There is an obvious correlation between ulcerative colitis and colorectal cancer, and the risk of colorectal cancer in patients with ulcerative colitis is increasing. Therefore, the combination therapy of anti-inflammatory and anti-tumor drugs may show promising to inhibit colon cancer. 5-aminosalicylic acid (5-ASA) with anti-inflammatory function is effective for maintaining remission in patients with ulcerative colitis and may also reduce colorectal cancer risk. Histone deacetylase (HDAC) plays an essential role in the progression of colon cancer. Butyric acid (BA) is a kind of HDAC inhibitor and thus shows tumor suppression to colon cancer. However, the volatile and corrosive nature of BA presents challenges in practical application. In addition, its clinical application is limited due to its non-targeting ability and low bioavailability. We aimed to synthesize a novel dual-prodrug of 5-ASA and BA, referred as BBA, to synergistically inhibit colon cancer. Further, based on the fact that folate receptor (FR) is over-expressed in most solid tumors and it has been identified to be a cancer stem cell surface marker in colon cancer, we took folate as the targeting ligand and used carboxymethyl-β-cyclodextrin (CM-β-CD) to carry BBA and thus prepared a novel inclusion complex of BBA/FA-PEG-CM-β-CD.It was found that BBA/FA-PEG-CM-β-CD showed significant inhibition in cell proliferation against colon cancer cells SW620. It showed a pro-longed in vivo circulation and mainly accumulated in tumor tissue. More importantly, BBA/FA-PEG-CM-β-CD gave great tumor suppression effect against nude mice bearing SW620 xenografts.Therefore, BBA/FA-PEG-CM-β-CD may have clinical potential in colon cancer therapy.The online version contains supplementary material available at 10.1186/s12951-021-01064-3. Colon cancer is a common malignant tumor of the digestive tract that tends to occur at the junction of the rectum and sigmoid colon. The pathogenesis of colonic cancer is concerned with living habits, genetic factors, and inflammatory bowel disease , 2. The At present, the treatment of colon cancer mainly includes surgery and drug treatment , 6. The It was reported that 5-ASA could inhibit NF-κB and scavenges free radicals to treat inflammation . It is eThe poor pharmacokinetic characteristics of BBA may limit its application with the traditional dosage form. It was reported that cyclodextrins (CDs) could be used as drug carrier due to the particularity of the cavity , 23, whiMoreover, some studies have shown that cyclodextrins are known to be barely hydrolyzed and only slightly absorbed in passage through the stomach and small intestine. This unique property makes cyclodextrins useful as a targeting carrier to the colon.A folic acid receptor is a glycoinositol-conjugated protein that is highly expressed in a variety of tumor cells, including ovarian, colon, liver, kidney, lung, and breast epithelial malignancies –28. FoliTaking together, this project firstly intended to synthesize the dual-prodrug BBA of 5-ASA and BA through the two-step chemical reaction. Then taking the functional PEG as the coupling agent, FA was connected to CM-β-CD through the amide reaction principle. The novel inclusion complex of BBA/FA-PEG-CM-β-CD was prepared by a saturated aqueous solution method. MTT assay was used to determine the inhibitory effect of BBA/FA-PEG-CM-β-CD on cell proliferation. Flow cytometry was taken to detect the cell cycle and cell apoptosis. Further, we established the colon cancer model in BALB/C nude mice, performed real-time fluorescence imaging analysis to detect the distribution of BBA/FA-PEG-CM-β-CD in vivo, and conducted in vivo pharmacodynamics detection in nude mice to evaluate the tumor inhibitory effect of BBA/FA-PEG-CM-β-CD.2000-NH2 and coumarin-6 were provided by Ruixi Biotechnology . DID was obtained from Keygen Biotechnology  and 5-fluorouracil from Tianjin Jinyao Medicine Pharmaceutical Co., LTD .5-ASA was provided by Runze Bentu Chemical Co., Ltd . CM-β-CD was from Solarbio Life Sciences . Butyryl chloride, butyric