Microbial production of 7-DHC from simple carbon has been recognized as an attractive complement to the traditional sources. Even though our previous work realized 7-DHC biosynthesis in Saccharomyces cerevisiae, the current productivity of 7-DHC is still too low to satisfy the demand of following industrialization. As increasing the compatibility between heterologous pathway and host cell is crucial to realize microbial overproduction of natural products with complex structure and relative long pathway, in this study, combined efforts in tuning the heterologous Δ24-dehydrocholesterol reductase (DHCR24) and manipulating host cell were applied to promote 7-DHC accumulation.7-Dehydrocholesterol (7-DHC) has attracted increasing attentions due to its great medical value and the enlarging market demand of its ultraviolet-catalyzed product vitamin DERG10, ERG13, tHMG1, ERG12, ERG8, ERG19, IDI1, ERG20). Through screening DHCR24s from eleven tested sources, it was found that DHCR24 from Gallus gallus (Gg_DHCR24) achieved the highest 7-DHC production. Then 7-DHC accumulation was increased by 27.5% through stepwise fine-tuning the transcription level of Gg_DHCR24 in terms of altering its induction strategy, integration position, and the used promoter. By blocking the competitive path (ΔERG6) and supplementing another copy of Gg_DHCR24 in locus ERG6, 7-DHC accumulation was further enhanced by 1.07-fold. Afterward, 7-DHC production was improved by 48.3% (to 250.8 mg/L) by means of deleting NEM1 that was involved in lipids metabolism. Eventually, 7-DHC production reached to 1.07 g/L in 5-L bioreactor, which is the highest reported microbial titer as yet known.In order to decouple 7-DHC production with cell growth, inducible GAL promoters was employed to control 7-DHC synthesis. Meanwhile, the precursor pool was increased via overexpressing all the mevalonate (MVA) pathway genes  contains supplementary material, which is available to authorized users. Intake  cancers , 3. Nowaery year , 4. Micrery year . Through24) Fig. a, heteroERG2,3 from Mus musculus), has been proven to be efficient to promote 7-DHC productivity [S. cerevisiae [FLD1  deletion strain, along with the enlarged lipid droplets. It was also reported by Park et al. [PAH1  led to striking changes in triacylglycerol and phospholipid metabolism, along with a significant increase on ergosterol synthesis. Therefore, manipulation of lipids metabolism to increase the flux flow toward sterol synthesis pathway would be helpful to improve 7-DHC accumulation in yeast. Besides that, introducing heterologous modules always upsets the original intracellular balance [7-DHC biosynthesis pathway covers eight genes in mevalonate (MVA) pathway and nine genes in post-squalene pathway Fig. a. In orductivity –7. Howevuctivity . For insrevisiae . As reporevisiae , sterolsk et al.  that the balance , 13. And balance –16. In t balance  or regul balance  might alIn this article, combined efforts in manipulating host and 7-DHC synthesis pathway were conducted to promote 7-DHC output base on our previous study [ERG10, ERG13, ERG12, ERG8, ERG19, IDI1, and ERG20 as well as three copies of tHMG1  as well as the overexpressed MVA pathway genes  revealed that disruption of ERG5 significantly activated the transcription of all of the MVA genes  [Moreover, our previous study has demonstrated that blocking competitive ergosterol biosynthesis pathway was essential for 7-DHC accumulation in yeast [rol Fig. a, obtainrol Fig. b. Excess_DHCR24)  generateH. sapiens, M. musculus (Mm_DHCR24) [Danio rerio (Dr_DHCR24) [Equus caballus (Ec_DHCR24) [Gallus gallus (Gg_DHCR24) [Xenopus tropicalis (Xt_DHCR24) [Bos taurus (Bt_DHCR24) [Arabidopsis thaliana (At_DHCR24) [Gossypium hirsutum (Gh_DHCR24); one invertebrate DHCR24 from Trypanosoma grayi (Tg_DHCR24) [Cryptococcus gattii (Cg_DHCR24) [24(28) bond, and then deoxidize the Δ24(25) bond in sterol substrate [24-reduction step for 7-DHC synthesis. In contrast, vertebrate DHCR24s, activities of which do not cover the initial isomerization reaction, could achieve 7-DHC synthesis in yeast at different levels   , and Dan_DHCR24) , have be_DHCR24) , 6. Howe_DHCR24) , Gallus _DHCR24) , Xenopus_DHCR24) , and Bos_DHCR24) ; two pla_DHCR24)  and Goss_DHCR24) ; and one_DHCR24)  were selubstrate . TherefoGAL7,1,10 were knocked out to eliminate galactose utilization [GAL80 was applied to avoid addition of the inducer [GAL7,1,10 and leaving GAL80 untouched [DHCR24 and even 7-DHC production. In brief, in the control strain SyBE_Sc01250009, DHCR24 expression cassette (PGAL7-DHCR24) was integrated into locus GAL80; whereas in strain SyBE_Sc0125XJ02, the same DHCR24 expression cassette was inserted into locus GAL7,1,10, leaving a wild-type GAL80  than that of ΔGAL80 strain (SyBE_Sc01250009) . As it was reported that the activity of promoter GAL1 (PGAL1) was stronger than that of PGAL7 [DHCR24 in strain SyBE_Sc0125XJ02 was replaced by PGAL1, generating strain SyBE_Sc0125XJ03  into locus ERG6  and SyBE_Sc0125XJ08 (ΔNEM1), respectively (Table FLD1 and ΔNEM1 achieved 15.7% (to 195.7 mg/L) and 48.3% (to 250.8 mg/L) improvement on 7-DHC production, respectively. However, further combination of ΔFLD1/ΔNEM reduced the 7-DHC titer to 109.0 mg/L  under YPD medium when harvested was only 1.25-fold higher than that under SC medium  in ergosterol synthetic yeast [FLD1. Meanwhile, as reported by Arendt et al. [PAH1 stimulated a dramatic expansion of the endoplasmic reticulum (ER), which resulted in overproduction of triterpenoids  probably by functional overproduction of ER-localized proteins. However, among those ER-located genes  [ERG25 and ERG27 was significantly activated by ΔNEM1, indicating that the enhanced 7-DHC productivity by ΔNEM1 might not bring by ER-engineering effect. Thus, it is probably required further global transcription analysis by RNA-sequencing to expose its functional mechanism toward 7-DHC synthesis.Deletion of Δmembrane , 11. Altmembrane . Howeveric yeast , which mt et al. , ΔPAH1 s ERG2-3) , only ERS. cerevisiae was an oxygen consumption process and too much oxygen supply would inhibit sterol synthesis [Fed-batch fermentation of strain SyBE_Sc0125XJ08 was carried out in 5-L bioreactor under glucose restriction strategy. Glucose concentration was controlled below 5 g/L for reducing the ethanol and glycerol produced during the fermentation process Fig. . Air floynthesis . As the ERG5-6 only in cell growth stage would compensate for the necessary block of ergosterol biosynthesis during 7-DHC accumulation period. One the other hand, sterols stored in S. cerevisiae in their esterified forms, and overexpression of two endogenous sterol acyltransferases (ARE1 and ARE2) could promotes sterols accumulation [However, 7-DHC synthesis could be further enhanced via host engineering in yeast. On the one hand, ergosterol is essential to maintain a normal structure and function of cellular membranes , and ergmulation , 44. Howmulation . Even thS. cerevisiae. A modified host cell was constructed to appeal to the increased 7-DHC accumulation via decoupling 7-DHC production with cell growth, enhancing MVA pools, totally blocking the competitive path , as well as deleting lipids metabolism gene (ΔNEM1). In the meanwhile, the optimal DHCR24 sources (Gg_DHCR24) were obtained by screening the enzymes from diversity species. And through fine-tuning the transcription level of Gg_DHCR24 in terms of adjusting its induction strategy , integration position , used promoter type (PGAL1), and copy numbers, 7-DHC production were stepwise improved accordingly. Eventually, the highest 7-DHC titer, so far known (1.01 g/L), was achieved in 5-L bioreactor, which is 26.9-fold higher than that of the starting strain. This work not only opens large opportunities to realizes downstream the de novo synthesis of other steroids, but also highlights the importance of the combinatorial engineering of heterologous pathway and host to obtain microbial overproduction of many other natural products.In this work, combined engineering of the host cell and the heterologous enzyme DHCR24 significantly improved 7-DHC productivity in Escherichia coli DH5α, which was used for plasmids construction, was cultivated at 37 °C in Luria–Bertani (LB) medium supplemented with 50 μg/mL kanamycin. The yeast strains used in this study were derived from S. cerevisiae CEN.PK2-1D and summarized in Table d-(+)-galactose) at 30 °C.H. sapiens, M. musculus, D. rerio, E. caballus, G. gallus, X. tropicalis, B. taurus, A. thaliana, G. hirsutum, C. gattii, T. grayi were obtained from NCBI database . Heterologous DHCR24 genes were codon-optimized  and listed in Additional file 600 of 0.2. The initial glucose concentration in either YPD medium or SC medium was 40 g/L. Before cultivation, 10 g/L d-galactose was supplemented into the media to induce the gene expressions controlled by GAL promoters. Yeast cells were harvested after 100-h growth.For shake flask fermentation, glycerol-stock yeasts were rejuvenated on solid YPD plate . Then a 600 of 0.2–8.0 in YPD medium. Then 200 mL seed cultures were transferred into a 5-L bioreactor  with an initial OD600 of 0.8. Fermentation was performed in 2-L cultures at 30 °C. PH and air flow were controlled at 5.8 and 1 vvm, respectively. The dissolved oxygen (DO) was maintained around 40% through adjusting the agitation speed. 