Background: In China, the incidence of ulcerative colitis (UC) is increasing every year, but the etiology of UC remains unclear. UC is known to increase the risk of colorectal cancer (CRC). The aim of this study was to investigate the protective effects of crocin against UC and CRC in mouse models.Methods: Crocin was used to treat the dextran sodium sulfate (DSS)-induced UC mice for 3 weeks, and ApcMinC/Gpt mice with colorectal cancer for 8 weeks. Proteomics screening was used to detect changes in the protein profiles of colon tissues of UC mice. Enzyme-linked immunosorbent assays and western blot were used to verify these changes.Results: Crocin strongly reduced the disease activity index scores of UC mice, and improved the pathological symptoms of the colonic epithelium. The anti-inflammatory effects of crocin were indicated by its regulation of the activity of various cytokines, such as interleukins, via the modulation of nuclear factor kappa-B (NF-κB) signaling. Crocin significantly suppressed tumor growth in ApcMinC/Gpt mice and ameliorated pathological alterations in the colon and liver, but had no effects on spleen and kidney. Additionally, crocin significantly decreased the concentrations of interleukins and tumor necrosis factor-α in the sera and colon tissues, suggesting its anti-inflammatory effects related to NF-κB signaling. Finally, 12-h incubation of SW480 cells with crocin caused cell cycle arrest, enhanced the apoptotic rate, promoted the dissipation of mitochondrial membrane potential, and the over-accumulation of reactive oxygen species. From the theoretical analyses, phosphorylated residues on S536 may enhance the protein-protein interactions which may influence the conformational changes in the secondary structure of NF-κB.Conclusion: The protective effects of crocin on UC and CRC were due to its suppression of NF-κB-mediated inflammation. Ulcerative colitis (UC) is a chronic and relapsing inflammatory intestinal disease, and typical UC symptoms are bloody diarrhea, mucus discharge, acute pain and weight loss . NotablyIn China, the incidence of UC is increasing every year, statistical data show that the prevalence rates of Heilongjiang, Xian, Guangzhou, Zhongshan, Hong Kong and Taiwan are 1.77/100,000, 0.42/100,000, 3.44/100,000, 3.14/100,000, 3.06/100,000 and 4.59/100,000 . With moAt present, surgery, radiotherapy and chemotherapy are the common therapeutic strategies for CRC . In addiAmong the therapeutic agents approved by the Food and Drug Administration (FDA), 40% are natural ingredients or their derivatives . Due to 44H64O24), the main biologically active compound of saffron, has been reported to show various pharmacological activities -induced UC and suppress the expression of various cytokines such as tumor necrosis factor-α (TNF-α) and NF-κB . Crocin MinC/Gpt mice, where the latter were obtained by point-mutating the adenomatous polyposis coil (APC) gene. Our experiments confirmed that the anti-inflammatory effects of crocin are related to its regulation of NF-κB signaling.In this study, the anti-UC and anti-CRC effects of crocin were successfully confirmed by experiments in a human colon adenocarcinoma cell line, SW480, and in DSS-induced UC mice and Apc2 incubator.SW480 cells, a human colon adenocarcinoma cell line , were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 1% 100 μg/ml streptomycin and 100 units/ml penicillin  at 37 C in a CO3 cells/well. After incubation at 37 C overnight, cells were treated with 0.4, 0.8, 2, 3 and 6 mM of crocin  for 24 h at 37 C. Thereafter, 5 μl of 3--2,5-diphenyl-2-H-tetrazolium bromide (MTT)  was added to each well, to obtain a final concentration of 5 mg/ml MTT. The plates were then incubated at 37 C for a further 4 h, at which point the supernatant was aspirated, 100 μl of dimethyl sulfoxide  was added to each well, and finally the absorbance of the plates was measured at 490 nm using a Synergy™ 4 Microplate Reader .SW480 cells were seeded into 96-well plates at a density of 8 × 105 cells/well, and the plates were incubated at 37 C overnight. Thereafter, cells were treated with 2.5 mM or 5 mM crocin and incubated at 37 C for a further 12 h.SW480 cells were seeded into six-well plates at a density of 2 × 10For cell cycle detection, collected cells were washed with pre-cooled phosphate buffered saline (PBS) and then incubated with 70% pre-cooled ethanol for more than 3 h. The cells were then exposed to Muse™ Cell Cycle Reagent  at room temperature for 30 min in darkness. A Muse™ Cell Analyzer  was then used to detect the cell cycle conditions of the cells.6 cells/ml. Cells were incubated with the Muse™ Annexin V and Dead Cell Reagent  at room temperature for 20 min in darkness. A Muse™ cell analyzer was then used to detect the apoptotic status of the cells.For cell apoptosis detection, collected cells were resuspended in DMEM containing 1% FBS and 1% bovine serum albumin (BSA) at a final concentration of 1 × 10For MMP detection, collected cells were treated with mitopotential working fluid at a final concentration of 10 μM  and then incubated at 37 C in darkness for 20 min. Thereafter, cells were washed three times with PBS, and then the MMP changes of SW480 cells were analyzed using a Muse™ cell analyzer.MinC/Gpt mice   purchased from GemPharmatech Co., Ltd.  were kept in a temperature- and humidity-controlled room at 22 ± 1 C and 50–65% humidity, with no convective wind and guaranteed sunshine for 12 h each day. Mice were provided with clean water and food each day, and their litter was cleaned daily.The experiment was approved by the Institutional Animal Ethics Committee of Jilin University (SY201905009 and SY201905004). C57BL/6 J mice   purchased from Liaoning Changsheng Biotechnology Co., Ltd  and Apcn = 12 per group). All 48 treatment-group mice were allowed ad libitum access to 3.0% solution of DSS in water  for 1 week. Subsequently, model mice (n = 12) were orally treated with normal saline, positive control mice (n = 12) were orally treated with 0.6 g/kg of sulfasalazine (SASP) , and crocin-treated mice were orally treated with 10 mg/kg (n = 12) and 30 mg/kg (n = 12) crocin once per day for 3 weeks. All mice continued to have ad libitum access to 3.0% DSS every second day for a further 3 weeks. The control mice (n = 12) (entitled CTRL) were orally treated with normal saline once per day for 3 weeks.Sixty C57BL/6 J mice were adaptively fed for 1 week, and then randomly divided into five groups . One of these was designated the control group, and these mice were treated with normal saline (0 mg/kg of crocin); the other was designed the treatment group, and these mice were treated orally with 30 mg/kg of crocin once per day for 8 weeks.Twenty ApcAt the conclusion of the experiment, all mice were euthanized by sodium barbiturate injection. Blood samples were collected from mouse caudal veins, and organs  were collected for subsequent biochemical and pathological analysis.The body weight, stool consistency and bleeding of mice with UC were recorded daily, and their DAI scores were measured according to the Hamamoto standard .A protein sample solution was obtained from the colon of mice with UC, quantified by using a bicinchoninic acid (BCA) kit, and then hydrolyzed with lyase. The resulting peptide samples were detached and then analyzed by liquid chromatography-tandem mass spectrometry, and the results were processed using MaxQuant (1.5.6.0). The UNIPROT database (Uniprot_mouse_1206_09) was used to obtain protein sequences, and the protein sequences and their respective reverse bait sequences were used in the MaxQuant search. Corresponding differentially expressed proteins were identified from statistical analysis of standardized quantitative results. Proteins with different expression folds (ratio A/B > 1.5 or ratio A/B < 0.66) were defined as significantly different. Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to determine protein interactions.The colon tissues of mice with UC and CRC were homogenized with physiological saline, and the protein concentration of homogenates was measured using a Pierce™ BCA Protein Assay Kit . The concentrations of IL-1β , IL-2 , IL-4 , IL-6 , IL-15 , IL-17 , IL-18 , inducible nitric oxide synthase (iNOS) , cyclooxygenase-2 (COX-2) , TNF-α  and interferon-α (IFN-α)  in the colon and serum samples from mice with UC and CRC were determined according to the manufacturer’s instructions.Fresh samples of colon, liver, kidney, and spleen tissues from mice with UC and CRC were fixed in 10% formalin, and the fixed tissues were then dehydrated by successive exposure to 30, 50, 70, 80, 95 and 100% ethanol. The dehydrated tissues were then immersed in xylene, embedded in wax, and cut into 5 μm sections. The sections were dewaxed using xylene, and then rehydrated by successive treatment with 100, 95, 80, 70, 50 and 30% ethanol. Finally, the rehydrated sections were stained by H&E and observed under a medical biological microscope .Proteins were extracted from colon tissues using radio immunoprecipitation assay  buffer containing 1% protease inhibitor cocktail  and 2% phenylmethanesulfonyl fluoride . Protein concentrations were measured using the Pierce™ BCA Protein Assay Kit. Protein samples (40 μg/μl) were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to a 0.45 μm polyvinylidene difluoride membrane . The loaded membranes were incubated with 5% BSA solution at 4 C for 5 h, and then incubated at 4 C overnight with the following primary antibodies: total- (T-) inhibitor of nuclear factor kappa-B kinase subunit (α/β) [IKK (α/β)] (75/87 kDa) (dilution: 1:2000) , B-cell lymphoma-2 (Bcl-2) (26 kDa) (dilution: 1:2000) , and phospho- (P-) inhibitor of nuclear factor kappa-B-α (IκBα) (40 kDa) (dilution: 1:2000) , P-IKK (α/β) (85/87 kDa) (dilution: 1:1000) , Bcl-2 associated X protein (Bax) (21 kDa) (dilution: 1:2000) , T-IκBα (35 kDa) (dilution: 1:2000) , T-NF-κB p65 (65 kDa) (dilution: 1:2000) , P-NF-κB p65 (65 kDa) (dilution: 1:4000) , UBX domain-containing protein 1 (UBXN1) (35 kDa) (dilution: 1:2000) , Dedicator of cytokinesis 1 (DOCK1) (215 kDa) (dilution: 1:2000) , and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (37 kDa) (dilution: 