount of nitroglycerin was used during coronary intervention. Extremities arterial intervention consisted of extremities arteriography, balloon dilation, stent implantation, and peripheral aneurysm repair, using iodixanol as contrast agent.A detailed headache questionnaire was designed by our team based on the ICHD-3 diagnostic criteria. The questionnaire consists of three parts: 1. Basic demographic information and medical history of the patient. 2. Information of interventional procedure. 3. Headache information: (a) Preoperative visit (information of previous headache history): headache age, attack frequency, duration, headache location, quality, intensity, accompanying symptoms, alleviating factors, family headache history, treatment, etc. (b) Postoperative visit (postoperative follow-up time points): headache occurrence, onset time and duration of headache, headache location, quality, intensity, accompanying symptoms, treatment, etc. Intensity was assessed with a numeric rating scale, which is a segmented numeric version of the visual analogue scale (VAS), in which the patients select a whole number from 0 to 10 that best reflects their pain level .1. Demographic information and related medical history such as: age, gender, body mass index (BMI), education level ≥ high school or not, history of tobacco and alcohol, allergy history, history of intervention, history of hypertension, diabetes, hyperlipidemia, cerebral infarction and information of previous headache history were collected before procedure after informed consent was obtained from patients and their family members. 2. Interventional information, such as type of intervention, type and dose of contrast agent, exposure time in X-ray and intervention procedure pathway were collected through electronic medical record system. 3. The patients were investigated immediately and 24 h, 72 h, 1 week and 2 weeks after the procedure. If discharged, patients were telephoned to follow up on their headaches. It is well known that contrast injection frequently causes a temporary sense of heat in the head and face called “flushing” [t test or and analysis of variance. Median and interquartile range were used to represent continuous data that did not conform to normal distribution, and rank sum test was used for comparison between groups. Categorical data were reported as numbers and percentages. Relationships between categorical variables were analyzed by Fisher’s exact test or Pearson’s chi-square test. Multiple logistic regression analyses were performed on variables that considered significant clinically and with a p value < 0.1 on univariate analysis. Using a two-tailed testing, p < 0.05 was considered statistically significant.Statistics were analyzed with the help of IBM SPSS statistics software, version 25.0. Continuous data consistent with normal distribution are reported as the mean ± standard deviation (SD). Hypotheses on differences in means between groups were tested by the independent samples Cerebral angiography group- In total, cerebral angiography was performed in 221 consecutive patients. Twenty-three had headache before interventional procedure, 12 refused to participate in the investigation, 1 was excluded due to renal failure, and 2 patients were unable to cooperate due to aphasia. A total of 183 patients were included. During the 2-week follow-up, 10 patients were lost to follow-up, 2 patients complained of unclear recall, and 64 patients were prematurely terminated due to intracranial stent, aneurysm embolization, or surgical procedures. Ultimately, 107 patients  completed the 2-week follow-up  completed the 2-week follow-up  completed the 2-week follow-up  years old, 61.7% (66/107) were male and 38.3% (41/107) were female. The mean length of hospital stay was (9.29 ± 6.73) days, with a median of 7 days. The mean length of hospital stay after cerebral angiography was (4.88 ± 5.10) days, with a median of 3 days. Of the 101 patients in coronary intervention group, 71.3% (72/101) were male, 28.7% (29/101) were female, and their mean age was (62.45 ± 12.79) years old. The mean length of hospital stay was (11.08 ± 7.08) days with a median of 9 days, and the mean length of hospital stay after procedure was (4.31 ± 4.72) days with a median of 3 days. Of the 71 patients in extremities arterial intervention group, 77.5% (55/71) were male, 28.7% (16/71) were female, and their mean age was (66.52 ± 10.77) years old. The mean length of hospital stay was (7.86 ± 2.71) days with a median of 8 days, and the mean length of hospital stay after procedure was (2.75 ± 1.04) days with a median of 2 days.p = 0.535) (Table Twenty-four of 107 22.4%) patients in cerebral angiography group developed headache within two weeks. The incidence of headache was 7.5% (8/107) during, 6.5% (7/107) within 24 h and 12.1% (13/107) within 2–14 days respectively after the procedure in cerebral angiography group. Of the 24 patients with headache, 29.2% (7/24) were male, 70.8% (17/24) were female and 41.7% (10/24) of patients had more than one headache attack. Twenty-four of 101 (23.8%) patients in coronary intervention group developed headache within two weeks. The incidence of headache was 6.9% (7/101) during, 6.9% (7/101) within 24 h and 14.9% (15/101) within 2–14 days respectively after the procedure in coronary intervention group. Of the 24 patients with coronary intervention related headache, 58.3% (14/24) were male, 41.7% (10/24) were female and 41.7% (10/24) of patients had more than one headache attack. Twelve of 71 (16.9%) patients in extremities arterial intervention group developed headache within two weeks. The incidence of headache was 8.5% (6/71) within 24 h and 11.3% (8/71) within 2–14 days respectively after the procedure in extremities arterial intervention group. Of the 12 patients with extremities arterial intervention related headache, 66.7% (8/12) were male, 33.3% (4/12) were female and 25.0% (3/12) of patients had more than one headache attack. There was no statistical significance in the incidence of headache among the three groups  patients had a history of headache, including 16 migraine, 6 tension-type headache (TTH) and 6 other types of headache. Of the 24 patients who developed angiography headache, 14 had a history of headache . Of the 8 patients with headache during angiography, 4 had a history of headache (2 migraine and 2 other types of headache), but the characteristics of headache in 75% (3/4) patients were different from their previous headache. Within the first 24 h after the procedure, headache was seen in 7 patients. Five of the 7 patients had a history of headache (4 migraine and 1 TTH), but the headaches in 60% (3/5) patients were different from their previous headaches. Between days 2–14 after angiography, headache was seen in 13 patients, of which 7 had a history of headache  and 29% (2/7) patients had different headache characteristics from their previous headaches  patients had a history of headache, including 7 migraine, 4 TTH and 1 other type headache. Among the 24 patients with coronary intervention headache, 7 patients had a history of headache, including 3 migraine, 3 TTH, and 1 other types of headache. Headache occurred in 7 patients during coronary intervention and none of the 7 patients had a history of headache. Within the first 24 h after coronary intervention, headache was seen in 7 patients. Three of the 7 patients had a history of headache , but the headache in 1 (33%) patient with other type of headache was different from the previous headache. Between days 2–14 after the procedure, headache was seen in 15 patients, of which 5 had a history of headache (3 migraine and 2 TTH), and 1 (20%) patient with migraine had different headache characteristics from the previous headache  had a history of headache . Of the 12 patients who developed headache after the procedure, 4 had a history of headache . None of the patients had headache during the procedure. Within the first 24 h after the procedure, headache was seen