50% (v/v) glucose was fed periodically into the culture to keep the glucose concentration under 2 g/L. When cells entered post-log phase (OD600 around 140), glucose feeding was stopped, and galactose solution was added into the bioreactor with a final concentration of 20 g/L. At the same time, ethanol was fed into the culture until the end of the fermentation. At least independent duplicate samples were collected to determine the cell density, glucose concentration, ethanol concentration, and 7-DHC production [Fed-batch fermentation was conducted in YPD medium supplemented with 20 g/L glucose as the initial carbon source. Seed cultures were obtained via overnight culture from an OD600 around 20) were harvested and resuspended in 200 μL 0.1 M NaOH for 5 min incubation at room temperature. Then cell pellets were harvested, resuspended in 50 μL SDS sample buffer (60 mM Tris–HCl (pH 6.8), 5% glycerol, 2% SDS, 4% β-mercaptoethanol, 0.0025% bromophenol blue), and boiled for 10 min. 20 μL cell lysis were loaded onto 10% SDS-PAGE gel. After electrophorescence, proteins were transferred to PVDF membranes. Membranes were blocked with 5% BSA in TBST buffer (10 mM Tris (pH 8.0), 150 mM NaCl, 0.05% Tween 20), then incubated with primary anti-polyhistidine , or anti-GAPDH (HRP)  overnight at 4 °C with shaking. Afterward, membranes were repeatedly washed with TBST buffer. The membrane probed to anti-polyhistidine required further incubation with secondary HRP-conjugated goat anti-mouse antibody . Signals were detected following the of SuperSignal™ West Pico PLUS Chemiluminescent Substrate Kit  by using Azure Biosystems C280 Chemiluminescent Blot Imaging System (USA). The intensities of the bands in western-blot pictures were quantified with Quantity One . The relative expression level of each DHCR24 was determined as the gray scale of anti-polyhistidine band divided by that of anti-GAPDH.In order to determine the expression levels of DHCR24s employed in this study, polyhistidine-tag was attached to the C-terminal of each tested DHCR24 in its particular expression cassette by PCR amplification with the primers listed in Table S1. Then the cassette was integrated into chromosome of strain syBE_Sc0125XJ01 with the same procedure to generate strain SyBE_Sc01250050  trifluoroacetamide (MSTFA) at 30 °C for 2 h to gain the sample ready for analysis.Extraction and analysis of sterols were applied according to Su et al.  with somm/z. The ion source temperature was 250 °C, and the injection site temperature was 260 °C. The temperature was initially 70 °C for 2 min, then it was increased at 30 °C/min to 250 °C, and finally followed by an increase to 280 °C at 10 °C/min. 280 °C was kept for 15 min, and was increased to the final temperature 290 °C at 5 °C/min. The final temperature was maintained for 5 min. Sterol standards  were purchased from Sigma-Aldrich (USA).The sterols were separated on an Agilent 6890 gas chromatograph (GC) (USA) coupled to Waters time-of-flight mass spectrometry (TOF–MS) (USA). The gas chromatograph was equipped with a DB-5 fused-silica capillary column . Ions were generated by a 70 eV electron beam in EI mode at an ionization current of 40 μA. Mass spectra were acquired in a range of 50–800 −ΔΔCt method [ALG9 was used for normalization [T test) was conducted using the SPSS 19.0 package to demonstrate variations between the tested groups. The level of significance was set at P < 0.05.Transcription levels of genes in 7-DHC biosynthesis pathway were analyzed by Real-Time PCR. Strains were cultured in shake flask for 10 h (glucose consumption phase) and 30 h (ethanol consumption phase), respectively, and then harvested. Total RNA extraction, reverse transcription, and quantitative PCR were carried out by Apexbio Inc. (China) based on Wang et al. . The relt method . Gene ALlization . All datAdditional file 1: Table S1. Oligonucleotides used in this study. Table S2. The Codon-optimized sequences of DHCR24s involved in this study. Table S3 Plasmids used in this study. Figure S1. Relative transcription level of the MVA pathway genes (a–h) and the post squalene pathway genes (i–r) in control and ΔERG5 strain. Cells were harvested at 10 h (glucose consumption phase) and 30 h (ethanol consumption phase). The relative transcription level for each gene was determined as 2−ΔΔCt using gene ALG9 for normalization. All data were from at least triplicate experiments. Significance levels of t-test were determined as “*” is for P < 0.05 and “**” is for P < 0.01. Figure S2. Cell growths of strains with DHCR24s from diversity species in YPD medium. The error bars represent standard deviation calculated from triplicate experiments. Hs, Homo sapiens; Mm, Mus musculus; Dr, Danio rerio; Ec, Equus caballus; Gg, Gallus gallus; Xt, Xenopus tropicalis; Bt, Bos Taurus; At, Arabidopsis thaliana; Gh, Gossypium hirsutum; Cg, Cryptococcus gattii; Tg, Trypanosoma grayi. Figure S3. Cell growths of strains SyBE_Sc01250009, SyBE_Sc0125XJ02 and SyBE_Sc0125XJ03 in YPD medium. Figure S4. Cell growths of strains SyBE_Sc0125XJ03, SyBE_Sc0125XJ04 and SyBE_Sc0125XJ06 in YPD medium. Figure S5. Effect of deleting lipids metabolism associated gene(s) on biomass building-up and 7-DHC production. a Cell growths of strains SyBE_Sc0125XJ06, SyBE_Sc0125XJ07, SyBE_Sc0125XJ08 and SyBE_Sc0125XJ09 in YPD medium as well as that of SyBE_Sc0125XJ08 in SC medium. b 7-DHC production of strain SyBE_Sc0125XJ08 in YPD medium and SC medium.


The COF-K exhibited high BET surface area and narrow pore size of 1.25 nm and gave a CO Covalent organic frameworks (COFs) are a class of porous crystalline materials constructed by rigid building blocks through covalent bonds ,2,3. It 2 adsorption remains to be disclosed. Porphyrin-based COFs have been extensively fabricated and studied due to the intriguing trait of their highly conjugated π-electron macrocycles and metal coordination sites. Specifically, for CO2 adsorption, the presence of basic pyrrole containing macrocyclic cavity is highly desired on account of the strong interaction with Lewis acid CO2  COFs, for example, COF-366 (2 nm) -TAPH-COF -T m2 g−1)  and seve m2 g−1) , COF-103 m2 g−1) ,42.−1 resolution using a Bruker ALPHA-T. Powder X-ray diffraction (PXRD) measurements were carried out on a Bruker D8 ADVANCE X-ray with a Cu-Kα sealed tube (λ = 1.5406 Å) from 2θ = 2° to 30° with 0.02° increment. Field emission scanning electron microscopy (FE-SEM) was performed on a Hitachi SU 8010 with accelerating voltage of 5.0 kV. The samples were sputtered with Pt (nano-sized film) prior to imaging by a SCD 040 Balzers Union. Transmission electron microscopy (TEM) images were obtained on a JEOL JEM-2100F electron microscope operated at 100 kV. The samples were prepared by dropcasting the sample from ethanol on copper grids. Thermogravimetric analysis (TGA) were taken on a Perkin Elmer Diamond by heating the samples at 10 °C min−1 to 1000 °C in an air atmosphere. The Brunauer–Emmett–Teller (BET) surface area and gas adsorption measurements were carried out at 77K with Micromeritics ASAP 2020 after being degassed at 100 °C for 12 h. 1H NMR spectra were recorded on Bruker AVANCE300, where chemical shifts were dependent on a residual proton of the solvent as standard. Solid-state 13C CP/MAS NMR measurements were measured on Bruker AVANCE III HD at a MAS rate of 5 kHz and a CP contact time of 2 ms.Fourier transform infrared (FT-IR) spectra were collected in KBr pellets with 2 cm2·2H2O, NH3·H2O were obtained from Sinopharm Chemical Reagent Limited Company. Pyridine, THF, n-hexane, dimethylformamide, 1,2-dichloroethane, chloroform, Tianjin Fuyu Fine Chemical Limited Company. All other reagents and solvents were commercially available and used as purchased without further purification. Soxhlet extraction was conducted through Soxhlet apparatus with THF.Pyrrol, p-nitrobenzaldehyde, phosphorus oxychloride, N-alkyl carbazole were obtained from Aladdin. Propionic acid, acetic anhydride, conc. HCl, SnClMaterials Studio 7. Unit cell parameters were first manually calculated from the observed PXRD peak positions using the Bragg equation. We utilized Pawley refinement to optimize the lattice parameters iteratively until the Rwp value converges. The pseudo-Voigt profile function was used for whole profile fitting and Berrar–Baldinozzi function was used for asymmetry correction during the refinement processes. The final Rwp and Rp values were Rwp = 1.97% and Rp = 1.02%.Molecular modeling and Pawley refinement were implemented using Reflex, a module in 1H NMR : δ (ppm)–2.80, 8.33 , 8.606 , 8.755 .A 250-mL three-neck round bottom flask was contained the mixture of propionic acid (110 mL) and acetic anhydride (5 mL), p-nitrobenzaldehyde  was heated to 173 °C, then freshly distilled pyrrole  was added dropwise. After dropping, the solution was stirred at 173 °C for 1 h. After the reaction completed, the dark violet precipitate was collected by filtration and washed with methanol. The crude product was dissolved in 100 mL pyridine and refluxed with stirring for 4 h, after cooling to room temperature, the product was collected by filtration to give 5,10,15,20-tetrakis(4′-nitrophenyl) porphyrin as violet powder (0.98 g) in 13% yield. The product was used directly for the next step without further purification. 2·2H2O  was added. The mixture was stirred at 70 °C for 30 min and then cooled to 0 °C. Aqueous NH3 was added slowly to neutralize HCl, the precipitate was collected by filtration and dissolved in THF. Then the solution was collected by filtration and recrystallized with n-hexane, to give 5,10,15,20-tetrakis(4′-aminophenyl)porphyrin as violet crystal (408 mg) in 40% yield. 1H NMR : δ (ppm)–2.731 , 5.541 , 7.013 , 7.858 , 8.874 .The compound was prepared according to the literature.  A mixturv/v dichloromethane/methanol) provided a white solid (1.8g) in 61% yield. 1H NMR : δ(ppm) 1.522 , 4.485 , 7.758 , 8.115 , 8.683 , 10.145 .The compound was prepared according to the literature.  A 250-mL2 and length 200 mm) was charged with 5,10,15,20-tetrakis(4′-aminophenyl) porphyrin , 3,6-diformyl-9-ethylcarbazole , 1 mL of o-dichlorobenzene, 1 mL of n-butanol, and 0.2 mL of 6 M aqueous acetic acid. This mixture was sonicated for 10 min in order to get a homogenous dispersion. The tube was flash frozen at 77 K (liquid N2 bath) and degassed by three freeze-pump-thaw cycles and then sealed off. The tube was heated to 120 °C for 3 days. After that, a dark violet precipitate was formed and collected by centrifugation and washed with acetone (10 mL × 3). The powder was purified by Soxhlet extraction with tetrahydrofuran for 3 days and then dried at 120 °C under vacuum for 12 h to give a dark violet powder in 69% (81 mg). Elemental analysis (%) calculated for C 82.34, H 4.85, N 12.81; found: C 78.22, H 5.06, N 12.05.A Pyrex tube (o.d. × i.d. = 10 × 8 mm2 adsorption isotherm and a narrow pore size distribution for 1.25 nm, but with a relatively weak PXRD pattern owing to lack of π–π interaction from the two-dimensional layers. Different from the previously two-dimensional COFs is that each porphyrin connects four linear building blocks to form two-dimensional networks; each porphyrin links with four nonlinear building blocks to form one-dimensional line in the COF-K. This strategy gives a new direction to build one-dimensional COFs and expands greatly the scope of the emerging materials. The new topology structure may show potential applications in woven materials, flexible conductive materials and super light aerogel.In summary, it is the first time that we successfully synthesized a novel one-dimensional COF-K from porphyrin and nonlinear building units. The COF-K is an ordered microporous material with a Type-I N