anhydride. FA-PEGDulbecco’s Modified Eagle’s Medium (DMEM), streptomycin, and penicillin were bought from Hammer Flew Biochemical . Fetal bovine serum (FBS) was provided by TianHang Biotechnology , and thiazolyl blue tetrazolium bromide (MTT) was bought from Beyotime Biotechnology .BALB/C nude mice (18–25 g) aged 4–6 weeks were from SPF Biotechnology Co, Ltd . Male SPF Kunming mice (19 ± 25 g) were provided by Dashuo Experimental Animal Company . All experimental procedures were approved by the Animal Ethics Committee of Southwest Medical University (No. 20200010) and were performed in strict accordance with the regulations of the Animal Protection and Use Committee of Southwest Medical University.2.The human colon adenocarcinoma cell line CaCo-2, and human colon cancer SW620 cells were purchased from the Shanghai Cell Institute of the Chinese Academy of Sciences. CaCo-2 and SW620 cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C with 5% COThe dual-prodrug of 2-butyryl oxy-5-butylaminyl benzoic acid (BBA) was synthesized as shown in Fig. 3 solution (16 mL) saturated with CO2, a solution of butyryl chloride (0.27 mL) in acetone was added slowly at 0 °C, then the mixture was stirred at room temperature for 4 h [Firstly, it is the synthesis of 2-Hydroxyl-5-Butylamino Benzoic Acid. To 5-aminosalicylic acid (1.31 mmol) in NaHCO for 4 h . After e for 4 h .Secondly, 2-hydroxyl-5-butylaminobenzoic acid (0.546 mmol) was added to butyric anhydride, and a few drops of concentrated sulfuric acid were added slowly . After s2000-NH2 (0.01 mmol) was added into the mixture and stirred at room temperature for 24 h in a dark room, dialyzed against water for 10 h, and freeze-dried to obtain FA-PEG-CM-β-CD [To prepare FA-PEG-CM-β-CD, N-hydroxysuccinimide  and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride  were mixed with CM-β-CD  in ultra-pure water to activate the carboxyl group of CM-β-CD and stirred for 15 min. Then FA-PEG-CM-β-CD .The inclusion complexes of BBA/CM-β-CD or BBA/FA-PEG-CM-β-CD were prepared by the saturated aqueous solution method at 1:2 molar ratio of BBA and CM-β-CD or FA-PEG-CM-β-CD. Briefly, BBA/CM-β-CD or FA-PEG-CM-β-CD dissolved in ultra-pure water (4 mL) was mixed with BBA which was dissolved in methanol, and sonicated for 40 min at 40 °C. After evaporation of the solvent at 35 °C, the product was freeze-dried to obtain BBA/CM-β-CD or BBA/FA-PEG-CM-β-CD , 35.−1.Fourier transformed infrared  was applied to analyze the formation of BBA/FA-PEG-CM-β-CD. Briefly, the specimens were blended with KBr at a ratio of 1:100 (w/w), taking BBA, blank inclusion complex of FA-PEG-CM-β-CD, and the physical mixture of BBA with FA-PEG-CM-β-CD as the controls. Then, KBr discs were prepared under 10,000 psi hydraulic pressure. FTIR spectra were carried out with a resolution of 4 mm/s on KBr pellets with a wavenumber range of 500–4000 cmX-ray diffraction (XRD) analysis was performed for BBA, FA-PEG-CM-β-CD, BBA/FA-PEG-CM-β-CD using X-ray powder diffractometer , where Cu Kα radiation was applied in the 2θ range of 3° − 50°. Thermal gravimetric analysis (TGA) measurements and differential scanning calorimetry (DSC) analysis were performed for the inclusion complex under nitrogen atmosphere by heating the samples at the rate of 10 °C/min from 30 °C up to 600 °C by the synchronous thermal analyzer  . MorpholTo get the encapsulation efficiency  and drug-loading rate , BBA was detected by the high-performance liquid chromatography (HPLC) analysis. Briefly, BBA was quantified on an Agilent ZORBAXSB-C18 column  at 30 °C attached to an Agilent 1260 HPLC system . The mobile phase was mixture of acetic acid (0.1 mol/L) and acetonitrile (v: v = 50: 50) at a flow rate of 1.0 mL/min. It was monitored at 240 nm, and the injection volume was 20 µL. Moreover, the chromatographic conditions were validated through specificity, linearity, precision, and blank recovery. EE and DL were calculated using the formula:Release of BBA from BBA/CM-β-CD and BBA/FA-PEG-CM-β-CD was investigated in vitro using the dialysis method. Briefly, 1 mL