in 6 patients. Three of the 6 patients had a history of headache (2 migraine and 1 TTH), and 1 (33%) patient with a history of TTH had different headache characteristics from the previous headache. From 2 to 14 days after the procedure, 8 patients developed headache, 2 of whom had a history of headache (1 migraine and 1 other type of headache), and their headache were similar to the previous headache  min after the beginning of the angiography, lasted for an average of (8.27 ± 11.20) min, and the median VAS was 4. The headache occurred on unilateral sides (75.0%), on bilateral sides (25.0%), in the frontal region (25.0%), temporal region (50.0%), occipital region (25.0%), and parietal region (37.5%) with pain associated with throbbing (75.0%), burning (12.5%) or other pain (12.5%) and accompanied by visual aura (37.5%) (Table Headache that occurred during the coronary intervention started at a mean time of 15.4 ± 13.63) min after the beginning of the intervention, lasted for an average of (24.71 ± 19.51) min, and the median VAS was 4. The headache occurred on unilateral sides (28.6%), on bilateral sides (71.4%), in the frontal region (14.3%), temporal region (57.1%), occipital region (14.3%), parietal region (28.6%), full head (28.6%) with pain associated with throbbing (71.4%), pressing (28.6%) and accompanied by nausea (14.3%), photo (28.6%) - or phonophobia (28.6%) or visual aura (28.6%) (Table  ± 13.63 p = 0.067) (Table p = 0.068), duration (p = 0.054) and VAS (p = 0.613) in the two groups  in cerebral angiography group and 6.9% (7/101) in coronary intervention group. There was no statistical significance in the incidence of headache during the procedure between the two groups (7) Table . CompareHeadache occurring within 24 h after completion of cerebral angiography started at a mean time of 7.69 ± 7.86) h and the median onset time was 4.5 h, lasted for an average of (400.00 ± 363.90) min and the median duration was 420 min, and the median VAS was 3. The headache occurred on unilateral sides (28.6%), on bilateral sides (71.4%), in the frontal region (71.4%), temporal region (14.3%), parietal region (14.3%), full head (14.3%) and with pain associated with throbbing (57.1%), pressing (14.3%) stabbing (14.3%) or others (14.3%) and accompanied by photo (14.3%) - or phonophobia (14.3%) or visual aura (28.6%) (Table 9 ± 7.86 Headache occurring within 24 h after completion of coronary intervention started at a mean time of (4.14 ± 2.11) h and the median onset time was 4 h, lasted for an average of (789.29 ± 715.27) min and the median duration was 720 min, and the median VAS was 4. The headache occurred on unilateral sides (14.3%), on bilateral sides (85.7%), in the frontal region 28.6%), temporal region (71.4%), parietal region (14.3%) and with pain associated with throbbing (71.4%), pressing (14.3%), others (14.3%) and accompanied by nausea (28.6%), photo (14.3%) - or phonophobia (28.6%) or visual aura (14.3%)  h and the median onset time was 7.75 h, lasted for an average of (2170.00 ± 3900.91) min and the median duration was 750 min, and the median VAS was 5.5. The headache occurred on unilateral sides 16.7%), on bilateral sides (83.3%), in the frontal region (33.3%), temporal region (16.7%), parietal region (50.0%), full head (16.7%) and with pain associated with throbbing (66.7%), pressing (16.7%), burning (16.7%), others pain (16.7%) and accompanied by nausea (33.3%), photo (33.3%) - or phonophobia (33.3%) or visual aura (16.7%)  (Table p = 0.097), duration (p = 0.328) and VAS (p = 0.206) in the three groups (Table The incidence of headache within 24 h after the procedure in the three groups was 6.5% 7/107), 6.9% (7/101) and 8.5% (6/71) respectively in cerebral angiography group, coronary intervention group and extremities arterial intervention group and statistical results showed that it was no statistically significant  days and the median onset time was 5 days, lasted for an average of (123.72 ± 200.77) min and the median duration was 30 min, and the median VAS was 3. The headache occurred on unilateral sides 53.8%), on bilateral sides (46.2%), in the frontal region (30.8%), temporal region (38.5%), occipital region (23.1%), parietal region (53.8%), and with pain associated with throbbing (69.2%), pressing (23.1%), stabbing (15.4%) and accompanied by nausea (7.7%), photo (7.7%) - or phonophobia (15.4%)  days and the median onset time was 7 days, lasted for an average of (412.93 ± 760.60) min and the median duration was 60 min, and the median VAS was 3. The headache occurred on unilateral sides (53.3%), on bilateral sides (46.7%), in the frontal region (26.7%), temporal region (46.7%), occipital region (13.3%), parietal region (20.0%), full head (6.7%) with pain associated with throbbing (66.7%), pressing (6.7%), stabbing (6.7%), burning (13.3%), others (6.7%) and accompanied by nausea (6.7%), photo (13.3%) - or phonophobia (26.7%) or visual aura (6.7%)  days and the median onset time was 6 days, lasted for an average of (228.00 ± 439.52) min and the median duration was 60 min, and the median VAS was 4. The headache occurred on unilateral sides (62.5%), on bilateral sides (37.5%), in the temporal region (50.0%), occipital region (25.0%), parietal region (12.5%), full head (12.5%) and with pain associated with throbbing (87.5%), stabbing (12.5%) and accompanied by phonophobia (12.5%) or visual aura (12.5%) (Table 1 days anp = 0.754) (Table p = 0.816), duration time (p = 0.129) and VAS (p = 0.771) in the three groups (Table The incidence of headache in the three groups was 12.1% (13/107), 14.9% (15/101) and 11.3% (8/71) within 2 to 14 days after the procedure and it was also no statistical significance (n = 107) were shown in Table p<0.001, p = 0.006 and p<0.001). No statistical differences were found in age (p = 0.395), BMI (p = 0.217), education (p = 0.266), alcohol use (p = 0.132), hypertension (p = 0.092), diabetes (p = 0.707), cerebral infarction history (p = 0.350), hyperlipidaemia (p = 1.00) and coronary heart disease (p = 0.184), whether there was cerebral vascular stenosis (p = 0.407), cerebral aneurysm(p = 0.560) or current cerebral infarction (p = 0.149), previous history of intervention (p = 0.407), transfemoral or transradial pathway (p = 0.242) in the headache and no-headache groups.Patient demographics of cerebral angiography n = 7 were shn = 101) are shown in Table p = 0.007). No statistical differences were found in sex (p = 0.108), age (p = 0.192), BMI (p = 0.142), education (p = 0.205), allergy history (p = 0.292), smoking history (p = 0.565), alcohol history (p = 0.175), hypertension (p = 0.629), diabetes (p = 0.403), cerebral infarction (p = 1.000), hyperlipidaemia (p = 0.070) history of intervention (p = 0.493), transfemoral or transradial pathway (p = 0.062), type of contrast agent used (p = 0.138), quantity of contrast agent used (p = 0.055 and 0.495), X-ray exposure time during intervention (p = 0.050), indication for intervention (p = 0.804) or type of intervention (p = 0.482) in the headache and no-headache groups.Patient demographics of coronary intervention (n = 71) were shown in Table p = 0.024). No statistical differences were found in sex (p = 0.448), age (p = 0.143), BMI(p = 0.248), education (p = 1.000), allergy history (p = 0.213), smoking history (p = 0.507), alcohol history (p = 0.627), hypertension (p = 0.335), diabetes (p = 0.370), coronary heart disease (p = 0.125), cerebral infarction (p = 0.336), hyperlipidaemia (p = 1.000) history of intervention (p = 0.758), transfemoral or transradial pathway (p = 0.581) and quantity of contrast agent used (p = 0.416) in the headache and no-headache groups.Patient demographics of extremities arterial intervention , female had an increased risk of cerebral angiography headache . In coronary intervention group, after adjusting for sex, education level, history of headache, history of hypertension, hyperlipidemia, interventional pathway and X-ray exposure time in Logistic regression model, patients with history of