Structural variations of the benzylphenoxyacetamide (BPA) molecular skeleton were explored as a viable starting point for designing new anti-glioblastoma drug candidates. Hand-to-hand computational evaluation, chemical modifications, and cell viability testing were performed to explore the importance of some of the structural properties in order to generate, retain, and improve desired anti-glioblastoma characteristics. It was demonstrated that several structural features are required to retain the anti-glioblastoma activity, including a carbonyl group of the benzophenone moiety, as well as 4′-chloro and 2,2-dimethy substituents. In addition, the structure of the amide moiety can be modified in such a way that desirable anti-glioblastoma and physical properties can be improved. Via these structural modifications, more than 50 compounds were prepared and tested for anti-glioblastoma activity. Four compounds were identified  that in addition to HR40 (PP1) from our previous study, have been determined to have desirable physical and biological properties. These include high glioblastoma cytotoxicity at low μM concentrations, improved water solubility, and the ability to penetrate the blood brain barrier (BBB), which indicate a potential for becoming a new class of anti-glioblastoma drugs. Surprisingly, the most effective way to increase survival of glioblastoma patients is still extensive surgical resection. However, in many instances, this approach is not feasible due to the tumor’s location and its infiltration of highly specialized brain areas5. The current standard of care therapies include maximal surgical resection, followed by radiotherapy plus concomitant and maintenance treatment with temozolomide (TMZ), which is one of the few anticancer drugs capable of crossing the blood brain barrier (BBB)6. Unfortunately, TMZ-treated tumors develop TMZ-resistance, and recurrent glioblastomas are practically incurable9. Moreover, numerous clinical trials targeting a variety of glioblastoma-specific pathways, as well as, those testing immune checkpoint inhibitors, have been implemented, but have failed to produce a positive outcome in glioblastoma patients11.Glioblastoma is the most aggressive and prevalent malignancy of the central nervous system (CNS), with a median patient survival rate of about 18 months15. All of these processes require high cholesterol metabolism and uptake for a continuous biogenesis of cellular membranes. Therefore, it does not come as a surprise that a combination of cholesterol lowering drugs with TMZ might be a good approach for glioblastoma treatment16. One lipid-lowering drug that has attracted attention as a possible candidate for an anticancer regimen is fenofibrate (FF)22. FF is a common lipid-lowering pro-drug that following its processing by blood and tissue esterases is converted to fenofibric acid (FAA), a potent agonist of peroxisome proliferator activated receptor alpha (PPARα) that upregulates fatty acid utilization and attenuates glycolysis26. Although activation of PPARα may explain some of the observed anticancer effects, glioblastoma cells treated with PPARα siRNA retain sensitivity to FF, indicating a PPARα-independent mechanism of FF anticancer action. Indeed, our previously published data demonstrate that unprocessed FF (ester) accumulates in mitochondrial membrane fraction triggering a severe and immediate inhibition of mitochondrial respiration, severe decline in intracellular ATP, and is followed by a massive tumor cell death27. In addition to FF, interesting anticancer effects of other lipid lowering drugs, fibrates and statins, have also been reported34. A ten-year all-cause mortality study involving 7,722 patients treated with different fibrates revealed that the use of these metabolic compounds was associated with a significantly lower total mortality and reduced probability of death from cancer35. In cell culture and in animal studies, various members of the fibrate family demonstrated a broad range of anticancer activities40. These multiple reports encouraged clinical trials in which chronic administration of low doses of FF was tested along with chemotherapeutic agents, minimizing their toxicity and acute side effects in patients with recurrent brain tumors and leukemia42.Therapeutic strategies that include TMZ in combination with other drugs have been also explored. For instance, glioblastomas are characterized by exaggerated lipogenesis, enhanced LDL and cholesterol uptake, and extensive phagocytosis and exosome formation23; effects of FF on cell membrane fluidity43; and the FF-induced inhibition of mitochondrial respiration in isolated cardiac and liver mitochondria45. Therefore, a growing line of evidence supports the interaction of unprocessed FF (ester) with biological membranes, which could be a reason for the observed strong anticancer activity of this lipid-lowering drug. However, FF does not cross the BBB, and is quickly processed by the blood and tissue esterases to form fenofibric acid (FFA). This acid functions as a potent peroxisome proliferator activated receptor α (PPARα) agonist, however, it is no longer effective in triggering tumor cell death46.Other groups also demonstrated that FF could have PPAR-independent cellular effects including: PPAR-independent activation of GDF15FF structure in order to improve the prospective anticancer drug stability, water solubility, tissue penetration, and ultimately, the anti-glioblastoma efficacy. One of the resulting compounds, PP1  varies between 5 and 6 μM and may not be sufficient to exert a highly effective anti-glioblastoma activity in vivo47. Therefore, we further explored the anti-tumoral contribution of the specific chemical moieties in the FF and PP1 structures, to improve the compound anticancer efficacy and its ability for more effective accumulation within the brain tumor tissue. As a result, 50 additional compounds were generated and analyzed in this study.We have made several chemical modifications to the FF) contains a benzylphenoxyacetate structural arrangement  by blood and tissue esterases46. An additional disadvantage of FF is that it has low water solubility, and a relatively high concentration is required for its anti-tumoral activity (50 μM)27. Therefore, we have selected the benzylphenoxyacetamide (BPA) molecular skeleton as a basis for designing new more potent anti-glioblastoma compounds due to its structural similarity to FF, as well as its increased water solubility and higher resistance to hydrolysis   Fig. . Selecti °C Fig. . In thisBPA, ammonium salts were prepared. Hydrochloric salts were prepared by simple mixing of the corresponding BPA with concentrated hydrochloric acid followed by water evaporation. Then, methylation of the basic BPA was performed with acetone as the solvent, and methyl iodide as the methylating reagent. The product, HR36, was crystalized directly from the reaction mixture  to evaluate their potential for reaching the intracranial tumor site at therapeutically relevant concentrations. Prior to the preparation of all BPA variant compounds in this study, we performed extensive molecular modeling to describe their physicochemical properties. A cell viability (CV) assay was then performed using the LN229 human glioblastoma cell line, and the cells were treated with BPA variants at 25 μM for 72 hours. The results of this computational characterization and cell viability testing are outlined in Figs. One fundamental challenge for the design of CNS penetrant drugs is the need to cross the blood-brain barrier (BBB). But, BBB-permeable compounds form a very small subset of oral drugs currently in existence, and experimental models for testing BBB penetration is quite complex. Therefore, an independent indicator of the BBB penetration was needed for the initial screening and selection of a large number of compounds  that indicate relative BBB penetration. The CNS-MPO scale is between 0 and 6.0, with scores ≥4.0 widely used as a cut-off to select compounds for hit CNS therapeutic drug discovery programs53. The validation of this approach utilizes a library of 616 compounds to evaluate the experimental distribution of the computed parameters incorporated into CNS-MPO scores53. It was found that CNS-MPO scores of 1–2 (0%), 2–3 (11.6%), 3–4 (40.8%), 4–5 (53.8%) and 5–6 (81.1%) correlate with the increased probability (%) of drugs to be found in the brain54.One currently accepted way to define physicochemical properties is by using a weighted scoring approach, known as the Central Nervous System – Multiparameter Optimization (CNS-MPO)BPA-derived compound analyzed in this study. Comparing these values is a valuable tool for understanding how different substituents may change polarization- and dispersion-type interactions with the active sites of their interacting proteins55.In addition, parameters that are routinely used for a Quantitative Structure Activity Relationship (QSAR) study are molecular polarizability (MP), minimal molecular projection area (MPA), and water solubility (LogS). These parameters are not incorporated into the CNS-MPO score, however they are also considered as factors that affect BBB penetration. Therefore, in the associated figures below, these additional parameters were defined and calculated for each 56. The minimal projected area (MPA) is also very important for drug transport and ultimately for drug activity. For instance, in recent studies by Cha, Müller, and Pos, a distinct phenotypical pattern of drug recognition and transport for the G616N variant was reported, indicating that drug substrates with MPA over 70 Å2 are less well transported than the smaller substrates58. Finally, water solubility (LogS) of −4.5 and greater are indicators of acceptable water solubility60 of the studied compounds.Molecular Polarizability (MP) is a response of electron distribution to an externally-applied static electrical field. It was postulated that an MP between 30–40 is optimal for a molecule to bind to a biotarget61. Finally, the rate of passive diffusion is inversely proportionate to the square root of molecular size (Graham’s law62), which is also included in our compound analysis.The BBB permeation propensity of all studied compounds is also indicated by the decimal logarithm of brain to-plasma concentration ratio (logBB) value, which is derived from the modified Clark’s equation: logBB = 0.152 ClogP -0.0148PSA + 0.139 30. LogBB was also calculated for the compounds in this study, and is listed in the corresponding tables  has a very potent activity against glioblastoma tumor cells47, we turned our attention to larger molecules based on this BPA structure. To evaluate the products of the following structural modifications, we used PP1 as a standard of comparison. We proceeded, by investigating the importance of the second aromatic ring in BPA, the conjugation of the carbonyl group, and the presence of a chlorine atom. All computed parameters for resulting compounds (HR1-HR5)  should produce a new drug candidate, HR4, with similar activity to PP1. However, HR4 has only moderate anti-glioblastoma activity (CV = 58.5%), and another candidate in this group, HR5, lost its anti-glioblastoma activity (CV = 90.47%)  Fig. . HoweverR5) Fig. . Replaciity Fig. . This isity Fig. 64. LipopHR1 Fig. . HoweverHR1 Fig. . If the 7%) Fig. . These rBPA skeleton. It is well documented that substituting hydrogen with fluorine substantially changes molecular polarizability (MP) and lipophilicity, and increases the binding affinity to targeted proteins65. Also, halogen bonding is stronger between chloro-aryls and carbonyl compounds, than