of BBA/CM-β-CD, BBA/FA-PEG-CM-β-CD, and free BBA were respectively placed into dialysis bags with a molecular weight cut-off of 3 kDa  and dialyzed at 37 °C against phosphate-buffered saline (PBS) containing 0.5% (w/v) Tween 80 at pH 7.4 with gentle stirring .To evaluate the stability, BBA/FA-PEG-CM-β-CD was placed respectively at high temperature and high humidity conditions. For the high-temperature experiment, the inclusion complex of BBA/FA-PEG-CM-β-CD (50 mg) was placed in a weighing flask at 60 °C for 10 days. The samples were taken respectively at 0 d, 5 d, and 10 d to detect the encapsulation efficiency. If the content of effective ingredients decreased by 5%, the same operation was further conducted at 40 °C.3 saturated solution were respectively placed in the closed container. The samples were taken respectively at 0 d, 5 d, and 10 d to detect the encapsulation efficiency [For the high humidity conditions experiment, BBA/FA-PEG-CM-β-CD inclusion complex (50 mg) was placed openly in a closed container (25 °C) with a relative humidity of 75% ± 5% and 95% ± 5% for 10 d, in which NaCl and KNOficiency , 40.2. BBA, FA-PEG-CM-β-CD, BBA/CM-β-CD, BBA/FA-PEG-CM-β-CD, as well as sodium butyrate (NaB), 5-ASA, the physical mixture of NaB and 5-ASA (NaB + 5-ASA), and the positive control of 5-FU were added to the wells at different concentrations and incubated for 24 h, 48 h and 72 h at 37 °C with 5% CO2. The medium was removed, and cells were washed three times with PBS before incubation with MTT for 4 h at 37 °C. After the medium was removed, 100 μL of DMSO was added to the well for 10 min. The absorbance of the solution was measured at 570 nm by Multimode Reader (Bio Tek Synergy H1) [CaCo-2 and SW620 cells were used as model cells to evaluate the anticancer effects in vitro. Single-cell suspension was prepared with DMEM containing 10% Gibco FBS and 1% penicillin–streptomycin. The cells were seeded in a 96-well plate (3500 cells/well) and incubated respectively for 24 h, 48 h, and 72 h at 37 °C with 5% COergy H1) . The cel5 cells/well) in 24-well plates, and cultured for 24 h. After the original medium was removed, 1 mL of BBA/FA-PEG-CM-β-CD and BBA/CM-β-CD diluent were added separately, placed in an incubator for 2 h. Additionnally, both CaCo-2 cells and SW620 cells were treated with BBA/FA-PEG-CM-β-CD in blank medium containing different concentrations of free folic acid (FA). After incubation, the culture medium was removed, washed thoroughly twice with PBS, and fixed in 4% paraformaldehyde for 30 min, and then staining was performed with 150 μL of DAPI (5 mg/mL) for 5 min. Glycerol was used to seal coverslips, and a confocal laser-scanning microscope was utilized to observe them at an emission wavelength of 477 nm. To quantify cellular uptake, Image J (NIH Image-Pro Plus 6.0) was used [As a drug delivery system, whether BBA/FA-PEG-CM-β-CD inclusion complex could deliver drugs to target cells, is an important index to evaluate the targeting of the drug delivery system. In this study, CaCo-2 cells and SW620 cells as cell models were used to investigate the cell uptake of the inclusion complex. Coumarin 6 (20 μg/kg) was used together with BBA to prepare BBA/CM-β-CD (30 μM) and BBA/FA-PEG-CM-β-CD (30 μM), which could emit green fluorescence. CaCo-2 cells and SW620 cells in good growth condition and logarithmic growth phase were plated on the sterile coverslips (3 × 10was used .6 cells/well) and BBA (30 μM), BBA/FA-PEG-CM-β-CD (30 μM), and BBA/CM-β-CD (30 μM) was added to the well and incubated for 48 h at 37 °C with 5% CO2.SW620 cells were taken as the cell model to investigate the cell apoptosis and cell cycle effects of BBA/FA-PEG-CM-β-CD by flow cytometry technology. SW620 cells were seeded in the 6-well plates  and 25 μL of propidium iodide (20 ×) in 0.5 mL of buffer were added . FinallyTo determine the potential toxicity in vivo and guide the analysis of anti-tumor efficacy in vivo, an acute toxicity study of BBA/FA-PEG-CM-β-CD was conducted in Kunming mice by a single oral administration of the tested