headache had an increased risk of coronary intervention-related headache compared with those without history of headache . In extremities arterial intervention group, after adjusting for sex, education level, history of headache and history of hypertension in Logistic regression model, patients with history of headache had an increased risk of headache related to extremities arterial intervention compared with those without history of headache  , 23.8% (24/101) in coronary intervention and 16.9% (12/71) in extremities arterial intervention. The incidence of headache in the cerebral angiography and coronary intervention group was similar, slightly higher than that in extremities arterial intervention group, and there was no statistical significance in the incidence of headache in the three groups.10.2 dialysis headache and were excluded [In our study, the incidence of headache in coronary intervention group 23.8%, 24/101) and extremities arterial intervention group  was higher than the incidence of headache in peripheral angiography group of Aktan  /101 and , 16. We excluded , 17. TheIn our study, female was not a risk factor for headache in coronary intervention group and extremities arterial intervention group, but a risk factor for cerebral angiography headache, which is consistent with the research of Gil-Gouveia . The pos8.1.1.1 immediate NO donor-induced headache in ICHD-3 [Previous study had shown that the occurrence of migraine after cerebral angiography was related to meningeal vasodilation . Studiesn ICHD-3 , 25, 26.In ICHD-3, diagnostic criterion A in 6.7.2 is “Any new headache fulfilling criterion C”. criterion C1 is “headache has developed during or within 24 hours of the angiography”. Both our study and previous reports have confirmed the headache during the procedure [Criterion C2 defines as “headache has resolved within 72 hours after the angiography”. In our cerebral angiography group, the average duration of headache during the procedure was (8.27 ± 11.20) min, with a median of 3 min; The average duration of headache within 24 h after the procedure was (400.00 ± 363.90) min, with a median of 420 min. The average duration of headache within 2–14 days after the procedure was (123.72 ± 200.77) min, with a median of 30 min. Aktan reported that the mean duration of headache during angiography was 41.7 ± 65.4) min, with a median of 22.5 min. The mean duration of headache within 24 h after the procedure was (171.7 ± 208.1) min with a median of 75 min, and the mean duration within 2–7 days after the procedure was (122.1 ± 173.1) min with a median time of 60 min [.4 min, wCriterion C3a defines as “developing during contrast injection and lasting <1 hour”. In our study, a total of 8 patients developed headache during angiography, with an average duration of 8.27 ± 11.20) min and a median duration of 3 min. Aktan reported  that the ± 11.20 Criterion C3b defines as “developing a few hours after the angiography and lasting >24 hours”. The criterion C3b and the criterion C3a are in parallel relationship. The criterion C3a is for the characteristics of headache during angiography, while the criterion C3b is obviously for the characteristics of headache after angiography. As in the previous discussion of criterion C1 and C2, the description of criterion C3b that headache developing a few hours after the angiography and lasting >24 h is obviously not appropriate. It is suggested to be modified to “ developing a few hours to 7 days after angiography and a single headache lasting < 24 hours”.1. Migraine and having the features of 1.1 Migraine without aura or 1.2 Migraine with aura” In our study, some patients with a history of migraine developed angiography headache with the same characteristics as their previous migraine, and some patients had visual aura, which is supported by previous reports [Criterion C3c defines as “occurring in a patient with  reports . It is rIn our study, the incidence of headache during the procedure was no statistical difference between cerebral angiography group and coronary intervention group. The incidence of headache within 24 h or within 2–14 days after the procedure was also no statistical difference among the three groups Table . The heaThere were both 41.7% (10/24) patients of cerebral angiography group and coronary intervention group, and 25.0% (3/12) patients of extremities arterial intervention group had more than one headache occurred during the 2 weeks’ follow up.Analysis of potential risk factors that may lead to headache showed that previous headache history was a common risk factor among the three groups, and female was a risk factor in cerebral angiography group. By statistical analysis, age, BMI, education, history of allergy, history of intervention, history of tobacco and alcohol, hypertension, cerebral infarction, diabetes were not potential risk factors for headache occurrence among the three groups.Criterion B defines as “Intra-arterial carotid or vertebral angiography has been performed”. In our study, we found that:6.7.2 angiography headache in ICHD-3 belongs to classification 6, namely “Headache attributed to cranial and/or cervical vascular disorder”, coronary intervention and extremities arterial intervention do not belong to the category of Cranial and/or cervical vascular disorder. Therefore, we recommend that ICHD add categories of “headache due to coronary intervention” and “headache due to extremities arterial intervention”.Based on the above analysis, we believe that cerebral angiography headache, coronary intervention-associated headache and extremities arterial intervention -associated headache have similar headache characteristics, suggesting that the occurrence of this kind of headache is not limited to whether intra-arterial carotid or vertebral angiography is performed, coronary intervention and extremities arterial intervention can also lead to similar headache. Criterion B seems to be expanded from “intra-arterial carotid or vertebral angiography has been performed” to “intra-arterial carotid or vertebral angiography or coronary intervention or extremities arterial intervention has been performed”. Considering that 6.7.2 angiography headache in ICHD-3 states that “there are three recognized (but not separately coded) subforms of 6.7.2 Angiography headache”.occurring during angiography, and closely related to contrast injection. This note is an interpretation of criterion C3a and it has been discussed in criterion C3a which is recommended to be retained.occurring later, but within 24 h . This note is an interpretation of criterion C1 and it is discussed in criterion C1. We propose to change the definition of “occurring later, but within 24 hours” to “occurring later, but within 1 week after angiography”. Patients with history of headache are more likely to have angiography headache has been established in previous and our studies , 20. In 1. Migraine and triggered by angiography .a migraine attack, occurring in a person who has The note of This note is discussed in criterion C3c and we recommend keeping this note.Previous studies showed , 4 cerebAktan reported 31.0% 13/42) of patients had more than one headache attack after cerebral angiography, and Gil-Gouveia reported that 48.8% (40/82) of patients had recurrent headache [2 of pati6.7.2 angiography headache in Table Based on the above discussion, we suggest that the new diagnostic criteria for 6.7.2 angiography headache in ICHD-3, we believe that there are problems in the applicability of the diagnostic criteria and it is easy to miss diagnosis, so it should be revised according to the current literature and research. In addition, it is suggested to add two new definitions and diagnostic criteria of “headache due to coronary intervention” and “headache due to extremities arterial intervention” in ICHD-3.Angiography headache is common in clinical practice but has received little attention. In our study, the incidence of headache was 22.4% in cerebral angiography group, 23.8% in coronary intervention group, and 16.9% in extremities arterial intervention group. There was no statistical significance in the incidence of headache among the three groups. Female and history of headache were risk factors in cerebral angiography group, and history of headache was a risk factor in coronary intervention group and extremities arterial intervention group. There were no differences in headache onset time, duration and VAS of intraoperative headache between cerebral angiography group and coronary intervention group. The headache onset time, duration and VAS of postoperative headache were similar in three groups. By discussing the diagnosis criteria of