1:2000) .Subsequently, the membranes were washed and then incubated with goat anti-rabbit IgG (H + L) [peroxidase/horseradish peroxidase (HRP) conjugated]  or goat anti-mouse IgG (H + L) (peroxidase/HRP conjugated)  at 4 C for 3–4 h. The membranes were then washed again, and treated with NcmECL Ultra , a high–sensitivity electro-chemi-luminescence (ECL) reagent. The protein band was then visualized on an imaging system . The imaging results were analyzed using Image–J software .p value of <0.05 was considered to be statistically significant.All values are expressed as the means ± standard deviation (SD). A one-way analysis of variance (ANOVA) was used to detect statistical significance, and post-hoc multiple comparisons (Tukey’s test) were performed using SPSS 16.0 software . A 1) using AutoDock 4.2 software (The 3D structure of crocin was optimized by Gaussian 09 software  at 6-31Gsoftware . The cursoftware . Proteinsoftware .p = 0.000) of mice, and that this was reversed after 32 days treatment with crocin or SASP (p < 0.05) (A DAI score can be used to assess the severity of UC . We foun < 0.05) .The colonic epithelial tissues of CTRL mice were smooth and ductile; in contrast, ulcers and edema were apparent in the colonic epithelial tissues of vehicle-treated UC mice, and these were markedly improved after treatment with crocin or SASP . The colp = 0.000), IL-2 (p = 0.006), IL-6 (p = 0.000), IL-17 (p = 0.002), iNOS (p = 0.006) and COX-2 (p = 0.007) were observed, and the concentrations of all of these factors were substantially reduced by 21-days crocin treatment (p < 0.01) (p < 0.05), except for those of TNF-α (p < 0.01), IL-2 (p = 0.000), IL-6 (p = 0.000), iNOS (p < 0.01), TNF-α (p < 0.01) and COX-2 (p = 0.018). In addition, SASP reduced the serum concentrations of IL-1β (p = 0.000), IL-2 (p = 0.000), IL-6 (p = 0.000), iNOS (p = 0.003) and TNF-α (p = 0.000), but not those of IL-17 and COX-2, in UC mice  . Similarof TNF-α . Crocin  UC mice .p = 0.022), P-IκBα (p = 0.000), P-NF-κB (p = 0.024) and DOCK1 (p = 0.000) were noted in the colonic tissues of UC mice, as were reduced expression of UBXN1 (p = 0.000). Both SASP and crocin reversed these expression-level alterations (p < 0.05) (Increased expression of P-IKK (α/β) ( < 0.05) .50 of 3.68 mM (p < 0.05) (vs. 12.60% in 0 mM crocin) (p = 0.002) and 78.65% (5 mM) (p = 0.000) apoptosis in SW480 cells (vs. 11.05% in 0 mM crocin)  . Crocin  crocin) . Incubat crocin)  and resu crocin) .MinC/Gpt mice are therefore useful for studying the formation of intestinal tumors. Compared with vehicle-treated mice with CRC, crocin-treated mice displayed markedly increased body weights on day 56 (p = 0.044), and increased spleen index (p = 0.002), and reduced indexes of liver (p = 0.009), and kidney (p = 0.044)  .Far fewer colonic tumors and longer intestines were noted in crocin-treated mice compared with vehicle-treated mice . Patholop < 0.01), IL-6 (p < 0.01), IL-17 (p < 0.05) and TNF-α (p < 0.05) and increased those of IL-15 (p = 0.000) and IFN-α (p = 0.000) in both colon tissues and sera (p = 0.033) and reduced those of IL-18 (p = 0.032) in the colon tissues , IκBα (p = 0.000) and NF-κB (p = 0.000) and the expression of DOCK1 (p = 0.000), but up-regulated the expression of UBXN1 (p = 0.000). All these results were consistent with those detected in UC mice. Furthermore, crocin increased the expression of Bax (p = 0.000) and reduced the expression of Bcl-2 (p = 0.000) in the colon tissues of CRC mice   in HCT116 cells (50) in CT-26 cells  due to, crocin acting via the NF-κB signaling pathway to decrease the concentrations of IL-6. These data suggest that crocin or its structural analogs should be explored as possible therapeutic candidates for colorectal diseases, especially UC and CRC.Overall, it was demonstrated that the potent anti-inflammatory properties of crocin protected the mouse colons against DSS-induced UC and suppressed tumorigenesis in Apc


Auksis apple trees which was grafted on super dwarfing P 22 rootstock and planted at 3 × 0.75 m distances.Global demand for food is increasing each year, but the area of land suitable for farming is limited. Thus, there is a need to grow not only larger quantities of food but also higher quality food products in the same area. This study aimed to evaluate the influence of rootstock and high-density orchards on cv. Auksis fruit quality. Two rootstocks were selected for this experiment, P 22 super dwarfing and P 60 dwarfing. Apple trees cv. Auksis were planted in the year 2001 in single rows spaced 1.00 m, 0.75 m, and 0.50 m, apart with 3 m between rows. High-density planting and rootstock combination was found to have no significant effect on sugar accumulation and most of the elements in apple fruits. However, super dwarfing P 22 rootstock accumulated significantly higher (up to 45%) content of organic acids and up to 33%–44% lower DPPH free radical scavering activity compared to P 60 dwarfing rootstock. After summarizing the obtained results, apples which accumulated the most antioxidants (according to the activity of phenolic compounds, DPPH Global demand for food is increasing each year, but the area of land suitable for farming is limited. Land degradation leads to smaller areas suitable for good yields . Thus, tTo fill the area faster and get a higher yield per unit area, world growers have introduced high-density planting schemes in apple orchards ,5. IncreA good combination of planting scheme and rootstock can result in a high yield and high-quality apple fruits suitable for the consumer ,10. ApplIn previous studies of apple quality in high-density orchards the data are often narrow and do not describe changes in the chemical composition of the fruit. Therefore, the aim of this study was to evaluate the influence of rootstock and high-density orchards on cv. Auksis fruit quality.Basic fruit sugars were not significantly affected either by the rootstock or by the planting scheme, however, the sorbitol content in the fruit increased in P 22 super-dwarfing rootstock up to 45% compared to the P 60 dwarfing rootstock . Higher Significant differences in the amount of total phenols in apples were found in the interaction between rootstock and distance . Signifi−1. Malic acid was followed by oxalic (0.10—0.14 mg g−1), oxalacetic (0.05–0.10 mg g−1), ascorbic (0.03–0.04 mg g−1), succinic (83.6–143.4 µg g−1), citric (11.0–29.0 µg g−1), fumaric (2.5–4.3 µg g−1) and folic (1.2–1.7 µg g−1)  . The roo µg g−1) .−1), followed by calcium (0.094–0.155 mg g−1), magnesium (0.063–0.071 mg g−1) and sodium (0.017–0.022 mg g−1) (The main element found in apples was potassium (0.623–0.730 mg g mg g−1) . Signifi−1); followed by manganese (about 1 µg g−1); copper (0.609–0.890 µg g−1); and zinc (0.275–0.358 µg g−1) (The highest content of minor elements was found in the following order: iron (0.99–2.60 µg g µg g−1) . The manThe entry of the sun into the canopy has a strong effect on photosynthesis processes, the higher density of trees reduces the penetration of light into the lower and inner layers of the canopy, which results in a decrease in photosynthesis rates ,28. Prev−1 (depending on variety) to 11.7–3.6 mM Trolox g−1 (depending on variety) during fruit ripening, while ABTS scavenging activity from 4.5–2.6 mM Trolox g−1 to 1.4–0.9 mM Trolox g−1 [Competitive stress for light caused by the high-density canopy from mechanical pruning has been shown to result in an increase of total phenol content in the fruit ,31. On tolox g−1 . Signifi−1 to 7.07 mg L−1 of juice depending on the variety [−1 to 4.64 mg g−1 depending on planting density and rootstock . Each apple was divided into four parts, the seed hopper was removed, and a sample was taken from each part of the pulp. The composite sample of five apples was weighed for further analysis. The remaining apples were crushed and dried to air-dry weight to determine the elemental composition.A trial was carried out in an experimental intensive orchard in Lithuania,  in 2017–2019. Apple samples were collected from fully grown, canopy-formed, and root-covered trees that strongly feel competitive tension, not only due to light but also water and nutrients. The apple tree  cv. Auksis was grafted on P 60 dwarfing rootstock and P 22 super dwarfing rootstock. Apple trees were planted in single rows spaced 1.00 m, 0.75 m, and 0.50 m apart, with 3 m between rows. Pest and disease management was carried out according to integrated plant protection practices, and the orchard was not irrigated. The soil conditions of the experimental orchard were clay loam, pH 7.3, humus 2.8%, Pg rpm for 15 min. A cleanup step was performed before the chromatographic analysis: 1 mL of the supernatant was mixed with 1 mL 0.01% (w:v) ammonium acetate in acetonitrile and incubated for 30 min at 4° C. After incubation, samples were centrifuged at 14,000× g rpm for 15 min and filtered through 0.22 µm PTFE syringe filter . Analysis was performed on a Shimadzu Nexera UPLC  system. Separation was performed on a Supelcosil 250 × 4 mm NH2 column  using 77% acetonitrile as the mobile phase at 1 mL min−1 flow rate. The calibration method (R2 < 0.99) was used for each individual sugar quantification (mg g−1 in FW).Soluble sugar  contents were evaluated using the UFLC method with evaporative scattering detection (ELSD) . About 1The total starch content was determined using the total starch Megazyme assay kit , a total starch assay kit based on the use of thermostable a-amylase and amyloglucosidase method of determination of starch in samples which also contains D-glucose and/or maltodextrins.g rpm for 5 min. Next, 0.1 mL of extract was diluted with 0.2 mL 10% Folin-Ciocalteau reagent (Folin reagent diluted with bi-distilled water 1:10) and with 0.8 mL 7.5% Na2CO3 solution [Using a colorimetric method, the total content of phenolic compounds was determined using methanol extracts—1 g of plant tissues grounded with liquid nitrogen and diluted with 10 mL of 80% methanol. The extract was mixed and left for 24 h in the fridge ( + 4 °C), subsequently centrifuged at 5000× solution . The abs•) free radical scavenging capacity. 