formulations, and the treated mice were observed for 14 d.After fed adaptively for 5 d, the animals were randomly divided into 8 groups (n = 5) following as NaB (2.5 mmol/kg), 5-ASA (1.25 mmol/kg), FA-PEG-CM-β-CD (0.096 mmol/kg), BBA (1.25 mmol/kg), BBA/CM-β-CD (0.096 mmol/kg), BBA/FA-PEG-CM-β-CD (0.096 mmol/kg) with saline as negative control and 5-FU as a positive control. All animals were given a single oral gavage of 0.2 mL of different dosage forms, and their body weight was monitored every day. Meanwhile, the toxicity and mortality of all animals were observed every day. The conventional changes such as the cases of the diet, urine, and feces were recorded. On the 15th day after treatment, the blood of each group was collected from the heart and centrifuged at 1,000 × g for 10 min to collect serum. An automated biochemical analyzer was used to detect serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr). Meanwhile, the heart, liver, lung, and kidney were quickly excised, washed with normal saline, and weighed. The visceral index is estimated according to the following formula as Viscera Index (%) = Viscera Weight / Body Weight × 100. And then, all the tissues were fixed with 10% formalin and stained with hematoxylin & eosin (H&E) for pathological analysis.SW620 tumor-bearing mice were randomly divided into 3 groups (n = 5). BBA, BBA/CM-β-CD, and BBA/FA-PEG-CM-β-CD were administered by gavage at a dose of 0.366 g/kg. The blood was collected respectively from the heart at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 h after administration. The whole blood was quickly centrifuged at 2000 × g for 5 min, and the upper plasma was precisely aspirated. Then 0.35 mL of methanol was added, vortexed for 2 min, and centrifuged at 3350 × g for 15 min. The supernatant was used to detect butyric acid by HPLC . The phaThe nude mice bearing SW620 xenografts were treated as same as that in the pharmacokinetics experiments. The animals were sacrificed at 1, 3, 8, and 12 h after oral administration. Samples of blood, heart, spleen, liver, kidneys, lung, and tumor tissues were collected quickly (n = 5) to detect butyric acid using the HPLC method.Meanwhile, the in vivo small animal imaging system  was used to investigate the in vivo distribution and targeting ability of BBA/FA-PEG-CM-β-CD. The hydrophobic infrared fluorescent dye DID was encapsulated into the inclusion compound (0.8 μg DID per mice) to form DID-BBA/CM-β-CD and DID-BBA/FA-PEG-CM-β-CD. The nude mice bearing SW620 xenografts were randomized into four groups (n = 5) and given respectively DID-BBA/CM-β-CD and DID-BBA/FA-PEG-CM-β-CD with saline and free DID as controls. Based on the previous work, the animals were anesthetized at 3, 8, and 12 h, and visualized at an excitation wavelength of 644 nm and an emission wavelength of 665 nm. Images of each mouse were normalized to the same intensity range of lowest and highest values, and Carestream MI  was used to quantify the fluorescence intensity.After the in vivo small animal imaging detection, blood was immediately taken from the heart and the nude mice were sacrificed. The whole blood was centrifuged at 1500 × g for 5 min and placed in a 2 mL EP tube. The heart, liver, spleen, kidney, lung, and tumors of nude mice in each group were quickly separated, and the fluorescence intensity of the blood, organs, and tumor tissues was analyzed with the IVIS imaging system .3. The animals were weighed before treatment, and the measurement was continued every 2 days. The two axes of the tumor were measured with a vernier micrometer . Tumor volume (mm3) was calculated as ½ (L × W2). Anti-tumor activity was evaluated by tumor growth inhibition (TGI), which was the mean tumor weight (MTW) of the treated group (TG) relative to the saline-treated control group (CG) on day 15, as calculated according to the formula [TG − MTWCG) / MTWCG × 100.Ten days after tumor transplantation, the SW620 tumor-bearing mice were randomly divided into 8 groups with 5 mice in each group when the tumors reached about 100 mm formula . TGI (%)15 