In invivo tests, after HLJG0701-β was orally administered at doses of 0, 125, 250, and 500 mg/kg in an animal model of memory impairment, behavioral evaluation (Morris water maze test and Y-maze task test) was performed. The levels of AChE, acetylcholine (ACh), blood catalase (CAT), and malondialdehyde (MDA) in brain tissues were measured. The results showed that HLJG0701-β produced the best results at a dose of 250 mg/kg or more. The neuroprotective mechanism of HLJG0701-β was determined to involve the inhibition of AChE activity and a decrease in oxidative stress. In summary, both invitro and invivo tests confirmed that HJG0701-β administration can lead to memory improvement.Wild ginseng has better pharmacological effects than cultivated ginseng. However, its industrialization is limited by the inability to grow wild ginseng on a large scale. Herein, we demonstrate how to optimize ginseng production through cultivation, and how to enhance the concentrations of specific ginsenosides through fermentation. In the study, we also evaluated the ability of fermented cultured wild ginseng root extract (HLJG0701-β) to inhibit acetylcholinesterase (AChE), as well as its neuroprotective effects and antioxidant activity. In  Typicalin cells . Aβ25-35ng to AD .N-methyl-d-aspartate (NMDA) receptor antagonist, are cholinergic inhibitors that act in the brain [The degradation of ACh, a neurotransmitter, by AChE can lead to declines in cognitive function and memory as AChE plays an important role in learning and memory . Effortshe brain . Studieshe brain ,12. Howehe brain . Accordihe brain .Panax ginseng C.A. Meyer, which belongs to the family Araliaceae, is naturally distributed in Korea, China, Japan, Europe, and North America [ America ,15,16. T America , while c America ,19. The  America ,21,22,23 America ,25. Amon America . Previou America , improve America , and imp America .The conventional methods of growing ginseng in the cultivation field are time-consuming and labor-intensive, which translates into increased costs . The usein vitro culturing and to enhance the contents of specific ginsenosides in fermented cultured wild ginseng roots using HPLC analysis. We also evaluated the ability of a fermented cultured wild ginseng roots extract, HLJG0701-β, to inhibit AChE activity and assessed its neuroprotective and antioxidant effects using a scopolamine and ovariectomized (OVX) + d-galactose-induced animal model of aging and memory impairment.The objective of the present study is to optimize ginseng production through Quantitative analysis was performed on eight types of ginsenosides, divided into high-molecule and small-molecule ginsenosides. The total concentration of ginsenosides in cultured wild ginseng root treated with jasmonate was 188.06 ± 4.98 mg/g. Five high-molecule ginsenosides  were detected, but no small-molecule ginsenosides  were detected. The total concentration of ginsenosides in fermented HLJG0701-β was 117.96 ± 3.38 mg/g. The levels of high-molecule ginsenosides  were decreased in this specimen compared to the unfermented specimen, while the levels of small-molecule ginsenosides were detectable  in the fermented sample . Overallp < 0.01). Since HLJG0701-β exhibited AChE inhibitory effects, it may be able to enhance memory and prevent dementia, suggesting the possibility of its application as a therapeutic AChE inhibitor  and 40 mg/mL (87.65 ± 0.16%) (nhibitor .25-35 and H2O2 were separately added to SH-SY5Y cells (5 × 104cells/96-well), which were then incubated at 37 °C for 24 h, to investigate the potential neuroprotective effects of the ginsenosides. In the Aβ25-35 test, the treatment of SH-SY5Y cells with 5 µM Aβ25-35 alone led to a significant decrease in cell viability, to 77.31 ± 2.74%, versus treatment with 0.31 µg/mL ginsenoside Rk1 positive control (89.29 ± 5.62%) (p < 0.01). The treatment group showed more than 80% activity at concentrations of 0.04 µg/mL (82.48 ± 0.51%), 0.16 µg/mL (80.52 ± 2.45%), 0.63 µg/mL (96.01 ± 4.04%), 2.5 µg/mL (84.23 ± 1.51%), and 10 µg/mL (86.05 ± 4.96%) (p < 0.01) (Aβ < 0.01) A.2O2 test, the treatment of SH-SY5Y cells with 100 µM H2O2 alone led to a significant decrease in cell viability, to 72.61 ± 2.38%, versus treatment with 0.31 µg/mL ginsenoside Rk1 positive control (99.58 ± 3.63%) (p < 0.01). In the test in which oxidative stress was induced by treatment with H2O2, when a low concentration of HLJG0701-β was treated, it was confirmed that SH-SY5Y cells were statistically significantly protected by the test substance when compared to the control group  by treatment with H2O2 for 4 h. After staining with DCFDA, fluorescence expression was measured to compare ROS activity between samples.HLJG0701-β was then pretreated, followed by incubation at 37 °C for 24 h. ROS production was induced in SH-SY5Y cells . The test group treated with positive control (300 µM H2O2 + 50 µM ascorbic acid) showed a ROS activity level of 62.51 ± 0.31% versus the H2O2 treatment group (100%). All treatment groups showed a reduction in H2O2-induced ROS activity  =  (p < 0.01).As expected, H± 1.37%) A (p < 0.2O2 treatment group compared to the control (p < 0.01). The test group treated with positive control (300 µM H2O2 + 50 µM ascorbic acid) showed 163.04 ± 4.82% of CAT activity versus the H2O2treatment group (p < 0.01). All treatment groups showed changes in H2O2-induced CAT activity  =  (p < 0.01).CAT activity was significantly decreased in the 300 µM H± 8.16%) B  is reduced.p < 0.01, p < 0.05). Meanwhile, 250 and 500 mg/kg HLJG0701-β and 5mg/kg donepezil treatment significantly increased the escape latency on the fourth  and fifth  (p < 0.01) days of the training period, compared to the scopolamine group  . The swimming time in the target quadrant of the scopolamine group (7.7±0.9 s) was significantly less than that of the control group (19.8 ± 4.7 s) (p < 0.05). On the other hand, the swimming time in the target quadrant was extended in the positive control treatment groups . Although the differences were not significant, an increased swimming time was observed in mice exposed to 125 and 250 mg/kg HLJG0701-β (p < 0.05). In addition, the swimming distance of the treatment groups  was clearly less than that of the scopolamine group (1184.6 ± 42.0 cm).The performance of all groups in the MWM training task is shown in JG0701-β B,C. As sp < 0.05). Spontaneous alternation significantly decreased to 37.1 ± 7.1% in the scopolamine group, from 59.8 ± 3.1% in the control group. However, the scopolamine-induced decrease in the spontaneous alternation rate was significantly increased in the positive control group (60.8 ± 3.9%) (p < 0.05), 125 and 250 mg/kg HLJG0701-β treatment groups , and 500 mg/kg HLJG0701-β treatment group (54.7 ± 4.1%) (p < 0.05).Scopolamine was used to produce AD-like memory deficits in mice, and donepezil , an approved AChE inhibitor, was employed as a positive reference drug. The total number of entrances and exits for each arm of the maze was measured for all test animals; no statistically significant difference was observed among all test groups B.d-galactose group took more time to find the platform than did the control group from the fourth to the fifth day of the training period, showing that OVX + d-galactose-induced changes could cause significant cognitive dysfunction in mice . Meanwhile, the