1 g of plant tissues grounded with liquid nitrogen and diluted with 10 mL of 80% methanol. The extract was mixed and left for 24 h in the fridge (4 °C) and subsequently centrifuged at 5000× g rpm for 5 min. 1 mL DPPH• solution (60µ DPPH•) and 0.1 mL for extract add into a cuvette. The absorbance scanned after 16 min from the beginning of the reaction at 515 nm [The antioxidant activity of methanol extracts of the investigated plants was evaluated spectrophotometrically relating to the 2,2-diphenyl-1-picrylhydrazyl (DPPHt 515 nm .• (2.2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) scavenging activities of apple extracts were determined. The same extraction was used as for the determination of DPPH free radical scavenging capacity. ABTS• was dissolved in methanol at a concentration of 2 mM. The ABTS radical cation was produced by incubating the ABTS• stock solution with 200 μL K2S2O8 (0.1982 g/10 mL H2O) in the dark for 16 h. Following this, 100 μL of the diluted sample was mixed with 2 mL of ABTS• solution and the absorbance was scanned for 11 min (plateau phase) at 734 nm. The ABTS• scavenging activity of each extract was calculated as the difference between the initial absorbance and that after reacting for 10 min, which was expressed as mmol (ABTS•) scavenged per 1 g fresh sample (mmol g−1). Methanol was used as the blank solution [The ABTSsolution .2O 1:10 (w:v). Extraction was performed in a heated water bath (50 °C) for 30 min. The extract was clarified by centrifugation at 10,000 rpm for 15 min and filtered through a 0.22 µm PTFE syringe filter . Separation was performed on Lichrosorb RP-184.6 × 250 mm, 5µm column (Altech). Mobile phase—0.05 M sulphuric acid, flow rate 0.5 mL min−1, injection volume—10 µL. The calibration method (R2 < 0.99) was used for each organic acid quantification (mg g−1 in FW).Organic acid  contents were determined using the HPLC method  on Shima3 using microwave digestion system Multiwave GO . The digestion program was as follows: 170 °C was reached within 5 min, digested for 20 min; the mineralized samples were diluted to 50 mL with deionized water; and the elemental profile was analyzed using an ICP-OES spectrometer . The operating conditions employed for ICP-OES determination were 1300W RF power, 12 L min−1 plasma flow, 1.0 L min−1 auxiliary flow, 0.8 L min−1 nebulizer flow, 1.0 mL min−1 sample uptake rate. The analytical wavelengths (nm) chosen were: B + 249.773 nm, Ca2+ 445.478 nm, Cu+ 324.754 nm, Fe2+ 259.941 nm, K+ 766.491 nm, Mg2+ 279.079 nm, Mn2+ 259.373 nm, Na+ 589.592 nm, P+ 213.618 nm, S+ 182.034 nm, and Zn+ 213.856 nm. The calibration standards were prepared by diluting a stock multi-elemental standard solution (1000 mg L−1) in 6.5% (v/v) nitric acid, and by diluting stock phosphorus and sulfur standard solutions (1000 mg L−1) in deionized water. The calibration curves for all the studied elements were in the range of 0.01–400 mg L−1.The macro and microelements contents in microgreens were determined by microwave digestion technique combined with inductively coupled plasma optical emission spectrometry ,49. Compp ≤ 0.05.MS Excel Version 2010 and XLStat 2020 Data Analysis and Statistical Solution for Microsoft Excel  statistical software were used for data processing. Analysis of variance (ANOVA) was carried out along with Tukey multiple comparisons test for statistical analyses, The meteorological data were collected from “iMetos” meteorological station at the Institute of Horticulture, Lithuania research center for agriculture and forestry, Lithuania in 2017–2019. Meteorological conditions fluctuated around the average multiannual conditions during the study year . The preThe highest accumulation of phenolic compounds were found in fruits of cv. Auksis trees grafted on P 22 rootstock at planting distance 0.75 m and grafted on more vigorous P 60 rootstock at planting distance 1 m between trees. Also, less vigorous P 22 super dwarfing rootstock accumulated significantly higher (up to 45%) content of almost all identified organic acids compared to P 60 dwarfing rootstock. Planting density and rootstock combinations had no significant effect on the amount of sugars identified in fruits of cv. Auksis. Significantly the highest amounts of potassium and magnesium, also, were found in fruits of trees grafted on P 22 rootstock, planted at 3 × 0.75 m distances. However, fruits of apple trees grafted on P 60 dwarfing rootstock accumulated 2.5 times more iron and up to 46% more copper compared to P 22 super dwarfing rootstock. Generally, if seeking to obtain a high-quality fruit harvest in high-density apple cv. Auksis orchards, P 22 super dwarf rootstock at 3 × 0.75 m planting distances is recommended.


Its water stability was highly enhanced by the doping of CuII ions by the one-pot method. In addition, the detection performances of doping material Cu0.1/Zn-MOF for sixteen metal ions and thirteen antibiotics were well studied. It was found that Cu0.1/Zn-MOF displays high sensitivity, fast response, lower detection limit, and long-term stability for the detection of Fe3+, NFT, NFZ, FZD, and TC in the aqueous medium.Luminescent metal–organic frameworks (LMOFs) have been widely developed in the field of chemical sensing owing to their outstanding photoluminescence performance, high selectivity, anti-interference, high sensitivity, and fast response, and have become one of the research hotspots of emerging functional materials. However, in practical applications, many tests are carried out in the water environment, and fragile water stability greatly limits the application of MOFs in the field. Therefore, it is important to develop a method to enhance the water stability of MOFs. Herein, a new complex {[Zn(L)]·CH The selection of H2L ligand is based on the following considerations: 1) H2L ligand containing carboxylic acid groups, aromatic ring, and benzimidazol-1-yl fluorescence conjugated groups is beneficial to the formation of large Stokes shift, which may have potential in fluorescence detection  and emission wavelength after immersion in aqueous solutions for 7 days. Therefore, in order to realize the recycling of Zn-MOF as sensors in the water environment, it is necessary to improve its hydrostability without losing its detection performance. The method of doping inert metal ions into the existing skeleton was considered first. Considering that Zn-MOF has a binuclear paddle-wheel secondary building unit (SBU), we chose Cu(II) ion as the pre-doped metal ion. The main considerations are as follows: 1) The metal ions in binuclear paddle-wheel SBUs with D4h being symmetric can be CuII, ZnII, CoII, FeII, CdII, etc., but CuII-paddle wheel SBUs generally have better thermodynamic stability ]·CHetection  and the etection ; 2) the etection ; 3) the etection . Althougtability . 2) In pclusters . 3) Fluoclusters . Fortuna2L ligands were purchased from Jinan Henghua Sci. and Tec. Co. Ltd. PXRD patterns at diffraction angles from 5° to 55° were obtained with a D/MAX-3D diffractometer. Elemental analysis was performed by Perkin–Elmer Elementarvario elemental analysis instrument. Fourier transform infrared spectra (FT-IR) were recorded on Nicolet iS50 . Thermogravimetric analysis (TGA) was performed on a SDT 2960 thermal analyzer from room temperature to 800°C at a heating rate of 10°C/min under nitrogen flow. Ultraviolet-visible (UV-vis) absorption spectra were obtained by UV-2600 UV-vis spectrophotometer. Fluorescence detection was carried out on CARY Eclipse Fluorescence Spectrophotometer at room temperature. Energy dispersive spectrometer (EDS) was obtained by JSM-6490LV  electron microscope.Starting reagents, solvents, and materials were commercially available and at least of analytical grade. The HZn-MOF were collected by Oxford Diffraction SuperNova area-detector diffractometer with the program of CrysAlisPro. The crystal structure was solved by SHELXS-2016 and SHELXL-2016 software (Zn-MOF (CCDC No. 2143524) can be downloaded free of charge viahttp://www.ccdc.cam.ac.uk/conts/retrieving.html. Selected bond lengths and bond angles for the Zn-MOF complex are shown in Single-crystal X-ray diffraction data of software . The cryZn-MOF. In order to investigate the stability, Cu0.1/Zn-MOF powder was dispersed in a vial containing 3 ml aqueous solutions with different pH scales (pH = 1–14) for 1 h, and then the PXRD test was carried out. In addition, in order to explore the fluorescence performance of Cu2+ doped Zn-MOF at different pH values, the synthesized sample Cu0.1/Zn-MOF (2.0 mg) was added into aqueous solutions (3.0 ml) with different pH values, and ultrasonicated for 20 min; then the fluorescence spectra were immediately determined.Powder samples (120 mg) were dispersed in vials containing 10 ml of aqueous solution, respectively. The vials were kept at room temperature for 1–7 days, and 30 mg samples were taken out at 1, 3, and 7 days, respectively, for PXRD tests. The results demonstrated that the doping of Cu ions improved the water stability of the 3)x . For the detection of antibiotics, 2.0 mg samples were finely ground and added into 3.0 ml of varied selected antibiotics solutions (100 ppm), including tetracyclines , sulfonamides (sulfamethoxydiazine SM), chloramphenicols (chloramphenicol CHL), aminoglycosides , nitrofurans , and β-lactams (cefotaxime CEF). In order to maintain its homogeneity, the mixtures were ultrasonicated for 20 min to form a suspension. The luminescence data of the suspension were monitored under the same conditions.For the detection of metal ions, 2.0 mg samples were finely ground and added into 3.0 ml of deionized water of M(NO3)2·6H2O , H2L , CH3CN/H2O (2 ml/3 ml), and one drop of dilute HNO3 (1 M) was added to a 10 ml Pyrex vial and stirred for 20 min. Then it was transferred to a Teflon-lined stainless steel vessel and reacted at 120°C for 72 h. After being cooled to room temperature at a rate of 5°C min−1, the light yellow block crystals were obtained by filtration and collection, washed with CH3CN, and then air-dried naturally with a yield of 83.8%  for C17H11N3O4Zn (Mr = 386.68): C 52.80 H 2.87, N 10.87; found (%): C 52.85, H 2.91, N 10.82. IR : 3,468 (w), 3,105 (m), 3,074 (w), 2,246 (w), 1837 (w), 1786 (w), 1,654 (s), 1,589 (s), 1,511 (s), 1,458 (s), 1,426 (s), 