days post-administration, all nude mice were sacrificed. The tumor tissues were collected, stained with H&E, and observed under an inverted microscope (400 ×). In addition, the TUNEL test was performed to detect cell apoptosis . MeanwhiThe results were stated as mean ± standard deviation (SD) and analyzed utilizing GraphPad Prism 6.01 . One- or two-way analysis of variance was used to statistically compare various groups. The p-value of < 0.05 was considered as a significant difference between groups. The higher significance level was set at P < 0.01.1H-NMR and 13C-NMR spectra of 2-hydroxy-5-butylamino benzoic acid that confirmed the successful synthesis of it. Characteristic peaks of 2-hydroxy-5-butylamino benzoic acid appeared at (g) and (h). Its NMR data were: 1H NMR : δ 10.97 , 9.84 , 8.10 , 7.64 , 6.88 , 2.24 , 1.58 , 0.90 . 13C-NMR : δ 172.44 (s), 171.39 (s), 157.32 (s), 131.54 (s), 127.82 (s), 120.60 (s), 117.42 (s), 112.93 (s), 39.00 (s), 19.05 (s), 14.25 (s).Additional file 1H NMR  δ 9.29 , 8.29 , 7.96 , 7.08 , 2.54 , 2.36 , 1.70 , 0.98 . 13C NMR  δ 172.79 (s), 172.48 (s), 166.18 (s), 147.33 (s), 138.60 (s), 125.35 (s), 125.12 (s), 123.10 (s), 39.90 (s), 36.73 (s), 30.30 (s), 19.90 (s), 19.11 (s), 14.35 .Additional file 1H NMR spectra of CM-β-CD (A), FA-PEG-NH2 (B), and FA-PEG-CM-β-CD (C). The NMR data of FA-PEG-CM-β-CD were: 1H NMR  δ 8.67 , 8.28 , 7.59 , 6.69 , 5.24 , 4.23 , 4.03 , 3.79 , 2.79 , 1.99 , 1.81 , 1.28 , 1.09 , 0.98 .Additional file −1 and 1653 cm−1 responded to N–H and C = O of the amide bonds in FA-PEG-CM-β-CD (a). The characteristic absorption peak of the conjugated N–H around 3499 cm−1 overlapped with the hydroxyl group (O–H) in the spectra of FA-PEG-NH2 (c). Peaks due to vibrations of the conjugated O–H bond and C = O bond was detected around 3416 cm−1 and 1599 cm−1 in the spectra of CM-β-CD (d). These results further proved that FA-PEG-CM-β-CD was successfully synthesized.The infrared spectrum was shown in Additional file −1 in the spectra of BBA (a), and a strong absorption peak of benzene ranged near 1699 cm−1. The absorption peaks of BBA and FA-PEG-CM-β-CD (b) did not simply overlap in the infrared spectrum of the BBA/FA-PEG-CM-β-CD (d). Some characteristic peaks of BBA disappeared or weakened, in which the N–H stretching vibration peak near 3295 cm−1 disappeared, and a peak due to vibrations of the conjugated C–O–C bond around 1103 cm−1 was weakened in the spectra of FA-PEG-CM-β-CD (b). It was concluded that BBA and FA-PEG-CM-β-CD successfully formed the inclusion compound of BBA/FA-PEG-CM-β-CD.The infrared spectra were shown in Fig. The XRD pattern was shown in Fig. DSC results of BBA (a), FA-PEG-CM-β-CD (b), the mixture of BBA with FA-PEG-CM-β-CD (c), and BBA/FA-PEG-CM-β-CD (d) were shown in Fig. As shown in Fig. As shown in Fig. The in vitro accumulative release (%) of BBA/FA-PEG-CM-β-CD was investigated by dialysis. As shown in Fig. Through detecting the encapsulation efficiency of BBA/FA-PEG-CM-β-CD under high temperature and high humidity conditions, it could be seen Fig. H that asMTT assay was used to investigate the cell-growth inhibition effects of BBA/FA-PEG-CM-β-CD at various concentrations against CaCo-2 and SW620 cells at 24 h, 48 h, and 72 h post-treatment Fig. A.Fig. 3TCompared with NaB, the dual-prodrug BBA showed some cell-proliferation inhibition effect on both CaCo-2 and SW620 at the time points of 24 h, 48 h, and 72 h (P < 0.0001). Meanwhile, as prediction the treatment of 5-FU (20 μg/mL) drastically enhanced the cell proliferation inhibition rate compared with the BBA group ( P < 0.0001). BBA/FA-PEG-CM-β-CD showed much better inhibitory effects than BBA, BBA/CM-β-CD. The proliferation inhibition effect of BBA/FA-PEG-CM-β-CD on SW620 cells was better than that on CaCo-2 cells, so we took SW620 cells as the cell model for subsequent tumor-relative evaluation.As shown in Fig. As shown in Fig. To fully understand the inhibitive effect mechanism of BBA/FA-PEG-CM-β-CD on the SW620 cells, we further performed cell apoptosis and cell cycle assays. As shown in