between corresponding fluoro-aryls. Therefore, the computed data, as well as, the cell viability data of modified variants of PP1 in which the chlorine atom was replaced with fluorine (HR8-HR11) are presented in Fig. FF (HR8) has virtually the same activity (CV) as FF, other esters  are virtually inactive at the same concentration (25 μM)  Fig. . So, furPP1 (HR9) has a lower potency than PP1, and therefore, halogen bonding appears to be very important for anti-glioblastoma activity in BPAs  > HR11 > HR9. However, the computed physicochemical parameters do not include an indicator of the effect of halogen bonding, which therefore, underestimates the contribution of the chlorine bonding in PP1. In addition, electrostatic potential maps for PP1 and HR9  compared to PP1 (CV = 1.04%). Surprisingly, the molecular polarizability of HR11 (39.36 Å3) is quite close to that of PP1  are all more desirable than for PP1  for both HR21 and HR22 compared to PP1 (CV = 1.04%)  Fig. . So, in AA) structural skeleton for anti-glioblastoma activity, it is now essential to assess the nature of the amide moiety  and suggest that based on this property alone all of these compounds should have good cell permeability. On the other hand, based on the estimated PSA and CNS-MPO data, HR28 should be expected to have a lower probability of penetrating into the cell, and possibly lower accumulation in the CNS. This is in spite of the fact that HR28 is very potent in eliminating glioblastoma cells in vitro (CV = 4.31%) phenoxy-2,2-dimethylacetamide  Fig. .HR31 has two nitrogen atoms separated by four methylene groups. One nitrogen is part of an amide bond and other is part of a primary amine. This compound has a CV value similar to that of 25 μM FF27 , and is more potent than PP1  with the exception of HR36, have a good anti-glioblastoma activity, including the hydrochloric salt HR35, which in addition has a more promising CNS-MPO value (4.41). In fact, HR35 is more potent than its free base HR34   Fig. .HR34, the activity is substantially diminished because they cannot be deprotonated. If one examines the computed parameters for HR36, it is apparent that the compound is very hydrophilic  and has a very low estimated BBB penetration (logBB = −0.6). Surprisingly the CNS-MPO of HR36 is 4.0, which would suggest that this compound should be capable of accumulating in CNS, however it is completely inactive at 25 μM (CV = 98%)  Fig. . In conc66. One approach to increasing water solubility is to introduce hydroxy groups in the non-essential structural area of the compound. The amide moiety of new compounds, listed in Fig. PP1 starts by replacing the N-methyl group with N-H to make HR39. Although CNS-MPO values are virtually identical for PP1 and HR39, the latter has less desirable ClogP and LogBB values, indicating potentially lower cell penetration. The resulting cell viability agrees with these estimates because HR39 is significantly less potent than PP1  are also presented in Fig. HR42 and HR43) are structural isomers, but both are secondary amides, as is HR39. Interestingly, HR42 and HR43 activities (CV) are almost identical, indicating that the presence of the tertiary amide moiety is more important than the presence of the two hydroxy groups. Two tertiary amides with two hydroxy groups, HR44 and HR46, are also structural isomers. They are both more potent than the secondary amide, HR39, but the tertiary amide HR46, with two CH2CH2OH moieties, is almost as potent as PP1. However, introducing three (HR45) or more (HR57) hydroxy groups slightly decreases the potency, possibly because the compounds are becoming substantially more hydrophilic, as is demonstrated by lower Clog, MPO, and logBB estimated values  the best candidate is HR46 due to its low CV value and acceptable CNS-MPO values that are very similar to the prototype drug, PP1 (HR40).Four drug candidates containing two hydroxy groups , that have strong in vitro anti-glioblastoma activity, but have physical properties that may contribute to improved brain tumor penetration , along with testing of cell viability, it was demonstrated that two methyl groups in the 2-position of BPA are important for retaining anti-glioblastoma activity. Substitution of the chloro- substituent in the 4-position of the benzophenone moiety, resulted in a significant loss of anticancer activity of the modified compound. The molecular rigidity between the two aromatic rings of the benzophenone moiety is essential because both methylene and oxygen replacement of the carbonyl group resulted in lost or diminished anti-tumoral activity. It was also demonstrated that tertiary amides are more potent than secondary amides, which, in turn, are more potent than primary amides. In tertiary amides, it is important to keep one substituent small (methyl), while the other group can be a hydroxy or nitrogen substituted alkyl. In addition to the evaluated chemical modifications in the BPA structure, there is still a high degree of flexibility for additional structural variations predominantly to the amide moiety  was recorded on Waters LCT Premier XE (that’s a Tof MS) with an ESI source, scanning 100–2000 m/z with direct injections of 5 µl sample, using a 0.2 ml/min flow of acetonitrile. All molecular physical properties were calculated using Marvin Sketch software. All computed molecular descriptors were generated by ChemAxon MarvinSketch version 19.4. Electrostatic potential maps were calculated with PM3 semi-empirical method as implemented in Spartan ’18 v 1.1.0. NMR and MS spectra for all HR compounds generated in this study are included in Supplementary Materials.All starting materials were reagent grade and purchased from Sigma–Aldrich, ArkPharm, TCI America, and AbaChemScene. 2 atmosphere. Prior to treatment, cells were plated in 96-well plates  at initial density of 2 × 104 cells/cm2. Stock solutions of the compounds were prepared in DMSO, diluted in cell culture medium and added to the cells in triplicate for every experimental condition 24 h after plating . For the vehicle control, DMSO was used at 0.5%. MTT assay69 (measuring cell metabolic activity) was performed after a 72 h incubation in the presence of the compounds, as previously described. Following 1 h incubation with MTT, formazan crystals were dissolved in 5 mM HCl in isopropanol and absorbance read at 540 nm. Data represent mean values expressed as a percentage of the vehicle control ± SD. Phase contrast images of treated cells were taken 48 hours following the treatment with a BZ-X800 fluorescence microscope (Keyence) equipped with a 20x objective.Human glioblastoma LN-229 cells were maintained as semi-confluent monolayer culture in DMEM with 1 g/L glucose, sodium pyruvate, and L-glutamine (Corning) supplemented with 10% heat inactivated FBS (Gibco) and P/S (50 units/mL of penicillin and 50 µg/mL of streptomycin) at 37 °C in a 5% CO3n). A water (10 ml) solution of sodium hydroxide , (4-chlorophenyl)(4-hydroxyphenyl)methanone  and benzene (100 ml) was refluxed for 10 minutes and water was azeotropically removed by using a Deen-Stark distillation apparatus followed by removal of benzene under reduced pressure. The resulting white powdery sodium phenoxide was mixed with dry isopropanol (100 ml) and isopropyl bromoacetate . The resulting mixture was stirred with sonication for 1 hour and refluxed for 4 hours. Solvent was evaporated to solid residue and mixed with dichloromethane (100 ml) and water (100 ml). The water layer was discarded, and organic layer was washed with 5% sodium carbonate (3 × 50 ml) and dried over anhydrous sodium carbonate. After solvent evaporation, the resulting white residue was dried under vacuum to give a white solid product; isolated yield of 93% (3.1 g). 1H-NMR  δ 7.79 , 7.71 , 6.97 , 5.16 , 4.67 , and 1.28  ppm.Preparation of isopropyl 2-(4-(4-chlorobenzoyl)phenoxy)acetate (4f). An isopropanol (300 ml) suspension of sodium carbonate , (3-hydroxyphenyl)(phenyl)methanone  and isopropyl 2-bromo-2-isobutirate  was refluxed with stirring for 3 days. After cooling to room temperature, the resulting white solid was separated by filtration washed with isopropanol (3 × 20 ml). Filtrates were evaporated and evaporated to an oily residue. This residue was mixed with water/chloroform (100 ml/200 ml). The water layer was discarded, and the chloroform layer was washed with 5% sodium hydroxide (3 × 50 ml), water (3 × 50 ml), and dried over anhydrous sodium carbonate. After filtration the chloroform was evaporated to oily residue, that standing at 5 °C overnight, gave a white solid in 92% (6 g) isolated yield. 1H-NMR δ 7.77 , 7.68 , 7.48 , 7.5–7.3 , 7.27 , 7.07 , 5.06 , 1.60 , and 1.18 . 13C-NMR  δ 173.2, 155.4, 128.6, 137.5, 129.9, 129.0, 128.2, 127.5, 123.8, 123.1, 120.3, 79.4, 69.1, 25.3 and 21.5 ppmIsopropyl 2-(3-benzoylphenoxy)-2-methylpropanoate (1e). A water (10 ml) solution of sodium hydroxide , benzene (100 ml) and 4-phenoxyphenol  was stirred at room temperature for one hour. Water was evaporated away by Dean-Stark distillation. The remaining benzene was removed under reduced pressure, and the resulting white powdery residue was mixed with isopropanol (200 ml) and isopropyl 2-bromo-2-methylpropanoate . The resulting mixture was stirred by sonication at 60 °C for two hours followed by refluxing overnight. After cooling to room temperature, 5% sodium hydroxide (100 ml) was added and the resulting mixture was refluxed for 2 hours. The solvent was evaporated to solid residue mixed with water (150 ml) and acidified with concentrated hydrochloric acid to pH ~3. The resulting white suspension was mixed with chloroform (100 ml) and the chloroform layer was separated, washed with water (3 × 50 ml), and then washed with 10% sodium carbonate (100 ml). The sodium carbonate layer was acidified with concentrated hydrochloric acid to pH ~ 3. The white precipitate was separated by filtration, washed with water and dried at room temperature under a vacuum to give a pure product [92% yield (2.5 g)]. 1H-NMR  δ 7.31 , 7.08 , 6.96 , 6.93 , and 1.58  ppm.Acid preparation. Preparation of 2-methyl-2-(4-phenoxyphenoxy)propanoic acid (AA). A dichloromethane (10 ml) suspension of fenofibric acid  and two drops of DMF was stirred at room temperature for 3 hours. The resulting clear dichloromethane solution was evaporated at 30 °C under reduced pressure. The remaining white solid residue was dissolved in tetrahydrofuran (20 ml) and mixed with aqueous ammonia  and stirred at room temperature for one hour. The resulting mixture was mixed with dichloromethane (50 ml) and water (50 ml). Organic solvent was separated, washed with water (3 × 50 ml), 5% sodium carbonate (50 ml), and dried over anhydrous sodium carbonate. The solvent was evaporated to give white solid product that was recrystallized from dichloromethane-cyclohexane to give pure product [93% yield (295 mg)]. 