1,388 (s), 1,310 (s), 1,239 (s), 1,179 (m), 1,112 (m), 1,043 (w), 1,004 (w), 921 (s), 850 (w), 784 (s), 750 (s), 718 (s), 689 (m), 648 (w), 531 (m), 456 (s).A mixture of Zn: A mixture of Zn(NO3)2·6H2O , Cu(NO3)2·3H2O , H2L , CH3CN/H2O (2 ml/3 ml), and two drops of HNO3  was added to a 10 ml Pyrex vial and stirred for 20 min, and then transferred to a Teflon-lined stainless steel vessel and reacted at 120°C for 72 h. After being cooled to room temperature at a rate of 5°C min−1, the light blue block crystals were obtained by filtration and collection, washed with CH3CN, and then air-dried naturally with a yield of 81.2, 82.9, 81.1, and 82.7%, respectively (based on H2L).3CN}n crystallizes in the monoclinic system, P21/c space group. In Zn-MOF, the asymmetric unit contains one Zn(II) atom, one fully deprotonated L2- ligand, and one free acetonitrile molecule  clusters. As illustrated in Zn-MOF could be simplified as a 3,6-c network with the Schläfli symbol {4·62}2{42·610·83} with TOPOS ]·CHmolecule . Zn(II) molecule . Adjacenmolecule . From a th TOPOS .Zn-MOF and Cux/Zn-MOF were determined by EDS  were carried out and shown in 2L is 1720 cm−1, while it is not visible in the five complexes, which illustrates the coordination of carboxylic acids with metals. The vibration band at 1,589 cm−1 and 1,389 cm−1 of the five complexes can be associated with the asymmetrical stretching vibration and symmetrical stretching vibration of -COO-, respectively. The observed band at 1,653 cm−1 in the FTIR spectrum of Zn-MOF is assigned as the stretching vibration of the -C=N- bond , probably due to competitive coordination between Cu2+ and Zn2+. The signals at 455 cm−1 can be attributed to the stretching vibration of Zn–O. For the copper-doped Zn-MOF, slight shifts  were observed, suggesting the coordination of Cu with the carboxylic group of H2L ligand. The FTIR spectra of Zn-MOF and Cu-doped Zn-MOF are almost the same, which further indicates that the two materials exhibit an isomorphic structure.The FTIR spectra of =N- bond , which mx/Zn-MOF  were examined by TGA under a N2 atmosphere in the temperature range 25–800°C. As shown in Zn-MOF complex, the weight loss rate reached 9.82%  at 152–275°C, which is to the loss of one acetonitrile molecule. Then the skeleton of the compound began to collapse at 419 °C. The analysis of TGA curve of Cu0.01/Zn-MOF confirmed that the weight loss rate was 10.34%  at 167–275°C, and the skeleton of the compound began to collapse at 413°C. For Cu0.1/Zn-MOF, the weight loss rate was 10.22%  at 166–300°C, and the skeleton of the compound began to collapse at 381°C. For Cu0.2/Zn-MOF, the weight loss rate was 9.31%  at 167–295°C, and the skeleton of the compound began to collapse at 352°C. For Cu0.5/Zn-MOF, the weight loss rate was 9.27%  at 170–295°C, and the skeleton of the compound began to collapse at 348°C. We found that the doping of Cu(II) ions have a significant effect on the decomposition temperature of organic frames. With the increase of the Cu(II) ion doping ratio, the thermal stability decreases gradually, which probably due to copper divalent is easier to be reduced than zinc divalent at high temperature.The thermal stabilities of compounds Zn-MOF and CuZn-MOF were in good agreement with the simulation diagram, indicating the high degree of pure phase. When different proportions of CuII were doped into Zn-MOF, the positions of diffraction peaks were unchanged, suggesting that copper doping did not change the crystal structure of Zn-MOF. The doped Cu ion may be incorporated into the framework of Zn-MOF , and adjusted using HCl and NaOH. PXRD patterns showed that samples were highly resistant and matched well with the original sample in a pH range of 3–12  or ligand-to-metal charge transfer (LMCT) is difficult to occur  (2L ligand containing carboxylic acid and aromatic and N-containing heterocycle may exist in π*→π or/and π*→n electronic transitions.It is known that dto occur . Howeverr (LLCT) . H2L lig2L ligand and various complexes in water were detected. As shown in Zn-MOF was significantly enhanced at 397 nm relative to the ligand in aqueous solution. The increased fluorescence may probably be attributed to LLCT induced by ZnII ion  may cause MLCT, which causes the fluorescence quenching  of Cu0.01/Zn-MOF, the Stokes shift of Cu0.1/Zn-MOF is up to 140 nm . As the fluorescence sensing material with large Stokes shift is rare and attractive, Cu0.1/Zn-MOF was selected as the fluorescence sensor for subsequent fluorescence detection.For a qualified fluorescence sensor, the stability of its frame structure, the luminescence intensity, and the sensitivity of fluorescence detection should be fully considered. Based on the aforementioned situation, Cuin water . From thr choice . Second,sitivity . As show0.1/Zn-MOF samples in the water environment were measured. As shown in 0.1/Zn-MOF in the water at different time periods did not change significantly. In addition, the fluorescence spectra of Cu0.1/Zn-MOF at different pH values were also measured. As shown in 0.1/Zn-MOF at a pH range from 4 to 11 did not change significantly, which proved that the fluorescence of Cu0.1/Zn-MOF also has good acid–base stability.Subsequently, the time-varying fluorescence spectra of Cu+, Ca2+, K+, Li+, Zn2+, Mg2+, Co2+, Mn2+, Cd2+, Ni2+, Ag+, Pb2+, Cu2+, Al3+, Cr3+, and Fe3+ at the same concentration, were utilized for investigating the sensibility of Cu0.1/Zn-MOF. As shown in 3+ ion showed obvious fluorescence quenching behavior. The fluorescence intensity at 397 nm was impaired by 93.8% compared with the initial value. This indicated that Cu0.1/Zn-MOF can be used as a visual sensor for selective detection of Fe3+ ion in water. The selectivity experiment of Cu0.1/Zn-MOF was explored. As shown in 3+ ion detection, which further confirms the selectivity of Cu0.1/Zn-MOF for Fe3+ detection.For extensive purpose, sixteen metal ions, including Na0.1/Zn-MOF as a fluorescence sensor for the detection of Fe3+ ion, a fluorescence titration experiment was carried out. As shown in 3+ ion increased, the fluorescence intensity of the suspension decreased gradually, and the fluorescence quenching can be observed when the concentration of Fe3+ ion was 833 μM. The linear Stern–Volmer (S-V) equation, (I0/I) = KSV[C] + 1, can be used to explain the quenching efficiency . According to the slope and standard error of the fitting curve, the LOD for Fe3+ was calculated to be 0.76 μM according to 3σ/KSV (σ is the standard deviation for eleven repeated luminescent measurements (σ = 0.0070). The sensitivity of Cu0.1/Zn-MOF is a rival to most of the reported MOF-based sensors for Fe3+ (3+.In order to explore the limit of detection (LOD) of Cuficiency , where I1/Zn-MOF  in the co 3σ/KSV , where σfor Fe3+ , suggest3+ solution for 4 h, the PXRD patterns were unchanged, indicating that the fluorescence quenching is attributed to neither skeleton collapse nor cation exchange. In addition, the rapid response of Fe3+ detection also ruled out the possibility of fluorescence quenching caused by cation exchange because cation exchange cannot occur in a short period of time. As can be seen in 3+ ions have a wide absorption band from 200 to 450 nm, which overlaps with the emission spectra range 310–510 nm of Cu0.1/Zn-MOF. Therefore, the main reason for fluorescence quenching may be on account of the fluorescence resonance energy transfer (FRET)  . In addi0.1/Zn-MOF, the detection performances of Cu0.1/Zn-MOF on thirteen common antibiotics in six categories were studied. The results showed that NFT, NFZ, FZD, and TC had obvious quenching effect compared with other antibiotics  for NFT, 0.2447 ppm−1 (4.85×104 M−1) for NFZ, and 0.1660 ppm−1 (3.74×104 M−1) for FZD. Based on the KSV values, the detection limits of Cu0.1/Zn-MOF toward NFT, NFZ, and FZD were calculated to be 95.0 ppb (0.4 μM), 85.8 ppb (0.43 μM), and 126.5 ppb (0.56 μM), respectively.Considering the red-shift factor of fluorescence spectrum, the maximum emission peak corresponding to each titration concentration was selected in the calculation of the quenching rate of antibiotics and the value of fitting curve, instead of the fluorescence emission peak at a fixed wavelength. In this case, when the titrated concentration of NFT and NFZ reached 75 ppm, the fluorescence quenching efficiency rate was 90.9 and 94.1%, respectively. When the titrated concentration of FZD reached 100 ppm, the fluorescence quenching rate was 85.3%. To further understand the luminescent quenching degree, the quenching curves were quantitatively studied by Stern–Volmer equation. As shown in 0/I-1 = lg[KSV]+lg[C]. This means that the quenching of luminescence may be the result of both dynamic and static factors  for TC. Based on the KSV value, the detection limit of Cu0.1/Zn-MOF toward TC was calculated to be 169.8 ppb (0.35 μM). Compared with the reported MOF-based sensors for the detection of antibiotics, the result implied that Cu0.1/Zn-MOF has potential application value in the detection of NFT, NZF, FZD, and TC  is also a possible luminescence quenching mechanism  are excited to the lowest unoccupied orbital (LUMO), followed by the transfer to the antibiotic with a lower LUMO level. To explain the possibility of this process, we calculated and derived the band structures of Cu0.1/Zn-MOF and antibiotics. According to Kubelka–Munk, the bandgap is about 3.6 eV  for NFT, 1.12 ×105 ppm−1 (2.23×104 M−1) for NFZ, 4.74×104 ppm−1 (1.07×104 M−1) for FZD, and 4.96×104 ppm−1 (2.39×104 M−1) for TC. The data showed that compared with Cu0.1/Zn-MOF, the KSV of Zn-MOF for five pollutants are significantly reduced. In other words, proper CuII doping improves the sensitivity of Zn-MOF to detect analytes. The mechanism of the higher sensitivity of Cu0.1/Zn-MOF in the detection of analytes is still not completely clear. We speculate that it may be related to the following factors: First, the increase of Stokes shift reduces the background interference, which is beneficial to the strong penetration to the sample, and enhances the detection sensitivity. Second, the liquid UV spectrum displayed that the UV absorption of TC was similar at 257 and 283 nm, while the UV absorptions of Fe3+, NFT, NFZ, and FZD were stronger at 257 nm than at 283 nm (It has been proved that the doping of Cut 283 nm . In otheZn-MOF complex with superior photophysical property. Subsequently, we successfully doped CuII ions into Zn-MOF in a simple and feasible way to enhance its water stability. This strategy enables LMOFs, which were initially limited by water stability, to implement detection in aqueous solvents. Interestingly, subsequent detection showed that bimetallic LMOFs doped with an appropriate proportion of CuII ions had lower detection limits for Fe3+, NFT, NFZ, FZD, and TC. This study is of great significance to broaden the range of alternative LMOFs in practical application and may provide a new strategy for the design of LMOFs in the future.In summary, we constructed a