positive control treatment groups exhibited significant amelioration of the OVX + d-galactose treatment increase in escape latency on the third, fourth  and fifth  days of the training period compared to the OVX + d-galactose group .The performance of all groups in the MWM training task is shown in d-galactose group (9.8 ± 2.4 s) was significantly less than that of the control group (20.7 ± 2.5 s) (p < 0.05). On the other hand, the swimming time was extended in the positive control treatment groups  (p < 0.05). Swimming time was significantly increased in mice exposed to the treatment group . In addition, the swimming distance of the treatment groups  were clearly reduced in comparison with that of the OVX + d-galactose group (1234.9 ± 123.6 cm) (p < 0.05).As shown in d-galactose and HLJG0701-β  treatment on spontaneous alternation behavior and locomotor activity  in the Y-maze test. OVX + d-galactose-induced mice showed a significant decrease in short-term memory performance, as evidenced by a decreased spontaneous alternation percentage compared with the control group (p < 0.01). Spontaneous alternation significantly decreased to 46.5 ± 3.9% in the OVX + d-galactose group, from 63.3 ± 3.3% in the control group. However, the OVX + d-galactose-induced decrease in the spontaneous alternation rate was significantly increased in the positive control group (57.4 ± 2.5%) (p < 0.05), treatment  groups , and HLJG0701-β 500 mg/kg group (59.5 ± 4.6%) (p < 0.05). When the total number of entrances and exits for each arm was measured for each test animal, no statistically significant differences were observed among all test groups  (p < 0.01) compared to the control group (181.18 ± 4.73 mU/mL). When the OVX + d-galactose-induced mice were treated with HLJG0701-β , a marked inhibition of AChE activity  in the brain tissue was observed in comparison with the OVX + d-galactose group (p < 0.01). Furthermore, donepezil treatment (positive control group) significantly suppressed the increase in AChE activity (177.84 ± 9.20) (p < 0.05).The effects of HLJG0701-β on brain AChE and ACh contents are shown in d-galactose group (0.769 ± 0.025 nmol) than in the control group (0.921 ± 0.028 nmol) (p < 0.01). In addition, the treatment  groups  showed a clear reversal of the decrease in the OVX + d-galactose group (p < 0.05). These results indicate that administration of HLJG0701-β suppressed the increase in AChE activity and the reduction of ACh levels induced by OVX + d-galactose.Meanwhile, ACh activity was found to be lower in the OVX + d-galactose group, OVX + d-galactose-induced changes significantly increased the MDA contents (24.40 ± 0.77 nmol/mL) compared to that in the positive control treatment  groups  (p < 0.05). The changes in CAT activity in the blood tissues are shown in d-galactose group, from 684.00 ± 14.00 mU/mL in the control group (p < 0.01). However, the decrease in CAT contents induced by OVX + d-galactose was significantly ameliorated in the positive group (680.18 ± 35.66 mU/mL) and treatment  groups  (p < 0.05).in vitro culture are known to have a similar genetic DNA to wild ginseng, with high saponin contents [25-35 levels, memory, and cognitive function [The natural growth rate of wild ginseng is very low, making it difficult to commercialize. Luckily, cultured wild ginseng roots produced by contents ,51. In tcontents . In the contents . Among dfunction .in vitro AChE inhibitory activity was observed using increasing concentrations of HLJG0701-β. Donepezil, the positive control, inhibited AChE activity in a dose-dependent manner. The improvement in memory following treatment with HLJG0701-β was comparable to that produced by donepezil treatment. Interestingly, the AChE inhibition rate of HLJG0701-β at a concentration of 40 mg/mL was greater than that of donepezil, indicating that HLJG0701-β may enhance the production of ACh. Moreover, the ginseol K-g3, a constituent of ginseng roots, has been reported to reverse scopolamine-induced amnesia in treated mice [Many previous studies have shown that ACh is required for learning and memory ,56,57. Ited mice . A numbeted mice  and of eted mice , which hted mice ,67 and dted mice ,69. In oted mice ,70. It hted mice . Moreoveted mice . In the ted mice ,76,77,78ted mice ,79. Moreted mice ,82,83,84•−) and hydroxyl (•OH), play a crucial role in inducing these disorders in the human body [d-galactose-induced changes significantly increased the MDA levels in the treated mouse brain. In the present study, HLJG0701-β significantly inhibited MDA levels at all concentrations, suggesting that HLJG0701-β has superior protective abilities, possibly because of its antioxidant properties.Neurodegenerative diseases such as AD and Parkinson’s Disease are caused by oxidative stress-induced cell damage ,10,56. C2O2, which is essential for cellular signaling [in vitro test, an effective decrease in ROS concentration was observed following treatment with low concentrations of HLJG0701-β, whereas CAT remained inactivated at low concentrations. However, activation of CAT may have occurred following treatment with 300 µg/mL HLJG0701-β, which was the highest concentration used in the test. Though CAT activation was not observed at lower concentrations, CAT tended to be activated as the concentration of the test substance increased, which suggests the emergence of an antioxidative effect at higher concentrations. Taken together, these results suggest that the neuroprotective effects of HLJG0701-β against H2O2-induced oxidative damage may involve the inhibition of ROS production.Antioxidant enzymes such as CAT, GPx, and SOD, as well as non-enzymes such as glutathione and vitamins, are known to play important roles in intracellular ROS scavenging . CAT is ignaling ,87. In t25-35 plays an important role in the development of AD, by causing neuronal cell death [25-35-induced neuronal damage in a concentration-dependent manner. Moreover, HLJG0701-β treatment inhibited H2O2-induced oxidative damage of the treated neuronal cells in a concentration-dependent manner. It is possible that HLJG0701-β treatment may reverse H2O2-induced oxidative damage by scavenging ROS, which is supported by the finding of increased ROS inhibition. Moreover, ginsenosides such as Rk1, a smaller molecular compound, exhibited higher antioxidant effects [25-35-induced neuronal damage by up-regulating neprilysin (NEP) gene expression [2O2-induced cell death in primary rat cortical astrocytes through the up-regulation of antioxidant systems, including glutathione S-transferase (GST) and heme oxygenase-1 (HO-1) [The accumulation of Aβll death ,88. In t effects . Therefopression . In anotpression . Moreove1 (HO-1) .d-galactose-treated female mice. In the Morris water maze test, the mice treated with donepezil showed a rapid reduction in daily escape latency. HLJG0701-β administration at 250 and 500 mg/kg (treatment groups) and donepezil administration (positive control group) produced significant reductions in escape latency on day 5 compared to negative control treatment (p < 0.01). A similar result was observed in the OVX + d-galactose-treated female mice. Furthermore, HLJG0701-β treatment dramatically improved the swimming speed and time taken to cover the target quadrant compared to the negative control in both male and female mice. The transformation of ginsenosides in the cultured wild ginseng roots by the fermentation process may be a reason, as HLJG0701-β contains a higher concentration of small molecular compounds such as Rg3, Rk1 and Rg5. According to Kim et al. [Scopolamine, a non-selective muscarinic ACh receptor antagonist, is known to interfere with learning and short-term memory . Animal m et al. , ginsenoPlant cell tissues were obtained from wild ginseng collected from Yanggu-gun, Gangwon-do, Korea. A plant tissue method was used to culture wild ginseng roots . Prior tL:D) photoperiod. Records of root growth began 5 weeks after initiation. The roots were treated with methyl jasmonate (250 