1H-NMR  δ 7.69 , 7.58 , 7.57 , 7.32 , 6.97 , and 1.50  ppm. 13C-NMR  δ 193.7, 175.4, 159.8,137.5, 136.7, 132.1, 131.6, 130.0, 129.0, 118.5, 80.9, and 25.4 ppm.(nitrogen unsubstituted amides). Preparation of 2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanamide (N-methyl amides). Preparation of 2-(4-(4-chlorobenzoyl)phenoxy)-N,2-dimethylpropanamide (MA). An aqueous solution (20 ml) of methylamine hydrochloride  and sodium carbonate  were mixed with tetrahydrofuran (20 ml) solution of acid chloride prepared, as explained above for fenofibric acid , and stirred at room temperature for two hours. Dichloromethane was added (50 ml) and the organic layer washed with water (3 × 50 ml), 5% sodium carbonate (50 ml) and dried over anhydrous sodium carbonate. After evaporation, the resulting oily residue was mixed with cyclohexane (5 ml) and left at room temperature overnight. The white needle-like precipitates were separated by filtration and dried at 60 °C under vacuum to give pure product [90% yield (300 mg)]. 1H-NMR  δ 8.09 , 7.67 , 7.66 , 7.56 , 6.93 , 2.58 , and 1.47  ppm. 13C-NMR  δ 194.0, 174.0, 159.6, 137.6, 136.5, 132.2, 131.6, 130.2, 129.1, 118.9, 81.2, 30.7, and 25.4 ppm.. Preparation 2-(4-(4-chlorobenzoyl)phenoxy)-N,N,2-trimethylpropanamide (DMA). A mixture of 40% dimethylamine in water  and the acid chloride of fenofibric acid  was stirred at room temperature for two hours. The reaction mixture was worked up as described above (Method E). Product was purified by crystallization from cyclohexane. The isolated yield was 89% (310 mg). 1H-NMR  δ 7.71 , 7.69 , 6.89 , 3.02 , 2.82 , and 1.59 ppm. 13C-NMR  δ 193.9, 171.5, 159.6, 137.5, 136.7, 132.6, 131.6, 130.1, 129.1, 116.8, 81.6, 37.3, and 25.9 ppm.-2-methylpropanamide (HR5). A dry dichloromethane (20 ml) solution of acid 1k , oxalyl chloride  and two drops of DMF was stirred at room temperature for 4 hours. The solvent was evaporated, and residue was dissolved in dichloromethane and mixed with mixture of tetrahydrofuran (10 ml) in diethanolamine  and water (10 ml) with sodium carbonate . The resulting mixture was stirred at room temperature for 2 hours and the solvent was evaporated under reduced pressure. The solid residue was mixed with dichloromethane (100 ml) and water (100 ml). The water layer was discarded, and the organic layer was washed with 5% hydrochloric acid (3 × 50 ml), 5% sodium carbonate (3 × 50 ml), water (3 × 50 ml) and dried over anhydrous sodium carbonate. The solvent was evaporated under reduced pressure to give pure product yielding 84% (330 mg). 1H-NMR  δ 6.92 , 6.82 , 3.92 , 3.66 , 3.60 , and 1.65 (6H) ppm.. Preparation of 2-(4-(4-chlorophenoxy)phenoxy)-HR32). A tetrahydrofuran (20 ml) mixture of FFA , oxalyl chloride , and two drops of DMF was stirred at room temperature for 2 hours. The solvent was evaporated, and solid residue was mixed with dry tetrahydrofuran (20 ml) and cooled down to 5 °C. This solution was mixed at 5 °C with ice cold water solution (20 ml) of sodium carbonate  and N,N-diethylethylenediamine . Resulting mixture was stirred at room temperature for two hours and evaporated to an oily residue. This residue was mixed with dichloromethane (100 ml) and 5% sodium carbonate (50 ml). Water layer was discarded, and organic layer was extensively washed with water (10 × 50 ml) and dried over anhydrous sodium carbonate. After solvent evaporation, the product was further purified by silica gel filtration with 5% triethylamine in ethyl acetate as the solvent. The isolated yield was 75% (317 mg). 1H-NMR  δ 7.73 , 7.69 , 7.45 , 7.730 , 6.95 , 3.31 , 2.49 , 2.41 , 1.61 , and 0.87  ppm. 13C-NMR  δ 194.1, 173.8, 158.9, 138.5, 136.2, 131.8, 131.1, 128.5, 119.4, 119.0, 81.7, 51.3, 46.6, 36.8, 25.1, and 11.6 ppm.(basic nitrogen substituted aryloxyacetamides). Preparation of 2-(4-(4-chlorobenzoyl)phenoxy)-N-(2-(diethylamino)ethyl)-2-methylpropanamide (HR1). An ethanol (50 ml) suspension of amide HR40  and 3% Pd/C (150 mg) was stirred at room temperature overnight under atmospheric hydrogen pressure. The catalyst was removed by filtration and the solvent was evaporated under reduced pressure to give a pure product with isolated yield of 96% (320 mg) isolated yield. 1H-NMR  δ 6.99 , 6.74 , 3.54 , 3.24 , 3.21 , 2.39 , 1,64 , 1.62 , 1.45 , 1.17 , and 0.98  ppm. Preparation of 2-(4-(cyclohexylmethyl)phenoxy)-N-(2-hydroxyethyl)-N,2-dimethylpropanamide (N-(2-hydroxyethyl)-N,2-dimethylpropanamide (HR2). A dichloromethane (30 ml) suspension of HR40  and fine grinded sodium borohydride  was kept at −5 °C for one hour. While stirring this suspension at −5 °C, trifluoracetic acid was added dropwise (15 ml), over a period of half an hour. The resulting suspension was then stirred at 0 °C for one hour and at room temperature for an additional two hours. The suspension was filtered, solid was discarded and dichloromethane filtrate was washed with 5% sodium carbonate (3 × 30 ml), dried over anhydrous sodium carbonate and evaporated to oily residue to give crude product. The product was purified by silica gel column chromatography with ethyl acetate – dichloromethane (7:3). The isolated yield was 72%. 1H-NMR  δ 7.23 , 7.08 , 7.01 , 6.76 , 3.85 , 3.78 , 3.53 , 3.21 , 2.73 , and 1.62  pppm.(sodium borohydride – trifluoracetic acid reduction of benzolphenoxyacetamide carbonyl group). Preparation of 2-(4-(4-chlorobenzyl)phenoxy)-HR35). A mixture of concentrated hydrochloric (3 ml) and HR34  was sonicated at room temperature for five minutes. The clear water solution was left under stream of nitrogen in hood for several hours to result in a white powder product. The white powder was dissolved in dichloromethane (10 ml) and dried over 4 Å molecular sieve overnight. The solvent was evaporated, and white residue was dried in vacuum to give 208 mg (95% yield). 1H-NMR (CDCl3) δ 13.20 , 7.75 , 7.71 , 7.56 , 6.90 , 4.78 , 3.93 , 3.52 , 3.45 , 3.27 , 2.63 , 2.53 , 1.98  and 1.71  ppm.. Preparation of 4‐{2‐[4‐(4‐chlorobenzoyl)phenoxy]‐2‐methylpropanoyl}6-8,17-22,27,46,50,52,53,55,63,67‐1‐methylpiperazin‐1‐ium chloride . An acetone (20 ml) solution of HR34  and methyl Iodide  was left in the dark at room temperature for three days (~70 hours). During this procedure, a white solid precipitate was formed. This solid product was separated by filtration, washed with acetone (3 × 3 ml) and dried at 110 °C for two hours to give pure product in 96% yield (130 mg). 1H-NMR (DMSO-d6) δ 7.73 , 7.71 , 7.60 , 6.93 . 4.06 , 3.86 , 3.30 , 3.25 , 3.12  and 1.60  ppm. 13C-NMR (DMSO-d6) δ 193.7, 170.7, 159.1, 137.6, 136.5, 132.7,131.7, 130.6, 129.1, 117.7, 81.8, 60.7, 51.2, and 26.2 ppm.phenoxy]-2-methylpropanoyl}Supplementary Dataset 1


These observational results demonstrated that the Pt NWs/Ti0.7W0.3O2 electrocatalyst is an intriguing anodic electrocatalyst, which can alter the state-of-the-art Pt NPs/C catalyst. Compared with the conventional Pt NPs/C electrocatalyst, the Pt NWs/Ti0.7W0.3O2 electrocatalyst exhibited the lower onset potential (~0.1 V for MOR and ~0.2 for EOR), higher mass activity (~355.29 mA/mgPt for MOR and ~325.01 mA/mgPt for EOR) and much greater durability. The outperformance of the Pt NWs/Ti0.7W0.3O2 electrocatalyst is ascribable to the merits of the anisotropic one-dimensional Pt nanostructure and the mesoporous Ti0.7W0.3O2 support along with the synergistic effects between the Ti0.7W0.3O2 support and the Pt nanocatalyst. Furthermore, this approach may provide a promising catalytic platform for fuel cell technology and a variety of applications.Finding out robust active and sustainable catalyst towards alcohol electro-oxidation reaction is major challenges for large-scale commercialization of direct alcohol fuel cells. Herein, a robust Pt nanowires (NWs)/Ti Compared to hydrogen-oxidation fuel cells, direct alcohol fuel cells (DAFCs) exhibited many advantages; namely, high conversion efficiency, high power density, and readily storage as well as facile transportation1. Up to now, carbon-supported Pt catalysts have widely utilized for both anode and cathode2, however, electrochemical corrosion of the carbon support3 causes the dissolution/detachment, Ostwald ripening, aggregation5 of the Pt nanocatalysts and thus the fuel cell performance is drastically deteriorated in long-term operation. In view of the above issues, numerous efforts have been devoted to designing non-carbon catalyst supports, which possess high corrosion resistance and strong interplay with the Pt nanocatalyst. Recently, M-doped TiO2 materials have emerged as a robust catalyst support in fuel cell applications owing to the synergistic effects with platinum catalyst leading to the improvement of both electrocatalytic stability and activity of the Pt-based electrocatalysts10.Low-temperature fuel cell systems have been attracted more and more attention as a promising green power technology to overcome environmental and energy issues in the 2111, however, the zero-dimensional possess some restrictions; namely, a high number of low coordination atoms and surface defects12 that directly affect their electrocatalytic activity and durability. To date, tuning the morphology and structure of the Pt catalyst has been proven to be a forward-looking approach to enhance both the activity and durability of the electrocatalyst14. Compared with the zero-dimensional structures, the one-dimensional structure like nanowires possessed many advantages; namely, the high surface-area-to-volume ratio, low number of surface defects, smooth single-crystalline and ability to prevent the particle agglomeration and coalescence of the Pt nanocatalysts14, resulting in good sensitivity and activity of the Pt-based electrocatalyst.At this juncture, the Pt nanoparticles  structures were commonly utilized in fuel cell systems0.7W0.3O2 catalyst toward methanol electro-oxidation and ethanol electro-oxidation which was successfully fabricated via the simple chemical reduction route at room temperature, only using formic acid (HCOOH) as reducing agents. These observational results indicated that the Pt NWs/Ti0.7W0.3O2 is promising anodic catalysts for methanol electro-oxidation reaction (MOR) and ethanol electro-oxidation reaction (EOR), which can alter the conventional Pt NPs/C electrocatalysts. For instance, the robust Pt NWs/Ti0.7W0.3O2 electrocatalyst exhibited the lower onset potential (~0.1 V vs. NHE for MOR and ~0.2 V vs. NHE for EOR), higher mass activity (~355.29 mA/mgPt for MOR and ~325.01 mA/mgPt for EOR) and higher If/Ib ratio (~2.70 for MOR and ~1.35 for EOR) as well as much higher electrochemical stability relative to the Pt NPs/C catalyst. The high mass activity and superior stability of the robust Pt NWs/Ti0.7W0.3O2 electrocatalyst could be derived from combining the merits of the one-dimensional Pt nanostructures and the mesoporous Ti0.7W0.3O2 catalyst support, as well as the synergistic effect between the mesoporous Ti0.7W0.3O2 catalyst support to the Pt nanoforms. Finally, this research