Oxysterols are oxidized derivatives of cholesterol that play regulatory roles in lipid biosynthesis and homeostasis. How oxysterol signaling coordinates different lipid classes such as sterols and triglycerides remains incompletely understood. Here, we show that 4β-hydroxycholesterol (HC) (4β-HC), a liver and serum abundant oxysterol of poorly defined functions, is a potent and selective inducer of the master lipogenic transcription factor, SREBP1c, but not the related steroidogenic transcription factor SREBP2. By correlating tracing of lipid synthesis with lipogenic gene expression profiling, we found that 4β-HC acts as a putative agonist for the liver X receptor (LXR), a sterol sensor and transcriptional regulator previously linked to SREBP1c activation. Unique among the oxysterol agonists of the LXR, 4β-HC induced expression of the lipogenic program downstream of SREBP1c and triggered de novo lipogenesis both in primary hepatocytes and in the mouse liver. In addition, 4β-HC acted in parallel to insulin-PI3K–dependent signaling to stimulate triglyceride synthesis and lipid-droplet accumulation. Thus, 4β-HC is an endogenous regulator of de novo lipogenesis through the LXR-SREBP1c axis. All cells must achieve and maintain a balanced composition of their internal membranes to grow, proliferate, or adapt to sudden changes in external conditions and nutrient availability . DedicatLXRα exhibit impaired bile acid metabolism and defective cholesterol elimination (The liver X receptor (LXR) α and β are transcription factors belonging to the nuclear receptor superfamily that play key roles in maintaining lipid homeostasis in multiple cells and organs , 5, 6, 7mination , along wmination . Conversmination , 13, 14.Another key mediator of lipid homeostasis is the helix-loop-helix-leucine zipper transcription factor, SREBP1c. SREBP1c is a master regulator of biosynthesis of fatty acids and triglycerides [collectively referred to as de novo lipogenesis (DNL)] that is subject to tight transcriptional and posttranslational regulation. Along with its paralogue, the master steroidogenic transcription factor SREBP2, SREBP1c resides at the endoplasmic reticulum (ER) membrane, to which it is anchored via a single transmembrane helix. When cholesterol concentration in the ER membrane is low, SREBP1c and SREBP2 are transported to the Golgi apparatus via interaction with SREBP cleavage-activating protein, a cholesterol-sensing chaperone that favors their loading into COPII vesicles. At the Golgi membrane, resident proteases cleave the DNA-binding portion of SREBP1c and SREBP2 from the transmembrane portion, enabling their translocation to the nucleus and activation of downstream programs for DNL and de novo steroidogenesis, respectively.In addition to their homeostatic regulation by cholesterol levels, the SREBPs lie downstream of metabolic hormone signaling. For example, in the liver, both the expression and proteolytic activation of SREBP1c are stimulated by the insulin-phosphatidylinositol 3-kinase (PI3K)-mechanistic Target of Rapamycin (mTOR) pathway, as part of a mechanism that converts excess of glucose into lipids, which are required for energy storage , 16, 17.SREBP1c gene and trigger activation of its downstream lipogenic genes , a condition that has risen to epidemic proportions in recent years . Thus, uSREBP1c and its downstream lipogenic programs, whereas several oxysterols have been shown to promote LXR-dependent cholesterol efflux. In contrast, synthetic LXR ligands including T0901317 and GW3965 can induce both cholesterol efflux and SREBP1c-dependent DNL  (4β-HC) as an LXR activator that selectively triggers ABCA1, with variable potency -HC  was unable to induce SREBP1c mRNA expression even at the highest concentration used (20 μM) -, 25-, and 27-HC. By quantitative PCR, several oxysterols previously identified as LXR activators, including 4β-HC, 7β-HC, 24(S)-HC and 25-HC, induced the expression of a canonical LXR target gene,  potency A. In con potency B. A dosey (ABCA1 C and ano1 (ABCG1 D. Conver24(S)-HC E. 4β-HC– (20 μM) F. These Oxysterols such as 25- and 27-HC suppress SREBP1 and SREBP2 activation by blocking their trafficking to the Golgi, where proteolytic processing of the SREBPs to the mature nuclear form occurs , 27.In contrast to these oxysterols, 4β-HC significantly increased SREBP1c mRNA levels A and proThese data suggest that, unlike other oxysterols that function as inhibitors of both SREBP1c and SREBP2, 4β-HC is a specific inducer of SREBP1c expression and activation.Along with other oxysterols, 4β-HC was previously shown to activate LXRα-dependent transcription in luciferase assays in vitro, supporting its role as a putative LXR ligand . In turncis-retinoic acid. Moreover, coincubation of 4β-HC with the LXR agonist, GW3965, used at concentrations that activate the LXR maximally, caused no additional increase in SREBP1c expression over GW3965 alone , Stearoyl-CoA desaturase (SCD1) and Lipin1 (LPIN1) are validated SREBP1c downstream targets in Huh7 cells , 31. TrePrevious work had shown that GW3965 induces FASN to a greater extent than the 1.6-fold we observed in Huh7 . Huh7, aent-4HC) for 72 h resulted in marked accumulation of lipid droplets (LDs), as revealed by staining with the lipophilic dye BODIPY 493/503  was measured. Consistent with the results in Huh7 cells, 4β-HC significantly increased the size and number of LDs in liver sections D and livInsulin is a key hormone that drives SREBP1c transcription, proteolytic processing, and DNL in the postprandial state. Insulin regulates SREBP1c transcription via poorly understood mechanisms, which include AKT-dependent transcriptional downregulation of Insig-2a, the ER-retention factor that blocks translocation of SREBP cleavage-activating protein-SREBP1c to the Golgi , 36. TheTo interrogate the relationship between 4β-HC and insulin signaling in driving SREBP1c transcription and processing, we used an insulin-responsive primary mouse hepatocyte . In thesSimilar to their effects on transcriptional induction, insulin and 4β-HC stimulated proteolytic processing of SREBP1c in an additive manner B. 4β-HC Consistent with previous reports, we also detected a marked decrease in Insig-2a mRNA in insulin-stimulated hepatocytes C. In conTo further probe possible connections between insulin-PI3K and 4β-HC-LXR signaling, we tested whether insulin signaling promotes 4β-HC synthesis. Previous reports had shown that in humans, 4β-HC has a very slow kinetics, with an extremely long half-life in plasma (60 h) . PharmacHere we identify 4β-HC as a unique oxysterol that activates SREBP1c expression and promotes lipogenic gene programs, resulting in induction of fatty acid biosynthesis and cellular accumulation of triglycerides in LDs both in cell culture and in vivo. Our results are most consistent with a model in which 4β-HC acts in parallel to insulin-PI3K-mTOR signaling, and the two pathways have additive effects on SREBP1c activation. A simple mechanism that explains the additive effect is that the SREBP1c promoter contains both an LXR-binding element and an SREBP-binding element and that transcription can be stimulated by the two transcription factors independently . Howeveret al.  had all observed that 4β-HC levels increase when animals are fed a high cholesterol diet , 45, 46,t manner . Further agonist . This stSREBP1c promoter requires a specific LXR ligand, while recruitment of RNA polymerase II to the ABCA1 promoter is more promiscuous  and corepressors  to the promoters of target genes in a ligand-dependent manner , 52, 53,miscuous . Thus, 4SREBP1 expression  , as a crFrom a more clinical perspective, 4β-HC might have an aggravating effect on the development of NAFLD. NAFLD is characterized by elevated liver triglycerides not due to alcohol consumption or any other known causes . ElevateIn conclusion, this work highlights a role for 4β-HC, which was long viewed as an ‘orphan’ oxysterol, in regulating lipid metabolism in the liver together with insulin. Future work, dissecting the role of 4β-HC in other organs and in different pathological settings, will provide a full picture on the function and significance of this highly abundant oxysterol.Reagents were purchased from the following sources. Antibodies used are as follows: SREBP1 , SREBP2 , LXRα , LXRβ , phospho-T308 AKT (C31E5E), AKT (11E7) .cis-retinoic acid (Sigma) was used at 50 μM. The LXR antagonist GSK-2033 (Axon Medchem) was used at 500 nM. The LXR agonist GW3965 (Fisher Scientific) was used at 500 nM. PI3K inhibitor, LY294002 , was used at 10 μM. Rapamycin was used at 100 nM and received as a gift from David Sabatini. Methyl-beta-cyclodextrin was purchased from Sigma. All sterols except for custom-synthesized ent-4β-HC (see below) were purchased from Steraloids. C13 glucose was purchased from Cambridge Isotope Laboratories.Drugs used are as follows: Cycloheximide  was used at 10 μg/ml. 9-All sterols were made to 50 mM stocks in ethanol. To deliver the sterols to cells, 1.25 mM sterol was complexed with 25-mM methyl-beta-cyclodextrin and vortexed until the solution was clear. Sterols were added to the media in an indicated concentration and incubation time. Control samples were treated by adding the same volume of ethanol to methyl-beta-cyclodextrin, which then was delivered to cells in the same corresponding volume.5 cells were plates in 6 cm plates. On day 2, media was changed