Fig. As shown in Fig. We examined the in vivo toxicity of BBA/FA-PEG-CM-β-CD in normal Kunming mice. It was found that the hair color, diet, and activities of all mice remained normal. No abnormal behavior, toxic symptoms, bodyweight loss, and death were observed after all the treatments except the 5-FU group. As shown in Table As shown in Table In addition to the viscera index, we further conducted serum biochemistry analyses of ALT, AST, BUN, and Cr to assess the potential toxicities of BBA/FA-PEG-CM-β-CD to the liver and kidney of mice. As shown in Table We also performed histological studies of the heart, liver, kidney, and lung after treatment with the different formulation mentioned above. At least three sections for each mouse from each group (n = 5) were randomly and blindly analyzed by a pathologist who was blinded to the experimental protocol. The experimental results were shown in Fig. 2 modification did not affect the metabolism of BBA/CM-β-CD. Pharmacokinetic parameters  and 13C-NMR spectra (B) of 2-hydroxy-5-butylamino benzoic acid in DMSO. Figure S2. Structural characterization of BBA. 1H-NMR spectra (A) and 13C-NMR spectra (B) of BBA in (CD3)2CO. Figure S3. Structural characterization of FA-PEG-CM-β-CD. 1H-NMR spectra of CM-β-CD (A), FA-PEG-NH2 (B), and FA-PEG-CM-β-CD (C). Fourier transform infrared spectra (D) of FA-PEG-CM-β-CD (a), mixture of FA-PEG-NH2 and CM-β-CD (b), FA-PEG-NH2 (c), and CM-β-CD (d). Figure S4. Particle size distribution of BBA/FA-PEG-CM-β-CD (DOCX 35712 KB)


The compound was obtained by in-house synthesis. The crystals have triclinic symmetry at 173 K. The crystal structure is built up by Na—O chains, which arrange the anions in polar/non-polar double layers.The crystal structure of a DMSO monosolvate monohydrate of a previously unknown monosodium salt of the industrial intermediate Pigment Red 52 (P.R.52) with the formula Na 2[C18H11ClN2O6S], is an industrially produced hydrazone-laked pigment. It serves as an intermediate in the synthesis of the corresponding Ca2+ and Mn2+ salts, which are used commercially for printing inks and lacquers. Hitherto, no crystal structure of any salt of Pigment Red 52 is known. Now, single crystals have been obtained of a dimethyl sulfoxide solvate hydrate of the monosodium salt of Pigment Red 52, namely, monosodium 2-[2-hydrazin-1-yl]-5-chloro-4-methylbenzenesulfonate dimethyl sulfoxide monosolvate monohydrate, Na+·C18H12ClN2O6S−·H2O·C2H6OS, obtained from in-house synthesized Pigment Red 52. The crystal structure was determined by single-crystal X-ray diffraction at 173 K. In this monosodium salt, the SO3− group is deprotonated, whereas the COOH group is protonated. The residues form chains via ionic interactions and hydrogen bonds. The chains are arranged in polar/non-polar double layers.Pigment Red 52, Na The sixth coordination site is occupied by an O atom of a sulfonate group of a neighbouring anion, generated by the symmetry operation −1 + x, y, z. The coordination polyhedron is a distorted octahedron. The crystal packing is characterized by chains built via Na—O coordinations, running along the a-axis direction  with the shortest distance C6⋯C12 of 3.303 (5) Å. The N–NH unit is stacked above the naphthyl-COOH group with the shortest distance N1⋯C21  of 3.304 (4) Å benzenesulfonato]bis(dimethylformamide)calcium diazenyl]naphthalene-2-carboxylato)bistetrakis(aqua)didysprosium(III) N,N-dimethylformamide solvate, bis{3-oxido-4-[diazenyl]naphthalene-2-carboxylato}bistetrakis(aqua)dieuropium(III) N,N-dimethylformamide solvate, bis{3-oxido-4-[diazenyl]naphthalene-2-carboxylato}bistetrakis(aqua)diterbium(III) N,N-dimethylformamide solvate, bisdiazenyl]naphthalene-2-carboxylato)bistetrakis(aqua)disamarium(III) N,N-dimethylformamide solvate  was dissolved with sodium hydroxide (6.4 g) in water (500 ml). The temperature was set at 278 K and concentrated hydrochloric acid (40 ml) as well as sodium nitrite (7.2 g) in water (100 ml) were added. The suspension was stirred for 30 min. The suspension was treated with amidosulfonic acid until all excess