can provide robust catalysts platforms for fuel cell technologies and other applications such as solar cells, water splitting.To be the best of our knowledge, there are a limited number of researches on the coherency of the non-carbon catalyst support and one-dimensional Pt nanowires to develop a robust electrocatalyst towards alcohol electro-oxidation reaction. In this work, we demonstrated the Pt NWs/Ti0.7W0.3O2 support was prepared via the one-pot solvothermal route without employing surfactant/stabilizer or further heat treatment8  as reducing agents  measurement. As can be seen in Fig. 2) was detected in the XRD pattern  was implemented to investigate the morphology of the Pt nanoforms on the mesoporous Ti0.7W0.3O2 support. Figure 0.7W0.3O2 supports. Besides, TEM images  exhibited the catalyst morphology to be the rhombus and sphere that could be explained due to the agglomeration of support materials and one-dimensional (1D) Pt nanocatalyst which maybe covers overall the surface of Ti0.7W0.3O2 support due to the high Pt loading (50 wt%) on the support. Moreover, HR-TEM image  ions resulting in the formation of clustered particles. The very low reduction rate of the formic acid (HCOOH) at room temperature, which environmental favors for the anisotropic development of platinum nuclei along the (111) direction16. From this mechanism, Pt nuclei are deposited on the surface of Ti0.7W0.3O2 support during the reduction of H2PtCl6 by HCOOH to form Pt nuclei that act as sites for further nucleation via the continual absorption and reduction of Pt (IV) ions resulting in the formation of clustered particles and form the Pt NWs on the surface of Ti0.7W0.3O2 supports under the very low reduction rate and long-time reaction (72 hours) at room temperature. These outcomes suggest that the simple chemical reduction route using formic acid is a suitable approach to design the Pt NWs/Ti0.7W0.3O2 electrocatalyst.The mesoporous Tints Fig. . The forern Fig. , suggestsee Fig.  exhibite0.7W0.3O2, Pt NWs/C and Pt NPs/C electrocatalysts, the X-ray photoelectron spectroscopy (XPS) measurement was performed. These XPS results  of the Pt NWs/Ti0.7W0.3O2, Pt NWs/C, and conventional Pt NPs/C electrocatalysts, calculated from the charge of hydrogen adsorption, are around 63.48 m2/g; 56.73 m2/g, respectively, which is approximate half that of the conventional Pt/C electrocatalyst (~130.32 m2/g)  in N2-purged 0.5 M H2SO4 at a scan rate of 50 mV/s was also employed to investigate the electrochemical stability of as-prepared electrocatalysts. After the 5000 cycling test, the ECSA loss of the Pt NWs/Ti0.7W0.3O2 catalysts was estimated to be 11.89% of initial ECSA value, meanwhile, the ECSA value of Pt NWs/C and conventional Pt NPs/C was significantly degraded to be ~19.33% and ~27.45% of initial ECSA value, respectively  Fig. . The lowely Fig. . The enh2-purged 10 v/v % CH3OH/0.5 M H2SO4 at a scan rate of 50 mV/s to evaluate the electrocatalytic activity towards methanol electro-oxidation reaction (MOR) of the Pt NWs/Ti0.7W0.3O2 catalyst. Figure 0.7W0.3O2, the Pt NWs/C and the conventional Pt NPs/C electrocatalysts. Compared with other as-obtained electrocatalysts, the Pt NWs/Ti0.7W0.3O2 catalyst exhibited the highest mass activity (355.29 mA/mgPt), which is ~1.23-fold and ~1.57-times higher than those of the Pt NWs/C (288.79 mA/mgPt) and conventional Pt NPs/C (226.40 mA/mgPt) catalysts, respectively, albeit it’s the low electrochemical surface area (ECSA) value. The great electrocatalytic activity of the Pt NWs/Ti0.7W0.3O2 catalysts is ascribable to the formed Pt nanoforms along the (111) orientation, which possessed the most activity towards methanol electro-oxidation owing to the low poisoning rate24. Furthermore, the potential at which the methanol oxidation starts  of the Pt NWs/Ti0.7W0.3O2 catalyst was found to be the lowest (~0.1 V vs. NHE), which is negatively shifted about 200 mV and 350 mV with respect to the Pt NWs/C (~0.3 V vs. NHE) and the conventional Pt NPs/C (~0.45 V vs. NHE)  and a negative-going current density (Ib) is generally represented the resistance to the poisoning of the accumulation of carbonaceous species25. Interestingly, the Pt NWs/Ti0.7W0.3O2 catalyst exhibited the highest If/Ib values in comparison with the Pt NWs/C and the conventional Pt NPs/C electrocatalyst. The If/Ib ratio of three different catalysts are shown in order: Pt NWs/Ti0.7W0.3O2 (~2.70) > Pt NWs/C (~1.03) > Pt NPs/C (0.97), suggesting that the Pt NWs/Ti0.7W0.3O2 electrocatalysts possessed the best CO-tolerance in comparison with the Pt NWs/C and the conventional Pt NPs/C catalysts towards MOR. The high If/Ib value of the Pt NWs/Ti0.7W0.3O2 electrocatalyst compared to that of the Pt NWs/C electrocatalyst could be explained due to the strong interaction between Pt nanocatalyst and Ti0.7W0.3O2 support, resulting in making weak adsorption of CO-like species on the sites of Pt nanocatalyst26  structure of Pt nanocatalyst such as (i) long segments of smooth crystal planes, (ii) a low number of surface defects, leading to good sensitivity and and activity for methanol electro-oxidation reaction18 and (iii) unique one-dimensional (1D) Pt morphology, resulting in improving mass transport and electron transfers during electrocatalytic reactions28.The cyclic voltammetry measurement was carried out in Nsee Fig. , implyin0.7W0.3O2 catalyst towards MOR, the 5000 potential cycling measurement was conducted in N2-purged 10 v/v % CH3OH/0.5 M H2SO4 solution at a scan rate of 50 mV/s. Figure 0.7W0.3O2 electrocatalysts exhibited superior stability in comparison with the Pt NWs/C, and the conventional Pt NPs/C catalysts. Particularly, the Pt NWs/Ti0.7W0.3O2 catalyst demonstrated the deterioration of the mass activity to be around 12.05% of the initial mass activity, which was ~2.18-times and ~3.06-fold lower than those of the Pt NWs/C (~26.36%), and the conventional Pt NPs/C electrocatalysts (~36.83%), respectively , and the traditional Pt NPs/C (~137.98 mA/mgPt), respectively  > Pt NWs/C (~0.87) > Pt NPs/C (~0.83), suggesting that the Pt NWs/Ti0.7W0.3O2 possessed the high resistance to poisoning of the accumulation of carbonaceous species  making the downshift d-band center of the Pt nanocatalyst and lower the adsorption energy of COads, facilitating the oxidation of COads at a lower potentials24, resulting in the significant enhancement of both the electrocatalytic activity and stability of the Pt NWs/Ti0.7W0.3O2 catalyst.Until now, ethanol has emerged as green fuel sources, which can alter for methanol because of lower toxicity and market cost, however, one of the most major challenges is the development of the electrocatalyst with the great electrocatalytic stability and activity towards ethanol electro-oxidation reaction (EOR)see Fig. , implyinely Fig. , suggest0.7W0.3O2 towards EOR was further investigated via the accelerated durability test (ADT) in N2-purged 10 v/v % C2H5OH/0.5 M H2SO4 aqueous solution. These outcomes indicated that the Pt NWs/Ti0.7W0.3O2 catalyst possessed the superior stability towards ethanol electro-oxidation in comparison with the Pt NWs/C and the conventional Pt NPs/C electrocatalysts. For instance, after 5000 cycling test, the mass activity of the Pt NWs/Ti0.7W0.3O2 electrocatalyst was found to be around 283.89 mA/mgPt with respect to the deterioration to be ~12.65% of the initial mass activity (~325.01 mA/mgPt), meanwhile, the Pt NWs/C and conventional Pt NPs/C electrocatalysts showed the decay to be around ~30.09% (from 169.73 mA/mgPt dropped to 118.66 mA/mgPt) and ~45.36% (from 137.98 mA/mgPt to 75.41 mA/mgPt), respectively  and the conventional Pt NPs/C (~175.21 mA/mgPt). After 7200 s test, the mass activity of the Pt NWs/Ti0.7W0.3O2 electrocatalyst was remained to be around 116.80 mA/mgPt, which is ~1.54-fold and ~8.44-fold higher than those of the Pt NWs/C (75.90 mA/mgPt) and the Pt NPs/C (13.84 mA/mgPt) at the same time, respectively. The decay rate of the as-prepared catalysts is showed in order: Pt NWs/Ti0.7W0.3O2 (~0.59 mA/mgPt.min) < Pt NWs/C (~0.86 mA/cm2.min) < Pt NPs/C (~1.34 mA/mgPt.min). The superior durability of the Pt NWs/Ti0.7W0.3O2 electrocatalyst relative to the conventional Pt NPs/C could be interpreted due to the lower vulnerability to dissolution, Ostwald ripening and aggregation of the 1D Pt structure (nanowires) than 0D Pt structure (nanoparticles)14. In addition, the high corrosion resistance of TiO2-based oxide in acidic and oxidative environments14 as well as the electronic effect between the mesoporous Ti0.7W0.3O2 support and the Pt nanocatalysts, which results in the weak adsorption of carbonaceous intermediate species on the active sites of the Pt nanocatalysts and, consequently, improve the catalytic stability of the Pt NWs/Ti0.7W0.3O2 catalyst.The chronoamperometry measurement in N0.7W0.3O2 electrocatalyst, which was prepared via a simple chemical reduction route at room temperature without utilizing surfactant/stabilizers or template. The experimental outcomes demonstrated that the robust Pt NWs/Ti0.7W0.3O2 catalyst is a promising electrocatalyst towards the methanol electro-oxidation and ethanol electro-oxidation. For instance, the Pt NWs/Ti0.7W0.3O2 electrocatalyst exhibited the lower onset potential (~0.1 V vs. NHE for MOR and ~0.2 V vs. NHE for EOR), higher mass activity (~355.29 mA/mgPt for MOR and ~325.01 mA/mgPt for EOR), and the greater If/Ib ratio (~2.70 for MOR and ~1.35 for EOR) along with the superior stability in acidic and oxidative environment related to the conventional Pt/C catalyst. These enhancements of the Pt NWs/T0.7W0.3O2 catalyst are attributable to the merits of the one-dimensional Pt structure and the mesoporous Ti0.7W0.3O2 catalyst support, as well as the electron transfers from the Ti0.7W0.3O2 catalyst support to the Pt nanowires, which was evidenced via the XPS spectroscopy, leading to the weak linkage of intermediate carbonaceous species on the active surface of the Pt nanocatalyst. Furthermore, this approach may provide a robust catalytic platform for fuel cell technologies and a variety of applications.In brief, we introduce the robust Pt NWs/Ti0.7W0.3O2 catalyst consisted of using the chloroplatinic acid hydrate (H2PtCl6.xH2O) and the as-prepared Ti0.7W0.3O2 catalyst support as starting precursors as well as the formic acid (HCOOH) as reducing agent. Briefly, a mixture of 8 mL H2O and 0.62 mL 0.05 M H2PtCl6 aqueous solution and 1.2 mL HCOOH was stirred for 15 min to generate a homogenous solution. Next, 6 mg Ti0.7W0.3O2 supports were ultrasonically dissolved into the above solution for 15 min to create a homogeneous suspension. Afterward, the as-prepared suspension was stored at room temperature for 72 hours to fabricate the 50 wt % Pt NWs/Ti0.7W0.3O2 catalyst. Finally, the obtained product was rinsed copiously with purified water and then dried at 80 °C overnight for further analysis. For comparison, the 