to 1% LDS and 1 g/l glucose DMEM. On day 3; plates were spiked with precomplexed sterols for indicated times, concentrations, and additional compounds.Huh7 cells were maintained on DMEM  supplemented with 10% FBS (VMR) and p/s (Gibco). Lipid-depleted serum (LDS) was made as described . For ass5/well on 6-well collagen-coated plates (Corning) in DMEM supplemented with 10% FBS. Once cells adhere, the media was replaced to Medium 199 (GIBCO) containing 100 nM dexamethasone (Sigma), 100 nM 3,3,5-triiodo-L-thyronine , and Insulin-Transferrin-Selenium (Gibco). Next day, the same media was used without Insulin-transferrin-Selenium to assay insulin, 4β-HC, and inhibitors at indicated times and concentrations.Primary mouse hepatocytes were purchased from the UCSF liver center. The isolation protocol is based on the study by Li, Brown, and Goldstein  and adjuRNA was extracted using the RNeasy kit (Qiagen). One microgram of RNA was reverse-transcribed using Super Script III (Invitrogen). Quantitative PCR was performed using Ssoadvanced (Bio-Rad) in StepOnePlus (ABI). The list of primers is in Cells were harvested with the RIPA buffer supplemented with Phosphatase inhibitor and protease inhibitor (10 mM Tris Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 10 mM Na-PPi, 10 mM Na-Beta-glycerophosphate), sonicated with Bioruptor (Diagenode), and normalized using the BCA kit (Thermo Scientific).5 cells/6 cm plate) in regular media without penicillin streptomycin for 5 h followed by replacement to regular media for 72 h.siRNA ON-TARGET plus smart pool against LXRα (cat# L-003413-00-0005), LXRβ (cat# L-003412-02-0005), or nontargeted siRNA ON-TARGETplus Non-targeting Pool (cat# D-001810-10-05) was purchased from Dharmacon. Five micromolar siRNA was mixed with 5 μl Lipofectamine RNAiMAX (Life Technologies) in Opti-MEM (Gibco). siRNA is added to preplated Huh7 (10Huh7 were plated on a coverslip coated with fibronectin (Corning) and treated as indicated with sterols and drugs. Cells were fixed with paraformaldehyde and stained with 1 μg/ml BODIPY 493/503 for 1 h. Coverslips were mounted with VECTASHIELD with DAPI (Vector Laboratories) and imaged on a spinning disk confocal system (Andor Revolution on a Nikon Eclipse Ti microscope). The BODIPY signal was measured using ImageJ and normalized by the number of nuclei.Liver samples were powdered with a pestle and mortar and lysed in the RIPA buffer. Huh7 cells were also harvested in the RIPA buffer. Five microliters of the samples was used to measure triglyceride using Triglyceride Infinity (Thermo Fisher) or cholesterol using the Amplex red cholesterol measuring kit (Invitrogen) in a clear 96-well sample. The BCA kit (Thermo Scientific) was used for normalization of the protein level. Absorbance and fluorescence were measured by the PerkinElmer Envision Multilabel plate reader.Huh7 cells were seeded at 200K per 6-cm plates. The next day, DMEM media with glutamine, containing 5 mM C13 glucose (Cambridge Isotope Laboratories) and 1% LDS including oxysterols and the LXR agonist were added for 24 h. C12 glucose–treated plates were used as reference. Cells were washed twice with ice-cold PBS and scraped, and pellets were snap-frozen and kept in −80°C for later analysis. Lipid extraction and analysis by LC/MS was performed as described .2 and cervical dislocation.All mouse procedures were performed and approved under the University of California, Berkeley Animal Care and Use Committee. Ten-week-old C57BL/6J male mice were purchased from the Jackson Laboratory and housed for one week in our facility under standard conditions before experiments were performed. Free access to water and chow  was provided throughout this acclimation period. Afterward, mice were placed on a diet with 50 mg/kg/day 4β-HC or LXR agonist GW3965 10 mg/kg/day for 7 days. Powdered 10% by kCal fat diet (Research Diets Inc. #D12450J) was used as the base of each treatment food, forming pellets that were dried overnight at room temperature in a laminar flow hood. After 7 days, mice were euthanized using COLiver samples were fixed using 4% (v/v) paraformaldehyde overnight at 4°C. The next day, samples were cryopreserved using sterile-filtered 30% sucrose (w/v) in dulbecco’s phosphate buffered saline (DPBS) . After 3 days, each sample was placed in a 1:1 30% sucrose:Neg-50 (Richard-Allan Scientific) solution and incubated overnight at 4°C. The samples were frozen on dry ice using undiluted Neg-50 at −50°C and stored at −80°C until sectioning. Sequential 20 μM thick sections were obtained from each sample using a Leica CM3050S cryostat.For nuclei and LD labeling, sectioned tissue was washed three times at room temperature in DPBS for 5 min each. Afterward, DPBS containing 10 μM BODIPY  was placed on the samples and incubated for 30 min at room temperature in the dark. Next, the slides were washed with DPBS twice before incubating in DPBS containing 5 μg/ml DAPI  for 10 min at room temperature in the dark. After DAPI staining, the slides were washed three times in DPBS for 5 min each before being mounted using SlowFade Diamond antifade  and sealing with nail polish overnight. Slides were imaged immediately using a Zeiss LSM710 confocal microscope. Images were developed using the IMARIS (Bitplane) image analysis software suite.ent-Testosterone (1) was prepared as described previously D20 +41.7 ; 1H NMR  δ 5.69–5.68 , 4.15–4.14 , 3.58–3.55 , 2.20–0.78 (m), 1.19 , 0.69 ; 13C NMR  δ 142.7, 128.8, 77.3, 72.5, 56.9, 56.1, 50.2, 42.3, 39.7, 39.5, 36.9, 36.2, 36.0, 35.8, 32.1, 31.8, 28.2, 28.0, 25.4, 24.2, 23.8, 22.8, 22.5, 21.0, 20.5, 18.7, 11.8; IR  3406, 1455, 1366, 1072.To a solution of This article contains R. Z. is cofounder, scientific advisor, and stockholder with Frontier Medicines Corp. All other authors declare that they have no conflicts of interest with the contents of this article.


The synthesized nanoconjugates were found to be able to self-assemble in aqueous condition and form nano-aggregates of an average diameter size of around 35 and 120 nm for β-CD@Cur and (β-CD)2@Cur, respectively. The photophysical properties, water solubility and cell toxicity on rat embryonic cortical neurons of the designed nanoconjugates were investigated and compared to that of Cur alone. The findings revealed that both new nanoconjugates displayed better water solubility and in vitro biocompatibility than Cur alone, thus making it possible to envisage their use as future nano-systems for the prevention or risk reduction of neurodegenerative diseases.Self-aggregation of Curcumin (Cur) in aqueous biological environment decreases its bioavailability and in vivo therapeutic efficacy, which hampers its clinical use as candidate for reducing risk of neurodegenerative diseases. Here, we focused on the design of new Cur- β-Cyclodextrin nanoconjugates to improve the solubility and reduce cell toxicity of Cur. In this study, we described the synthesis, structural characterization, photophysical properties and neuron cell toxicity of two new water soluble β-CD/Cur nanoconjugates as new strategy for reducing risks of neurodegenerative diseases. Cur was coupled to one or two β-CD molecules via triazole rings using CuAAC click chemistry strategy to yield β-CD@Cur and (β-CD) Neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases, as well as spinocerebellar ataxias, are common causes of cognitive impairment and morbidity in older adults ,2. The hCurcumin (Cur) is the major polyphenolic compound of Curcuma longa, an herbaceous plant endemic to South Asia . There hRecently, Cur-based nano-systems, including liposomes, polymeric nanoparticles, micelles, conjugates, solid dispersion, emulsion, peptide carriers and cyclodextrins (CDs) have been proposed as suitable and promising systems for enhancing the overall bioavailability, biodistribution and controlled release of Cur, along with reducing Cur toxic effects, and increased transport across the BBB ,10,11,122@Cur, respectively , HRMS and HPLC techniques. The effect of the covalent binding on Cur disaggregation in aqueous conditions was investigated by spectroscopic measurements. Finally, cytotoxic studies were performed on primary cultures of rat embryonic cortical neurons to evaluate the behavior of the nanoconjugates in a biological environment.In this current study, we explored two new nanoconjugates based on the covalent binding of Cur to either one or two β-CD molecules identified as β-CD@Cur and (β-CD)ectively . The two4 and (β-CD)2@Cur 5, Cur was combined with β-CD via a triazole covalent linkage using a CuAAC click chemistry strategy. To achieve this goal, Cur mono-alkyne 1, Cur di-alkyne 2 and mono-6-azido-β-CD 3 were first synthesized as previously described + calculated 1566.5257, found 1566.5531, [M + Na]+ calculated 1588.5076, found 1588.5293 and [M + 2H]2+ calculated 783.7667, found 783.7844.β-CD@Cur 2@Cur 5: 1H NMR : δ 3.04–3.75 , 3.78 , 3.95–4.07 , 4.47–5.62 , 7.00 , 7.20 , 7.31 , 7.37 , 7.61 , 8.17 . 13C NMR : δ 50.4 (C11), 55.7 (C7-OMe), 59.0 (C6), 59.9 (C6), 61.5 (C1), 70.0 , 71.7 , 72.1 , 72.4 , 73.0 , 81.1 (C4), 81.5 (C4), 82.1 (C4), 83.4 (C4), 101.3 (C1), 102.0 (C1), 111.3 (C6), 112.9 (C9), 118.8 (C3), 124.1 (C10), 125.8 (C13), 127.0 (C5), 142.0 (C12), 144.7 (C4), 149.1 (C8), 150.5 (C7), 193.1 (C2). HRMS (ESI) for C111H162N6O74: [M + 2H]2+ calculated 1382.4627, found 1382.5022, [M + 2Na]2+ calculated 1404.4446, found 1404.4793 and [M + 2K]2+ calculated 1420.4186, found 1420.4437.