μM) during the 3 week of cultivation. The roots were then harvested and used as materials for fermentation.Briefly, adventitious roots (0.5–2.0 cm slices) were inoculated into a plant culture medium (67.1 g of Schenk and Hildebrandt (SH) medium mixed with 450 g sucrose and 15 L of purified water, with pH adjusted to 5.75 ± 0.10). The medium was placed into a bioreactor and sterilized at 121.0 °C for 60 min. The adventitious roots were inoculated in the medium under aseptic conditions and cultured for 8 weeks. The cultures were maintained at 15 °C and 30 °C during the day and night, respectively, with a 16:8  was used as the microorganism in this study. It was obtained from kimchi after separating, identifying, and sub culturing lactic acid bacteria [P. pentosaceus in microbial culture medium  medium, mixed with 1 L of purified water with pH adjusted to 6.50 ± 0.05). The medium was placed into a fermenter and sterilized at 121.0 °C for 30 min. After cooling to room temperature, P. pentosaceus HLJG0702 was inoculated into the medium under aseptic conditions and cultured  for more than 48 h. Thereafter, bacterial cells were recovered by centrifugation . The cells were then mixed with an anti-freezing agent (95% glycerol) and stored at −80 °C before use as the microbial material for fermentation.bacteria . A batchP. pentosaceus, followed by fermentation with some modification [P. pentosaceus was prepared by mixing cells harvested from centrifugation and purified water at a ratio of 0.31% to 99.69%. Then, the above-prepared cultured wild ginseng root extract and P. pentosaceus were mixed at a ratio of 80% to 20%, followed by fermentation at 120 ± 5 °C for 210 ± 10 min, filtration, and concentration (60 Brix or more) to prepare the final raw material, HLJG0701-β.HLJG0701-β was prepared by mixing cultured wild ginseng root extract with fication . To prepEight ginsenoside reference standards, Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rg5, and Rk1, were purchased from Sigma Aldrich, Inc. . The purities of all reference standards were above 98%. High-performance liquid chromatography (HPLC)-grade acetonitrile (AcN), water, and methanol (MeOH) were purchased from Thermo Fisher Scientific . All other reagents used in this study were of analytical grade. Ginsenosides were extracted three times from ginseng samples using a sonicator from Daihan Scientific Co., Ltd.  and analyzed using a Thermo Fisher Scientific Ultimate 3000 Series  liquid chromatography coupled with an ultraviolet detector, according to previous methods ,97. The Each test sample (2 g) was precisely weighed, added to a 50 mL flask, and completely dissolved after filling to 50 mL with 100% MeOH. The solution was filtered through a 0.2 μm syringe filter and used after diluting. The separation was based on a Capcell Pak C18  column  at a temperature of 30 °C, analyzed with an injection volume of 10 μL at a flow rate of 1.0 mL/min, and detected at a wavelength of 203 nm. The binary gradient elution solvent consisted of water (A) and acetonitrile (B). The gradient elution program was as follows: 0–5 min, 80% A; 5–20 min, 80%–77% A; 20–25 min, 77%–70% A; 25–30 min, 70%–60%; 30–35 min, 60%–50% A; 35–65 min, 50%–15% A; 65–75 min, 15%–80% A. The peak areas corresponding to ginsenosides from the samples, with the same retention time as authentic ginsenosides , were integrated by comparison with an external standard calibration curve.A: enzyme + substrate + absorbance after reaction of sample—control group absorbance; ODB: enzyme + absorbance after reaction of substrate—control group absorbance.The AChE activity of HLJG0701-β obtained from cultured wild ginseng roots was assessed using a colorimetric method . Initiall-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin and incubated at 37 °C with 5% CO2. Cell Counting Kit-8  reagent was added to each well of a 96-well plate. After reacting for 2 h at room temperature, the absorbance was measured at 450 nm using a microplate reader. All samples were checked three times. The concentration of each material was set based on the concentration that did not affect cell survival. The methods were performed as previously described [SH-SY5Y human neuroblastoma cells were used in this study to determine the effects of the test samples on cell viability. The cells were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM escribed .25-35  and H2O2  were used to induce nerve cell damage [4 cells/well. After incubation at 37 °C in a CO2 incubator for 24 h, 5 µM Aβ25-35 or 100 µM H2O2 was applied to the cells along with HLJG0701-β for 24 h. The CCK-8 assay was then performed as described above. Cell survival  was taken to reflect the cell-protective effects of the test materials against nerve cell damage.Aβl damage ,101. The6 cells into each well of a 6-well plate and incubating for 24 h, then treated with HLJG0701-β diluted in serum-free media at different concentrations for 24 h. As a positive control, 50 µM ascorbic acid (Vitamin C) was used. Oxidative stress was induced by adding 300 µM H2O2 and then incubating at 37 °C in a CO2 incubator for 4 h. The activity of ROS and CAT were measured according to the manufacturer’s instructions using a ROS detection assay kit  and CAT assay kit .After plating 2 × 10d-galactose-induced aging animal model [Male mice were used for the scopolamine-induced animal model and female mice were used for the al model ,102. C57d-galactose-induced aging, the skin around the lumbar spine was disinfected following inhalation anesthesia with isoflurane, and a 2 cm cut was made around the lumbar spine. Both ovaries were removed. After extraction, the skin was sutured and disinfected. In the normal control group, all procedures except for ovarian extraction were performed in the same manner. To induce aging, d-galactose was administered subcutaneously at a dose of 100 mg/kg to animals whose ovaries were removed. The test substance and positive control substance were administered once daily, 7 days/week for 8 weeks. During the period of the behavioral experiment, after 8 weeks of administration, the test substance or positive control was administered about 1 h before the start of the experiment. The aging-inducing substance, test substance or positive control substance was administered once daily, 3 days/week for 6 weeks followed by 7 days/week for 2 weeks, for a total of 8 weeks. Scopolamine hydrobromide and d-galactose were purchased from Sigma Aldrich Inc. . Donepezil was obtained from Aurobindo Pharma Ltd. .To prepare the animal model of d-galactose-induced control group, a positive control (donepezil) group, and three HLJG0701-β groups. The control and scopolamine or OVX + d-galactose groups were administered sterile distilled water. The test groups were administered the test substances orally by gastric gavage. The test substance was HLJG0701-β. It was administered at doses of 125, 250, and 500 mg/kg. Donepezil, the positive control, was injected intraperitoneally at a dose of 5 mg/kg : a control group, a scopolamine- or OVX +  5 mg/kg .The maze consisted of a stainless-steel circular pool with a diameter of 120 cm and a height of 50 cm. The pool was equally divided into four imaginary quadrants ; a visual cue of four unique shapes  was attached to the wall in each of the four quadrants. A transparent escape platform (6 cm in diameter and 29 cm in height) was located in the center of one quadrant (NE) and the pool was filled with water until the platform was submerged 1 cm below the surface. The water was made opaque with titanium oxide and maintained at a temperature of 22 ± 2 °C. All experimental conditions were maintained for the duration of the experimental period. During the two days before trial initiation, all animals were allowed to swim in the pool without a platform daily for 60 s in order to evaluate their locomotor activity and acclimation to the surrounding environmental conditions. Training sessions were conducted with two trials per day at an interval of 120 s