50 wt % Pt NWs were grown on Vulcan XC-72 support at the same condition.In this work, the simple chemical reduction route was utilized to produce the Pt NWs/Ti0.7W0.3O2 support was measured via the X-ray diffraction (XRD) measurement operated on a D2 PHASER-Brucker using Cu Kα radiation at 30 kV. The transmission electron microscopy (TEM) measurement was conducted on the JEOL-LEM 1400 microscope at an accelerating voltage of 3800 V to examine the morphology of the as-prepared Pt NWs/Ti0.7W0.3O2 catalyst. Furthermore, the X-ray photoelectron spectroscopy (XPS) was implemented to investigate the surface properties of the as-obtained Pt NWs/Ti0.7W0.3O2 electrocatalyst.The structure information of the formed Pt nanowires over the mesoporous Ti2-statured 10 v/v % CH3OH/0.5 M H2SO4 solution and N2-statured 10 v/v % C2H5OH/0.5 M H2SO4 solution, respectively. Furthermore, the ADT test was performed in the range of 0 V to 1.0 V (vs. NHE) for 5000 cycles at a scan rate of 50 mV/s at room temperature in N2-purged 10 v/v% CH3OH/0.5 M H2SO4 and in N2-purged 10 v/v% C2H5OH/0.5 M H2SO4 for methanol electro-oxidation reaction (MOR) and ethanol electro-oxidation reaction (EOR), respectively. All potential ranges in this work were reported with the normal hydrogen electrode (NHE) scale. The catalyst ink preparation: the catalyst powder was ultrasonicated in a solution comprising ethanol absolute and 0.5% Nafion within 30 min. Before the catalyst ink placement, the surface of the glassy carbon disk was polished with 0.5 µm BAS and then washed with ethanol as well as purified water. To start with, the catalyst electrode was activated by 100 cycles at a scan rate of 50 mV/s. In this work, the Pt loading onto the glassy carbon electrode was maintained at 0.13 mg/cm2 in all electrochemical tests.An EC-LAB Electrochemistry instrument (Bio-Logic SAS) with an Ag/AgCl/Sat. KCl electrode, and a Pt gauze, as well as glassy carbon electrode with 5 mm in diameter with respect to a reference electrode and the counter electrode as well as a working electrode, were used for investigating all electrochemical tests. The electrocatalytic activity towards methanol electro-oxidation reaction (MOR) and ethanol electro-oxidation reaction (EOR) of the as-obtained catalysts was recorded at a scan rate of 50 mV/s in NSupplementary informationSupplementary information


F. esculentum bee pollen oil by GC-MS analysis, and 16 compounds were isolated from F. esculentum bee pollen by column chromatography and identified. A high-performance liquid chromatography-diode array detector (HPLC-DAD) method was established for the quality control of F. esculentum bee pollen. A validated HPLC-DAD method was successfully applied to the simultaneous characterization and quantification of nine main constituents in seven samples collected from seven different areas in China. The results showed that all standard calibration curves exhibited good linearity (R2 > 0.999) in HPLC-DAD analysis with excellent precision, repeatability and stability. The total amount in the samples from the seven regions ranged from 23.50 to 46.05 mg/g. In addition, seven compounds were studied for their bioactivity using enzymic methods, whereby kaempferol (3) showed high α-glucosidase inhibitory activity (IC50: 80.35 μg/mL), ergosterol peroxide (8) showed high tyrosinase inhibitory activity (IC50: 202.37 μg/mL), and luteolin (1) had strong acetylcholinesterase inhibitory activity (IC50: 476.25 μg/mL). All results indicated that F. esculentum bee pollen could be a nutritious health food.Bee pollen contains all the essential amino acids needed by humans. China is the largest producer of bee pollen in the world. In the present study, we identified 11 fatty acids in  Bee pollen (BP), one of the hive products in addition to honey, royal jelly and propolis, is gaining attention due to the presence of bioactive compounds associated with beneficial health properties ,2. The Bα-Glucosidase (AG) is an important enzyme to maintain the normal metabolism of the human body. It hydrolyzes maltose and sucrose by catalyzing the hydrolysis of 1,4-glycosidic bonds in sugar, which is widely distributed in human body and plays a crucial role in human metabolism . AcetylcF. esculentum bee pollen.Melanin is one of the natural pigments responsible for skin pigmentation and hair color in mammals . The excF. esculentum pollens and quantitatively analyzed F. esculentum pollens collected from different areas in China. Nine compounds, including flavonoids, phenolic acids and polyphenols were analyzed by a validated HPLC-DAD method. We also carried out enzyme activity experiments to verify their α-glucosidase inhibitory activity, tyrosinase inhibitory activity and acetylcholinesterase inhibitory activity. The obtained quantitative data will be useful for quality control of F. esculentum pollens for their comprehensive use as a functional food.In the light of this, we studied the composition of Fagopyrum esculentum Moench from different production areas in China, were collected in 2016. These samples were identified by the corresponding author (NB). Their voucher specimens were deposited in a refrigerator at 4 °C at the Department of Pharmaceutical Engineering, Northwest University, China. BPSX was from Shannxi, BPAH was from Anhui, BPNM was from Neimeng, BPHN was from Heinan, BPGS was from Gansu, BPHB1 was from Hebei and BPHB2 was from Hubei.Seven batches of BPs, consisting of 2Cl2 and methanol used in this experiment were of analytical grade and purchased from Hengxing Chemical Reagent Co., Ltd. . The 4-nitrophenyl β-d-glucopyranoside (PNPG), acarbose, l-arginine, kojic acid, acetylthiocholine iodide (ACTI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and huperzine A used in the activity experiments were all from Shanghai Aladdin Biochemical Technology Co., Ltd. . We have isolated the standard substances 1–16 and their structures were fully characterized by chemical and spectroscopic methods (NMR and MS data are provided as 1), resveratrol (2), kaempferol (3), β-daucosterol (4), caffeic acid (5), chlorogenic acid (6), rutin (7), ergosterol peroxide (8), octacosanol (9), 2-β-d-glucopyranosyloxy-1-hydroxytrideca-3,5,7,9,11-pentayne (10), α-d-Alt-p-OMe (N2-6) α-d-Gle-p-OMe (11), O-α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside (12), catechin (13), quercitrin (14), oleanolic acid (15) and tyrosol (16). The purity of each compound was >98%, determined by HPLC analysis. The chemical structures of these reference compounds are shown in Silica gel (100–200 mesh), MCI GEL CHP-20P  and Sephadex LH-20  were used for column chromatographic separations. Thin layer chromatography (TLC) was performed on Sigma-Aldrich silica gel TLC plates . Acetonitrile was of HPLC grade from Merck . Water was purified with a sub boil high purity water still . Other reagents such as ethyl alcohol, CHHPLC analysis was performed on a 1260 LC Series system  using a Luna C-18 column  with a flow rate of 1.0 mL/min, and the column temperature was maintained at 20 °C. Injection volume were 10 µL for the standard solutions and 15 µL for sample solutions. The mobile phase was composed of C (methanol) and D (water) with a gradient elution: 0 min, 95% D; 0–6 min, 95–92% D; 6–11 min, 92–90% D; 11–17 min, 90–86% D; 17–25 min, 86–75% D; 25–30 min, 75–70% D; 30–40 min, 70–45% D; 40–47 min, 45–30% D; 47–55 min, 30–5% D; 55–70 min, 5–95% D. At the end of the run, 95% of acetonitrile was used to flush the column for 5 min and an additional 5 min of post run time were set to allow for equilibration of the column with the starting eluant. UV detector was set at 210 nm wavelength for the compounds during the experiment.F. esculentum pollen oils were obtained by a pressing method under low temperature, and processed by saponification and methylation reactions for further GC-MS analysis. Briefly, 0.5 g of F. esculentum pollens oil prepared above was dissolved in 4 mL methanol solution containing 0.5 mol/L of potassium hydroxide, stirred for 2 min, and kept in a 60 °C water bath for 1 h in a capped test tube to induce saponification for derivatization. Then, 3 mL of methanol solution containing 10% concentrated sulphuric acid was added, the sample was again placed in a 60 °C water bath for 1 h. After reactions, the sample was cooled to room temperature. Subsequently, 3 mL of deionized water and 6 mL of n-hexane were added for extraction. After centrifugation , the supernatant was obtained and dried using a nitrogen flow. The obtained solution was stored in a refrigerator at 4 °C and filtered through a 0.45 μm nylon-membrane filter prior to injection into the GC-MS analysis.The N Network System, M 5973N) instrument was coupled with a Rtx-5MS column (30 m × 0.25 mm × 0.25 μm (5% phenylmethylsiloxane)). Helium  was used as the carrier gas at a flow rate of 3.0 mL/min. The injection volume is 1 μL by split (1:30). Injector, source and oven temperatures were 250 °C, 200 °C and 120 °C, respectively. The initial oven temperature 120 °C held for 2.5 min and then ramped at 10 °C/min to 180 °C, ramped at 1.5 °C/min to 210 °C, ramped at 5 °C/min to 250 °C and last for 3 min.The FA profiles in the pollen oils were analyzed by GC-MS as their corresponding methyl ester. The oil sample was analyzed three times under the same conditions. The GC-MS (Agilent 65902O (6.5 L) and then extracted exhaustively with PE, EtOAc and n-BuOH (6.5 L each) 3 times. The n-BuOH solution containing 195.3 g of solid was chromatographed on a silica gel column  with CH2Cl2-MeOH (100:1–0:1) gradient to give seven crude fractions (Fr.1–7).The BPs (15 kg from Shanxi) was percolated with 90% EtOH at room temperature for three times, then the EtOH extracts were combined. The extract (6.32 kg) was suspended in H2Cl2-EtOAc, then they were repeatedly subjected to MCI GEL CHP-20P column with water-MeOH system (start from H2O and increased MeOH ratio to 10–100% MeOH) to give compound 7 , 16 , 3 , 5  and 6 . Fr. 5 and Fr. 6 were repeatedly subjected to MCI GEL CHP-20P column and/or Sephadex LH-20 column with water-MeOH system (start from H2O and increased MeOH ratio to 10–100% MeOH) to give compound 13 , 14 , 1 , 2  and 9 . The separation process of other compounds is also as described above. Compound 4 , 8 , 9 , 10 , 11 , 12  and 15 . Their structures are shown in Fr.2, Fr.3 and Fr.4 were repeatedly subjected to silica gel column with CHEach BP material was ground to powder and sifted through an 80 mesh sieve. Subsequently, 5 g of pulverized samples were accurately weighed, and extracted with ultrasonication using 40 mL of 90% methanol (volume fraction) for 1.5 h. After centrifugation , the supernatant was concentrated and transferred into a 5 mL volumetric flask. The obtained solution was stored in a refrigerator at 4 °C and filtered through a 0.45 μm filter prior to HPLC analysis.Nine reference standards were accurately weighed  respectively, then