(β-CD)The size, polydispersity index (PDI) and ζ-potential of the synthesized nanoconjugates were measured following their dilution with ultrapure water (1:200) using DLS . Nanoconjugates were examined by standard capillary electrophoresis cell. The size and ζ-potential were measured at room temperature (25 °C) using a 0.01 absorbance under a scattering angle and a refractive index of 173° and 1.59, respectively.The morphology of the synthesized nanoconjugates in water was monitored via TEM using a negative staining method. In brief, the nanoconjugates were first diluted in distilled water. A 2% ammonium molybdate solution was added with a ratio of 1:1. The mixtures were reserved at room temperature for 3 min. Then, one drop of the nanoconjugates suspension was deposited and dried on a grid type EMS CF200-Cu Formvar carbon-coated copper grid . The nanoconjugates morphology was studied with a Philips CM20 TEM equipped with an Olympus TEM CCD camera at 200 kV.4 and (β-CD)2@Cur 5 nanoconjugates in water at room temperature was studied by comparing their UV–vis absorption, fluorescence emission and fluorescence lifetime spectra with those of free Cur in the same conditions. Briefly, each product was dissolved in D2O. Fluorescence (Φf) quantum yield was determined using Quinine (Quin) solution in acetonitrile as fluorescence standard (Φf = 0.55) . Neurons were incubated at 35 °C in 6% CO2 with day (D) 0 being the day of the preparation of the cells. β-CD, Cur, β-CD@Cur 4 and (β-CD)2@Cur 5 were first dissolved in DMSO , and the solutions were diluted to the desired concentrations in M2 culture medium. On Day 3, the neuronal culture medium was treated with each compound separately at a final Cur concentration of 2–20 µM and incubated for 24 h at 35 °C in 6% CO2. Control cells were treated with the same concentrations of DMSO alone that are not toxic for cells.Cell culture studies were carried out at the Bioavailability–Bioactivity  platform. Primary cultures of cortical neurons were prepared from rat fetuses (day 16–17 of gestation) as previously reported ,32. Cell2. Media was removed and replaced with DMSO followed by incubation while shaking at room temperature for 10 min. The absorbance of formazan was measured at wavelength of 570 nm . Cell viability was expressed as percentage of living cells compared to values obtained from control untreated cells.The neuronal viability of the different molecules was assessed using the 3--2,5-diphenyltetrazolium bromide ) reduction activity assay. MTT solution  was added to each well and the plates were then incubated for 1 h at 35 °C in 6% COp-value greater than 0.05 was regarded as significant.Experiments were performed in triplicate, and results are shown as the mean ± SD, unless otherwise indicated. A variance analysis was done by ANOVA supplemented with a Scheffe’s post hoc test and a 2@Cur self-assembled in water to form nanoparticles with an average diameter size of around 35 and 120 nm, respectively. These nanoconjugates were successfully synthesized using CuAAC click chemistry strategy via a triazole covalent link between Cur and β-CD molecules. The chemical structure and purity of the synthesized nanoconjugates was checked by NMR spectroscopy , ESI-HRMS and HPLC analyses. Furthermore, the β-CD effect on the disaggregation of Cur in aqueous medium and cell cytotoxicity effect on rat cortical neurons were investigated.Two novel nanoconjugates denoted β-CD@Cur and (β-CD)2O revealed that, compared to free Cur, water solubility of the nanoconjugates increased with the number of β-CD resulting in an improvement of the absorption intensity and in the restoration of fluorescence. Secondly, the addition of β-CD reduced significantly the toxicity of Cur illustrated by around 90% and 80% rat cortical neurons viability for (β-CD)2@Cur and β-CD@Cur nanoconjugate, respectively, at 20 µM concentration, compared to only 48% viability with free Cur at the same concentration. These results showed the potential interest of these nanoconjugates as possible new Cur derivatives for application in neurodegenerative diseases prevention. The next step will be to determine the efficacy of curcumin’s neuroprotective effects of these two nanoconjugates, including protection against Aβ cell toxicity, and antioxidative properties using cell and animal models, with the goal towards developing prevention strategies to reduce risk of neurodegenerative diseases. The findings indicated that β-CD covalent binding with Cur led to an improvement of its solubility and photophysical properties in aqueous conditions preventing cytotoxic effects. Firstly, UV–vis absorption and fluorescence emission spectra in D


Lawsonia inermis L. (henna) seeds has been frequently recommended for the improvement of memory in Iranian Traditional Medicine (ITM). In this respect, different fractions of the plant were prepared and evaluated for their in vitro biological assays related to Alzheimer's disease (AD), including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity as well as metal chelating ability and DPPH antioxidant activity. The dichloromethane and ethyl acetate fractions were able to inhibit the BChE selectively with IC50 values of 113.47 and 124.90 μg/mL, respectively, compared with donepezil as the reference drug (IC50 = 1.52 μg/mL). However, all fractions were inactive toward AChE. Phytochemical analysis of the dichloromethane fraction indicated the presence of β-sitosterol (1), 3-O-β-acetyloleanolic acid (2), 3-O-(Z)-coumaroyl oleanolic acid (3), betulinic acid (4), and oleanolic acid (5). The inhibitory activity of isolated compounds was also evaluated toward AChE and BChE. Among them, compounds 2 and 5 showed potent inhibitory activity toward BChE with IC50 values of 77.13 and 72.20 μM, respectively. However, all compounds were inactive toward AChE. Moreover, molecular docking study confirmed desired interactions between those compounds and the BChE active site. The ability of fractions and compounds to chelate biometals  was also investigated. Finally, DPPH antioxidant assay revealed that the ethyl acetate (IC50 = 3.08 μg/mL) and methanol (IC50 = 3.64 μg/mL) fractions possessed excellent antioxidant activity in comparison to BHA as the positive control (IC50 = 3.79 μg/mL).Using  Alzheimer's disease (AD) is characterized as the most common neurodegenerative disease leading to a gradual decrease in memory, cognitive disorders, psychological and behavioral disturbances, and serious problems in daily activities. Also, it accounts for more than 70% of dementia cases in elderly people worldwide . The numβ-amyloid peptides [2+, Cu2+, and Zn2+) [AD is the consequence of multiple etiological factors including genetics, environment, and lifestyle . The exapeptides , dyshomend Zn2+) , calciumnd Zn2+) , and oxind Zn2+)  play impNow, it is clear that ACh levels in the hippocampus and cortex are crucial for the regulation of memory, attention, learning, and motivation . CholineβA plaques [Currently, there is no definite cure for AD, and most available drugs approved by FDA, such as donepezil, galantamine, and rivastigmine, are ChE inhibitors that improve cognitive disorders and the disease symptoms . In this plaques .Lawsonia inermis L., commonly known as “henna” [L. inermis is known for its valuable applications in the cosmetic industry [Trichophyton, Sporotrichum, and Cryptococcus have been recorded in the literature [L. inermis seeds to evaluate different biological activities related to AD including in vitro ChEs inhibitory activity as well as metal chelating ability  and DPPH antioxidant activity. Also, according to the efficacy of dichloromethane fraction, phytochemical analysis led to the isolation and identification of five compounds that have not been previously reported for henna seeds.The development of anti-AD drugs is directly affected by the multifactorial nature of the disease; hence, medicinal plants possessing a wide range of phytochemicals are the potent candidates for the AD drug discovery . Lawsoni “henna” , belongs “henna” . L. inerindustry  and tradindustry . Antidiaterature . Accorditerature . Herein,terature –32, we s1H-NMR and 100.63 MHz for 13C-NMR as well as 2D HMBC, COSY, and HSQC experiments. Silica gel for column chromatography (70–230 and 230–400 mesh) and precoated silica gel F254 (20 × 20 cm) plates for TLC were purchased from Merck. TLC plates were visualized under UV light (254 and 366 nm) as well as spraying anisaldehyde-sulfuric acid, followed by heating at 150°C.Melting points were determined on a Kofler hot stage apparatus and are uncorrected. NMR spectra were recorded on an Avance III spectrometer (Bruker) operating at 400.20 MHz for Acetylcholinesterase , butyrylcholinesterase , acetylthiocholine iodide (ATCI), and 5,5-dithiobis-2-nitrobenzoic acid (DTNB) were purchased from Sigma-Aldrich. Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium hydroxide, and sodium hydrogen carbonate were obtained from Fluka. Solvents for the extraction and column chromatography (CC) were of technical grade and redistilled before use. Deuterated solvents (100 atom % D) were obtained from Sigma-Aldrich.Lawsonia inermis L. were collected from the southern coast of Iran, Hormozgan Province, Bandar Abbas City, and the taxonomic identities of the plant were authenticated by Doctor Ajani, a taxonomist at the Research Institute of Forests and Rangelands, and deposited in the Herbarium of Faculty of Pharmacy, Tehran University of Medical Sciences (voucher specimen no. 7028-TEH).Fresh and healthy fruits of In vitro anti-ChEI activity was performed against acetylcholinesterase  and butyrylcholinesterase  using the modified Ellman's method [μg/mL) and compounds , while obtaining the final ratio of 50/50 DMSO/methanol. Each well consisted of 50 μL potassium phosphate buffer , 25 μL prepared sample as described above, and AChE enzyme (25 μL) with a final concentration of 0.22 units/mL in the buffer. They were preincubated for 15 min at room temperature, and then, 125 μL DTNB (3 mM in buffer) was added to the mixture. Following the addition of 25 μL substrate ATCI (3 mM in water), changes in the absorbance were spectrometrically measured at 405 nm using a UV Unico Double Beam spectrophotometer. In parallel, a blank containing all components without enzyme was used in order to account for the nonenzymatic reaction. A negative control was also used under the same conditions without inhibitor, and donepezil was used as the positive control. The IC50 values were determined graphically from log concentration vs. % of inhibition curves. All experiments were performed in triplicate. BChE inhibition assay was performed in the same method.s method . To obta2+, Cu2+, and Zn2+ ions were obtained from FeSO4.7H2O, CuCl2.2H2O, and ZnCl2, respectively. To investigate the biometal chelating ability, a solution of compound or fraction at distinct concentration (1 mL) was mixed with the metal solution (1 