for five consecutive days. Mice were semi-randomly released in one quadrant (without the platform) and the escape latency, the time it took the mouse to reach the hidden platform, was measured. The mice were allowed 60 s for exploration of the platform. It was acceptable for them to stay on the platform for 15 s once they found it. If the mice failed to locate the platform on their own, they were gently guided to it and allowed to remain on it for 15 s .The Y-maze consisted of three white arms connected at 120° angles . After placing a mouse on one arm and allowing it to move freely for 8 min, the order in which the mouse entered and exited each arm was recorded. If the tail of the animal fully entered an arm, it was considered to have entered that arm. The total number of times that a mouse accessed each arm was measured. The number of cases of successive entries to each arm  was measured and 1 point was given. Using this result, the alternation behavior was calculated as follows:The total number of arm entries was an indicator of the locomotor activity of the animals.Immediately after the last behavioral experiment, the mice were euthanized and their brains, excluding the cerebellum, were extracted. These tissues were homogenized in 1 mL of phosphate-buffered saline  and centrifuged at 5000 rpm for 10 min at 4 °C. The supernatant was used to determine AChE and ACh levels. Protein concentration was analyzed using a Pierce™ Rapid Gold BCA Protein assay kit . The same amount of protein for all samples was then used to measure AChE and ACh content using commercial kits  according to the manufacturer’s instructions .Blood samples collected from the abdominal aorta were incubated for 30 min at room temperature in a serum separating tube . Serum was obtained by centrifugation at 1500 rpm for 10 min .MDA has frequently been used to measure lipid peroxidation. MDA assay was performed by determining the reaction of malonaldehyde with two molecules of 1-methyl-2-phenylindole at 45 °C. The reaction mixture consisted of 0.64 mL of 10.3 mmol/L 1-methyl-2-phenylindole, 0.2 mL of sample and 10 μL of 2 μg/mL butylated hydroxyltoluene. After mixing by vortex, 0.15 mL of 37% HCl was added. The mixture was incubated at 45 °C for 60 min and centrifuged at 15,000 rpm/min for 20 min. The cleared supernatant absorbance was determined at 586 nm. An MDA assay kit  was used for the analysis. For CAT activity, a CAT assay kit  was used for the analysis.in vitro tests used to measure the degree of AChE inhibition by the test substance, a test case in which the added substrate did not react with AChE was set as having an inhibition rate of 100%. The absorbance value of the group treated with sterile distilled water was taken to represent 0% AChE inhibition, assuming that both the enzyme and the substrate reacted. If the value was negative, it was set at 0. To measure the neuroprotective effect of the treatment based on ROS level and CAT activity, the results of the control, Aβ25-35, and H2O2 control groups were subjected to Student’s t-test. Differences among the Aβ25-35, H2O2 control, and test substance-administered groups were analyzed by one-way analysis of variance (ANOVA) to determine the significance of the findings and test for equal variance. If the significance and equal variance were recognized, Duncan’s test was performed. If the equal variance was not recognized, Dunnett’s T3-test was used. p-values of <0.05 indicated statistical significance. All statistical analyses were performed in accordance with the standard operating guidelines for statistical processing of this laboratory. All analyses were performed using SPSS 12.0 K , a widely used commercial statistical package. All in vivo test data are presented as the average ± SEM. Differences between the control and negative control groups were compared with an independent samples t-test. Differences between the negative control group and the HLJG0701-β administered group were analyzed for significance through a one-way ANOVA and tested for equal variance. In the one-way ANOVA, if significance and equal variance were recognized, Duncan’s test was performed. However, if the equal variance was not recognized, Dunnett’s T3-test was used. All statistical analyses were performed using IBM SPSS software , with a p-value of less than 0.05 indicating statistical significance.In the in vitro culture. Small-molecule ginsenosides in the test substance, HLJG0701-β, were produced by bioconversion through steaming and fermentation. The total ginsenoside content of cultured wild ginseng root was increased after methyl jasmonate treatment (elicitor), and HLJG0701-β was developed by improving the manufacturing process. To verify the functionality of the new raw material, HLJG0701-β, we determined its AChE inhibitory effect and neuroprotective effects against Aβ25-35 and H2O2. Based on the results of in vitro and in vivo evaluations, HLJG0701-β may be considered as a potent candidate for the inhibition of AChE activity and oxidative stress, and thus may be involved in improving memory deficit in mice. It also improved memory by increasing the activity of cholinergic nerves. The efficacious and safe dose of HLJG0701-β needs to be determined through future clinical trials. The results of this study suggest that HLJG0701-β can be developed as a raw material for healthy functional foods.We established a culture technology that could artificially produce wild ginseng root through


Streptococcus pneumoniae strain Rx1, a Hex mismatch repair-deficient standard transformation recipient, was obtained by combining Nanopore and Illumina sequencing technologies. The genome consists of a 2.03-Mb circular chromosome, with 2,054 open reading frames and a GC content of 39.72%.The complete genome sequence of  Streptococcus pneumoniae is a human pathogen and the most important model organism for studying bacterial genetics and genomics. Widely used laboratory strains include type 2 Avery’s strain D39 and its derivatives Rx1 and R6, which are standard transformation recipients (590) of 0.8. Pneumococcal cells were harvested by centrifugation , and the cell pellet was dry vortex-mixed and lysed in 0.1% deoxycholate-0.008% SDS. High-molecular-weight DNA was purified three times with 1 volume of chloroform-isoamyl alcohol (24:1 [vol/vol]), precipitated in 0.6 volumes of ice-cold isopropanol, and spooled on a glass rod. DNA was resuspended in 10× saline-sodium citrate (SSC) buffer (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and then adjusted to 1× SSC and maintained at 4°C. The DNA solution was homogenized using a rotary mixer. Oxford Nanopore Technologies MinION and Illumina HiSeq 2500 instruments were used for DNA sequencing. DNA was not sheared; size selection was obtained with 0.8 volumes of AMPure XP beads (Beckman Coulter). The Nanopore sequencing library was prepared using the SQK-LSK108 kit (Oxford Nanopore Technologies) following the manufacturer’s instructions, and the sample was sequenced using an R9.4 flow cell (FLO-MIN106). Postsequencing high-accuracy base calling and adapter trimming of raw Nanopore reads were performed using Guppy v4.0.11 with configuration dna_r9.4.1_450bps_hac, and base-called reads were analyzed with NanoPlot v1.18.2 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Nanopore and Illumina sequencing generated 3,892 long reads  and 86,582 read pairs (2 × 250 bp), respectively, for Rx1, whereas 4,771 long reads  and 278,462 read pairs were obtained for R36A. Sequence coverage was 31.6× for Rx1 and 67.0× for R36A. A hybrid assembly of Nanopore and Illumina reads was obtained using Unicycler v0.4.712 (https://github.com/mw55309/ideel), respectively. Annotation was performed with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v5.1  accession no. SRR15216323 and SRR15216322, respectively, for R36A and SRA accession no. SRR15216380 and SRR15216379, respectively, for Rx1.The complete genome sequences of R36A and Rx1 are available under GenBank accession no.