dissolved in methanol and a small amount of dimethyl sulfoxide to final concentrations of 1.0 mg/mL. Every standard stock solution was diluted with methanol to five appropriate concentrations . All the standard solutions were stored at 4 °C in darkness for HPLC analysis.Identification of these analytes were carried out by comparing the HPLC retention time and UV spectra of target peaks with those of the standards. Quantification was performed on the basis of linear calibration plots of the peak areas versus the concentration. The results are presented in n = 3); Group B was added 40 μL of 0.1 mol/L PBS and 20 μL of the sample as the control group (n = 3); Group C was added 40 μL 0.1 mol/L PBS and 20 μL α-glucosidase as blank group (n = 3); after incubation for 15 min at 37 °C, then 2.5 mmol/L PNPG glycoside was added to each well and incubated at 37 °C. The reaction was stopped by adding 80 μL of a 0.2 mol/L Na2CO3 solution to each well after 15 min. The absorbance at a wavelength of 405 nm was measured with a microplate reader. Acarbose was used as a positive control. The specific grouping is shown in In a 96-well plate, group A was added 20 μL of 0.1 mol/L PBS ), 20 μL of the sample (standard solution dissolved in DMSO to the concentration of 1.0 mg/mL) and 20 μL of α-glucosidase as a sample group (n = 2); group B was added 130 μL of 0.1 mol/L PBS and 50 μL of solvent as blank background group (n = 2); group C was added with 80 μL of 0.1 mol/L PBS, 50 μL of the sample (standard solution dissolved in DMSO to the concentration of 1.0 mg/mL) and 50 μL of tyrosinase as the experimental group (n = 2); group D were added 130 μL of 0.1 mol/L PBS and 50 μL of the sample as experimental background group (n = 2); finally, 20 μL of substrate (l-tyrosine) was added to each well to trigger the reaction, and the 96-well plate was further incubated at 37 °C. After 30 min, the absorbance at a wavelength of 405 nm was measured by a microplate reader. Kojic acid was used as a positive control. The specific grouping is shown in In a 96-well plate, group A was added 80 μL of 0.1 mol/L PBS, 50 μL of solvent (solvent used to dissolve the sample: DMSO) and 50 μL of tyrosinase as a blank group (n = 3); Group B was added with 160 μL of 0.1 mol/L PBS and 20 μL of the sample as a control group (n = 3); Group C was added with 160 μL 0.1 mol/L PBS and 20 μL acetylcholinesterase as a blank group (n = 3), the all were incubated at 4 °C for 20 min, then 10 μL of 15 mM ACTI and 10 μL of 2 mM DTNB were added per well, continue incubated at 37 °C for 20 min, the absorbance at a wavelength of 412 nm was measured with a microplate reader. Huperzine A was used as a positive control. The specific grouping is shown in In a 96-well plate, group A was added 140 μL of 0.1 mol/L PBS, 20 μL of the sample (standard solution dissolved in DMSO to the concentration of 1.0 mg/mL) and 20 μL of acetylcholinesterase as a sample group . The components in F. esculentum pollen oil were listed according to their elution order on the non-polar Rtx-5MS column as shown in F. esculentum pollens oil sample led to identification of 11 constituents, which correspond to 85.20% of the total contents. In the present work, -octadecatrienoic acid α-linolenic acid, ALA)  was found to be dominant at the high level of 36.25%, which is higher than previously reported in the literature (35.58%) [Z,12Z)-octadecadienoic acid   (see cis-13-eicosenoic acid (C20:1Δ13), heptadecanoic acid (C17:0), eicosanoic acid (C20:0), octadecanoic acid (C18:0), tetradecanoic acid (C14:0) and docosanoic acid (C22:0) were also found. The methylated (35.58%) , followe.8%) see . ALA andTo determine the optimal extraction conditions and enhance the overall response of those investigated compounds in the HPLC analysis, extraction methods (refluxing and sonication), extraction solvents , number of extractions , and extraction times  were investigated individually by using a univariate approach  on the sample BPSX. The results suggested that the established extracted method (each sample was extracted three times with 50% methanol under ultrasonication condition for 90 min) was optimal for HPLC analysis. The results are shown in The HPLC method was validated by determining the linearity, limit of detection (LOD), limit of quantitation (LOQ), precision (inter-day and intra-day), repeatability, stability, and accuracy. Y) versus the corresponding concentration  of each compound. All calibration curves exhibited good linear regressions (R2 ≥ 0.999) within the tested concentration ranges (N) for each compound was approximately 3 for LOD and 10 for LOQ. The LODs and LOQs of all analytes were less than 0.23 and 0.64 µg/mL, respectively. Linear calibration curves were established by plotting the peak area  of peak area for each marker compound was taken as a measure . The RSDTo check the accuracy of the analytical method, a recovery test was performed. In the test, a known amount of standards was added into 0.1 g of sample BPSX previously quantified. The spiked samples were extracted, processed and quantified per the methods above. Three replicates were performed for the test. The recovery values ranged from 97.25 to 102.07%, and RSDs values were less than 3.49%. These results indicate that the established method is accurate enough for quantitative analysis.1 (luteolin) and analyte 2 (resveratrol) were detected in seven samples, the contents of analyte 1 varied from 6.23 to 10.94 mg/g and analyte 2 varied from 3.26 to 5.25 mg/g. In addition, compound 6 could not be detected in many analyzed samples, only be detected in BPSX and BPNM, and the contents were 1.45 and 1.12 mg/g, respectively.The proposed analytical method was then successfully applied to the simultaneous quantification of seven BPSs collected from different areas in China. The results  indicate6 (chlorogenic acid) varied from 1.12 to 1.45 mg/g, and analyte 5 (caffeic acid) varied from 3.47 to 6.00 mg/g. Look at all the data, the highest contents of analyte 1 is 10.94 mg/g in sample BPNM, the lowest contents of analyte 7 (rutin) is 1.05 mg/g in sample BPHB1. Chlorogenic acid and caffeic acid are common bioactive compounds ,35. ChloGlucosidase is an important member of the sugar metabolism pathway in vivo, and α-glucosidase is directly involved in the metabolic pathways of starch and glycogen . By inhi3 (kaempferol), 7 (rutin), 8 (ergosterol peroxide), 10 , 11 (α-d-Alt-p-OMe (N2-6) α-d-Gle-p-OMe) and 12 (O-α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→2)-β-d-fructofuranoside) had significant α-glucosidase inhibitory activity compared with the positive control group, the IC50 value of the acarbose as the positive control group, which was 515.98 μg/mL, and the IC50 value of the compound 3 was the lowest, which was 80.35 μg/mL, and the IC50 value of the compound 7 was 188.42 μg/mL, indicating that F. esculentum bee pollen has hypoglycemic effects. Notably, compounds 8, 10, 11 and 12 were tested for α-glucosidase inhibitory activity for the first time, and the difference in their IC50 value was not significant. The inhibition mechanism of kaempferol on α-glucosidase activity has been investigated by multispectroscopic methods and kaempferol had a significant inhibitory activity on α-glucosidase with an IC50 value of (1.16 ± 0.04) × 10−5 mol L−1 and Ki value of (1.31 ± 0.03) × 10−5 mol L−1 [From  mol L−1 .1 (luteolin), 8 (ergosterol peroxide), 11 (α-d-Alt-p-OMe (N2-6) α-d-Glep-OMe), and 12 (O-α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside) had significant tyrosinase inhibitory activity compared with the positive control group, the IC50 value of the kojic acid as the positive control group, which was 517.07 μg/mL, indicating that F. esculentum bee pollen has hypoglycemic effect. The IC50 value of the compound 8 was the lowest, which was 202.37 μg/mL, followed by the compound 8, which was 302.42 μg/mL, compared with the positive control group, their IC50 value were low. The phenolic extracts from rape bee pollen have been shown to have anti-tyrosinase activity, which could reduce the content of melanin and inhibit the production of melanin in B16 mouse melanoma cells through the cAMP/MITF/TYR pathway [Quercus mongolica revealed that the polyamine derivatives with coumaroyl and caffeoyl moieties showed tyrosinase inhibition with IC50 values of 19.5–85.8 μM [From  pathway , and the–85.8 μM .1 (luteolin), 3 (kaempferol), 7 (rutin), 8 (ergosterol peroxide), 10 , 11 (α-d-Alt-p-OMe (N2-6) α-d-Gle-p-OMe), and 12 (O-α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside) has strong acetylcholinesterase inhibitory activity, the difference between the positive group was not significant. IC50 value of the huperzine A as the positive control group, which was 502.98 μg/mL, compound 10 was the highest, with IC50 values of 521.14 μg/mL, compound 1 was the lowest, with IC50 values of 476.25 μg/mL. At present, there are few studies on the inhibitory activity of acetylcholinesterase in bee pollen. Acetylcholinesterase is involved in the development and maturation of cells and can promote the development of neurons and nerve regeneration [From neration .F. esculentum bee pollen, including flavone compounds, phenolic acids, terpenoids, and 11 fatty acids were analyzed in F. esculentum bee pollen oil. Saturated fatty acids were 30.86%, unsaturated fatty acids were 69.14%, of which -octadecatrienoic acid content was as high as 36.25%, indicating that the unsaturated fatty acid content of F. esculentum bee pollen is high, and it is a green and healthy food. A HPLC method with high stability, good repeatability and high precision was established, and the content of nine compounds in F. esculentum bee pollen from seven different habitats was quantitatively analyzed. From the results, the content of bee pollen collected in Neimeng was the highest (46.05 mg/g), and the content of bee pollen collected in Gansu was the lowest (23.50 mg/g). Kaempferol (3) showed high α-glucosidase inhibitory activity (IC50: 80.35 μg/mL), ergosterol peroxide (8) showed high tyrosinase inhibitory activity (IC50: 202.37 μg/mL), and luteolin (1) had strongly acetylcholinesterase inhibitory activity (IC50: 476.25 μg/mL). Furthermore, bee pollen has been used as a health food supplement for many years due to its abundant nutrient properties including proteins, polysaccharide, and lipids [F. esculentum bee pollen is a kind of natural nutrition and health food for anti-aging, beauty and improving human immunity. In this study, 16 compounds were isolated from d lipids . The aved lipids . Polysacd lipids . Lipids d lipids . Other t highest .05 mg/g,d lipids ,49. Our At present, most of reports on bee pollen are aimed at the study of extracts. Therefore, the innovation of this paper is the separation and identification of extracts. The quantitative analyses of bee pollen from seven different places of origin were also carried out.