mL) at the same concentration in a 1 cm quartz cuvette, and the mixture was left at room temperature for 30 min. Then, the absorbance of the solution was read in the wavelength range of 260–500 nm [All solutions used in metal chelating study were prepared in methanol. The solutions of Fe0–500 nm .Several concentrations of the test fraction in MeOH were prepared. Aliquots of different concentrations of the fraction (1 mL) were added to the DPPH methanolic solution , and the mixtures were shaken vigorously and left for 30 min at room temperature in the dark. The absorbance was then measured at 517 nm using a UV⁄visible spectrophotometer. The percent scavenging activity was calculated using the following formula:50 (μg/mL), which is obtained from linear regression plot between concentrations of the test fractions and percent inhibitions.In the same procedure, butylated hydroxyanisole (BHA) was used as the positive control. The DPPH radical scavenging activity of the fractions was expressed as IC2 and 5. The crystal structure of apo BChE (PDB ID: 4TPK) was retrieved from the Protein Data Bank (http://www.rcsb.org). The 3D structure of donepezil (positive control) was obtained from the DrugBank database (https://go.drugbank.com). The 2D structures of the desired compounds were drawn on ChemDraw Professional 16.0, and 3D structures were generated using Chem3D suite, saved in .pdb format, and converted to .pdbqt format coordinate by AutoDockTools (ADT). Moreover, for preparation of the pdbqt form of the enzymes, polar hydrogen atoms were added to amino acid residues and Kollman charges were assigned to all atoms using ADT, and the obtained enzyme .pdbqt was used as an input for the Autogrid program. In Autogrid for each atom type, the inhibitor's maps were calculated with 0.375 A° spacing between grid points, and the center of the grid box was placed x = 1.176, y = 11.017, and z = 11.175 for BChE. The dimensions of the active site box were set at 40 × 40 × 40 × A° for BChE [A molecular docking study was carried out using the Autodock 4.2.6 program for compounds for BChE . FlexiblAfter drying the fruits of the plant in the shade and away from the sun, the seeds, a brown cone, were carefully separated from the capsule-like fruits, and then, the skin of the fruits and other plant organs were removed. Next, 400 g seeds were milled using a laboratory-scale mill and extracted with methanol (MeOH) 100% (5 × 2 L) for 72 h at room temperature. The collected extract was concentrated under vacuum at 40°C using a rotary evaporator, and finally, 84.20 g dry extract was obtained, yielding 21.05%. This extract was subjected to silica gel-column chromatography (CC)  and washed with petroleum ether (PE), dichloromethane (DCM), ethyl acetate (EtOAc), and MeOH (4 L per solvent), respectively.The DCM fraction was loaded onto a silica gel vacuum liquid chromatography (VLC) , eluted with a gradient of the PE : DCM, and then DCM : acetone (ACE). Totally, 13 fractions (F1–F13) were combined with the aid of TLC analysis (bands were detected on TLC under UV (254 nm and 360 nm) and spraying anisaldehyde-sulfuric acid, followed by heating at 150°C.1 (4 mg). Fraction F5 ) was subjected to another silica gel CC , eluted with a gradient of the PE : ACE (90 : 10 to 65 : 35) to afford three subfractions (SFs. 5a–5c). Subfraction 5b was further separated on silica gel CC , eluted with PE : ACE (85 : 15) to give compound 2 (10 mg). Fraction F8 ) was separated over a silica gel CC  with a gradient mixture of PE : ACE (80 : 20 to 0 : 100) as eluent, to afford thirteen subfractions (SFs. 8a–8m). Subfraction 8f was triturated with EtOAc to give an insoluble solid, which was recrystallized from MeOH to afford compound 3 (8 mg). Subfraction 8d was loaded onto silica gel CC , eluted with a gradient mixture of n-hexane : ethyl acetate (80 : 20) to give five subfractions (SFs. 8d1–8d5). Two subfractions, 8d1 and 8d3, were recrystallized from MeOH to afford compound 4 (7 mg) and compound 5 (8 mg), respectively.Fraction F4  was applied on silica gel CC  and eluted with a gradient of the PE : ACE (95 : 5 to 80 : 20) to afford six subfractions (SFs. 4a-4f). Subfraction 4d was recrystallized from chloroform to give compound L. inermis seeds was conducted using silica gel column chromatography to isolate and characterize five compounds 1–5 for the first time for henna seeds : colorless needles (4 mg), mp: 134–137°C. 1H NMR  δ = 5.35 , 3.52 , 1.01 , 0.92 , 0.79–0.87 , 0.68 . 13C NMR : colorless crystal; m.p. >250°C. 1H NMR  δ = 5.27 , 4.49 , 2.82 , 2.05 , 0.74, 0.85, 0.86, 0.90, 0.93, 0.94, 1.12 . 13C NMR -Coumaroyl oleanolic acid (3): white powder, m.p. >250°C. 1H NMR : δ = 7.61 , 7.60 , 6.86 , 6.43 , 5.24 , 4.59 , 1.19, 0.99, 0.97, 0.95, 0.95, 0.92, 0.81 . 13C NMR ; see 3-4): white amorphous powder, m.p. >250°C. 1H NMR  δ = 12.07 , 4.69 , 4.78 , 4.28 , 2.96 , 1.65, 0.94, 0.88, 0.88, 0.77, 0.65 . 13C NMR : white amorphous powder, m.p. >250°C. 1H NMR  δ = 12.05 , 5.19 , 4.32 , 3.03 , 2.77 , 1.84 , 1.26, 1.12, 0.92, 0.90, 0.88, 0.74, 0.70 , 0.69 . 13C NMR   .1–5 demonstrated no AChEI activity; however, results related to the selective anti-BChE activity were worth considering. As can be seen in 50 values of 113.47 ± 1.25 and 124.90 ± 1.15 μg/mL, respectively, whereas the other fractions were not active. In the case of isolated compounds, compounds 2 and 5 depicted selective BChE inhibitory activity with IC50 values of 77.13 ± 0.01 and 72.20 ± 0.42 μM, respectively.It should be noted that all fractions and compounds β plaques and senile plaques. It seems that inhibition of BChE can be considered as a therapeutic target in the treatment of AD [Gelsemium elegans and Aglaia odorata, paeonol and hydroperoxy-24-vinylcholesterol exhibited selective BChE inhibitory activity [Ganoderma lucidum were selective inhibitors of AChE [The compensatory role of BChE in late-stage or advanced AD has been fully proven and the enzyme is responsible for the reduction of the ACh levels in the brain. Also, BChE plays an important role in the transformation of nonfibrillary to fibrillar Ant of AD . In thisnt of AD . Terpenent of AD . Among factivity . Also, s of AChE . All theL. inermis has been investigated from different points of view. Amat-ur-Rasool et al. reported that the methanolic extract of henna leaves had strong in vitro AChEI and BChEI activity with IC50 values of 0.33 and 0.41 mg/mL, respectively [in vivo investigation of the ethyl acetate and chloroform extracts of L. inermis leaves at a dose of 25 mg/kg indicated their memory-enhancing potentials using two methods including “without inducing amnesia” and “induction of amnesia” by administration of diazepam as well as the cognitive improvements using behavioral models including elevated plus maze (EPM) and the passive shock avoidance (PSA) paradigm [The effect of ectively . Also, sectively . As repoparadigm .2+, Zn2+, and Cu2+ can increase the toxicity of Aβ plaques by forming coordinate bonds. Also, excessive accumulation of metal ions and interaction with β-amyloid plaques leads to oxidative stress due to an increased formation of reactive oxygen species (ROS). It seems that the biometal chelation hypothesis can be considered as a strong therapeutic tool in the treatment of AD [1–5 was investigated. For this purpose, the UV-visible absorption spectra were recorded in the wavelength range of 260–500 nm and compared with those obtained from the treated solution of fractions or compounds with Fe2+, Zn2+, and Cu2+ ions with final concentrations of 20 μg/mL and 20 μM, respectively. Those findings indicated the desired capability of phytochemicals present in the fractions to form complexes with biometals.Redox-active metals such as Fent of AD . Thus, t2+ ions more strongly than Fe2+ and Zn2+ ions. The absorbance peak of MeOH fraction  at 286.4 nm changed to 284.3 nm after 30 min interaction with Cu2+ ions (The MeOH fraction was able to chelate Cuu2+ ions .μg/mL) showed three absorption peaks at 294.9, 290.7, and 282.1 nm. It was capable of Cu2+ chelating as a peak at 288.5 nm appeared after interaction with Cu2+ ions. The interaction with Fe2+ ions led to a change in the absorption wavelength (288.5 and 297.1 nm). However, the fraction could not chelate Zn2+ ions  showed several absorption peaks at 326.9, 320.5, 316.3, 303.5, 292.8, and 286.4 nm. Interaction of the fraction with Cu2+ ions led to the appearance of new peaks at 307.7 nm, a red shift from 326.9 to 331.2 nm, and a blue shift from 316.3 nm to 314.1 nm. In the case of interaction with Zn2+ ions, a blue shift from 303.5 to 297.1 was clear. Interaction of the sample with Fe2+ ions demonstrated absorption peaks at 318.4, 314.1, 309.9, 301.3, 294.9, 290.7, and 286.4 nm  showed an absorbance peak at 275.7 nm. After the interaction of the fraction with Cu2+ ions, a red shift from 275.7 nm to 288.5 nm was confirmed. When it was treated with Zn2+ and Fe2+ ions, a red shift from 275.7 nm to 282.1 nm was also observed  was able to form chelates with biometals  scavenging activity comparing with butylated hydroxyanisole (BHA) as the standard drug (IC50 = 3.79 ± 0.37 μg/mL). As shown in 50 = 3.08 ± 0.10 μg/mL and 3.79 ± 0.37 μg/mL, respectively) as potent as the positive control. However, both PE and DCM fractions showed lower antioxidant activity than BHA.DPPH is a stable free radical that can accept an electron or hydrogen radical to become a stable molecule. All fractions  of 2 and 5) with BChE (PDB ID: 4TPK), molecular docking experiments were performed using the Glide module (autodock suite). Docking scores in kJ/mol are shown in To gain an insight into the binding interaction of the isolated compounds , 3-O-β-acetyloleanolic acid (2), 3-O-(Z)-coumaroyl oleanolic acid (3), betulinic acid (4), and oleanolic acid (5). Evaluation of compounds 1–5 toward AChE and BChE revealed that compounds 2 and 5 were the most potent and selective inhibitors of BChE with IC50 values of 77.13 and 72.20 μM, respectively. Furthermore, evaluation of the antioxidant and metal chelating ability of fractions and compounds confirmed the capability of L. inermis seeds to be considered in the treatment of AD.In this study, different biological activities of