Solenopsis  fire ants are a range of compounds possessing various biological activities. A series of racemic 2-methyl-6-alkyl-Δ1,6-piperideines were synthesized for chemical confirmation of the natural products found in fire ant venom, and the evaluation of their biological activity. Synthetic Δ1,6-piperideines and the natural compounds in the cis-alkaloid fraction of Solenopsis invicta had identical mass spectra and retention times. Their insecticidal activities against the third-instar larvae of cotton bollworm (Helicoverpa armigera) were evaluated by using injection and topical application methods. All three compounds exhibited no lethal effect at concentrations of 0.05–0.4 mol/L by topical treatment, but moderate lethal effect at 0.4 mol/L through injection treatment. Compound 6a showed significantly higher activity than the natural insecticide nicotine. The differences in activity among compounds 6b, 6c and nicotine were not significant. The elongation of the carbon chain at the 6-position of the piperideine ring appears to decrease insecticidal activity.2,6-dialkylpiperideines found in the venom of  Solenopsis invicta, known as one of the most notorious invasive species in the world + = 252.2691, found 252.2685.2-Methyl-6-undecyl-Δ from 5a . 1H NMR 1,6-piperideine (6b): Yield 77% from 5b. 1H NMR : δ 0.67 , 0.90–1.06 , 1.24–1.38 , 1.51–1.54 , 1.83–1.96 , 3.21 ; 13C NMR : δ 13.9 (CH3), 18.4 (CH3), 22.5 (CH2), 23.1 (CH2), 26.8 (CH2), 28.2 (CH2), 29.16 (CH2), 29.19 (CH2), 29.31 (CH2), 29.38 (CH2), 29.49 (CH2), 29.52 (CH2), 31.7 (CH2), 41.0 (CH2), 52.7 (CH), 170.1 (C=N); HRMS calculated for C19H37N [M + H]+ = 280.3004, found 280.2997.2-Methyl-6-tridecyl-Δ1,6-piperideine (6c): Yield 52% from 5c + = 308.3317, found 308.3311.2-Methyl-6-pentadecyl-Δ from 5c . 1H NMR S. invicta, were collected from Guangzhou, China . The colony was maintained under laboratory conditions  in a 1-gallon plastic jar coated with a Teflon suspension to prevent escape, and was fed sugar water and crickets. Ant workers were subjected to extraction within one month of collection.Workers of the red imported fire ant, S. invicta workers were conducted following previously reported procedures [cis- and trans-alkaloid fractions were obtained by pooling collections containing specific groups of alkaloids. A mixture of synthetic Δ1,6-piperideines 6a, 6b and 6c in equal amounts was dissolved in hexane. To confirm the identities of Δ1,6-piperideines, both the cis-alkaloid fraction with a final volume of 1.5 mL and the solution of synthetic standards were subjected to GC-MS analysis using a Shimadzu GCMS-TQ8050 NX equipped with a Shimadzu SH-Rxi-5Sil MS column with exactly the same dimensions as the HP-5MS column above. The oven temperature was programmed as follows: 90 °C for 1 min, increased at 10 °C/min to 160 °C, then to 250 °C at 3 °C/min, and held for 2 min. The transfer line temperature was set at 250 °C. Mass spectra were obtained using electron impact . Helium was used as the carrier gas (1 mL/min).The extraction and isolation of venom alkaloids from ocedures ,16,48. BHelicoverpa armigera were purchased from a commercial supplier, Keyun Industry Co., Ltd. . Newly hatched H. armigera larvae were reared in groups of 50–100 in 200 mL plastic cups at 27 ± 1 °C, relative humidity 60% and 16L:8D h photoperiod, until the third instar. Small cubes of artificial diet [6a–c was determined through topical and injection applications.Eggs of ial diet  were ref6a–c and nicotine were dissolved in acetone and then diluted to obtain the following concentrations: 0.4, 0.2, 0.1 and 0.05 mol/L. Acetone was used as a negative control, whereas nicotine was used as a positive control. A 2 μL drop of acetone solution was applied with a micropipette onto the thoracic dorsum of an anesthetized early third-instar larva placed in a metal bath at 4 °C. Twenty larvae were treated for each treatment. Treated larvae were placed individually in a 10 mL glass tube and maintained on an artificial diet during the post-treatment observation period under the standard rearing conditions describe above. Larvae were considered dead if they were unable to move in a coordinated manner when touched several times with a hairbrush. The numbers of dead larvae were recorded 24 h and 48 h after treatment for the calculation of mortality.Piperideines 6a–c and nicotine solutions at 0.4 mol/L were performed using the Nanoject II Injector . The needle of the injector was carefully inserted between the second and third abdominal segments of an anesthetized third-instar larva placed in the icy metal bath at a vertical angle of about 30° under a dissecting microscope. A total of 202.4 nL (50.6 nL × 4) of the sample was applied to a single larva. Sixteen larvae were treated in each treatment. The tests were replicated five times over 5 consecutive days. Acetone was served as the control. The treated larvae were maintained separately under standard conditions. Mortality was recorded 24 h and 48 h after treatment.Injections of 1,6-piperideines were synthesized and used to confirm the identification of the corresponding natural products. Their retention times and mass spectra data coincided with those in the cis-alkaloid fraction purified from the hexane extract of S. invicta workers. Their insecticidal activities were further evaluated with topical application and injection methods. The results of the bioassays indicated that these compounds displayed insecticidal properties against the early third-instar larvae of H. armigera. In particular, compound 6a, with an undecyl side chain, displayed promising injection activity as compared to the natural alkaloid nicotine at 0.4 mol/L. The activity of 2-methyl-6-alkyl-Δ1,6-piperideines is negatively correlated to the length of the carbon chain at the 6-position of the piperideine ring. Interestingly, the piperideines were found to be almost inactive when applied topically. The cuticle of the cotton bollworm may prevent the penetration of piperideines into the haemolymph, resulting in such inactivity by topical application. The insecticidal activities of compounds 6a–c provide valuable information for further research on biological activities.In summary, three minor components of the fire ant venom and 2-methyl-6-alkyl-Δ