nitrous acid was destroyed. The suspension was then added dropwise to a solution of β-oxynaphthoic acid  with NaOH (20.1 g) in water (550 ml). The pH was kept at alkaline conditions, around 11 to 9, maintained by the addition of 2 M NaOH solution as required, and the temperature was maintained at 278 K. When the dropwise addition of the suspension was finished, the solution was allowed to accommodate to room temperature and subsequently heated to 353 K for half an hour. The red suspension was then neutralized with 2 M HCl, filtered off and the obtained red powder was washed with water and dried at 323 K. The yield of the crude product was about 98%, but X-ray powder diffraction revealed the presence of some sodium chloride as impurity.2-Amino-5-chloro-via the gas phase. Single crystals of the title compound were picked from the solution.The crude in-house synthesized P.R.52 (0.59 g) was dissolved in DMSO (60 ml). The solution was transferred to a glass vessel, which in turn was placed into a further, larger vessel with water (100 ml). The outer vessel was closed with a plastic lid and stored for 20 days at room temperature, allowing the water to diffuse into the DMSO Uiso(H) = 1.2Ueq(C) or with Cmethyl—H = 0.98 Å and with Uiso(H) = 1.5Ueq(C). The methyl group attached to the phenyl ring was allowed to rotate but not to tip. The H atoms bonded to N and O were found in the difference-Fourier synthesis and freely refined.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021002577/yk2145sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021002577/yk2145Isup2.hklStructure factors: contains datablock(s) I. DOI: 2068733CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Genkwa Flos. Through this method, we discovered the toxicity was enhanced through the formation of a supramolecular complex from Genkwa Flos/glycyrrhizic acid. The morphology and size distribution of the self-assembled nanoparticles were characterized by scanning electron microscopy and dynamic light scattering Furthermore, a total of 58 constituents  consisted from the supramolecular complex were identified through accurate-mass measurements in full-scan MS/data-dependent MS/MS mode. Based on the hydrophobic interaction of glycyrrhizic acid with yuanhuacine (one of main ingredients from Genkwa Flos), the supramolecular self-assembly mechanism was revealed with proton nuclear magnetic resonance (1H-NMR) and NOESY 2D NMR. The toxicity of Genkwa Flos and Genkwa Flos/glycyrrhizic acid supramolecular complex were compared through in vitro studies on L-02 cells using a 3--2,5-diphenyltetrazolium bromide (MTT) assay; and 4′,6-diamidino-2-phenylindole (DAPI) staining was performed to further confirm the enhancement inhibition of Genkwa Flos/glycyrrhizic acid supramolecular complex than Genkwa Flos. This study provides fundamental scientific evidence of the formation of a self-assembled phytochemical supramolecular when Genkwa Flos and glycyrrhizic acid are combined, enabling to understand their clinical incompatibility and contraindication.Researchers often focus on the mechanisms of synergistic agents, a few explore drug combinations that enhance toxicity, while few have studied the internal mechanism of compatibility enhancement in chemical level. Herein, we present a comprehensive analysis based on ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) and a self-assembled supramolecular strategy, which reveals the toxicity-enhancing essence of glycyrrhizic acid originated in licorice when combined with  This study provides fundamental scientific evidence of the presence of a Genkwa Flos/glycyrrhizic acid self-assembled phytochemical supramolecular, enabling better understanding of their clinical incompatibility and contraindication.In this study, we utilized UHPLC-Q-Orbitrap HRMS to analyze the composition of a self-assembled supramolecular structure. In combination with proton nuclear magnetic resonance . Glycyrrhizic acid was supplied by Shanghai Yuanye Bio-Technology Co., Ltd. , with the purity greater than 98%. MS-grade methanol was purchased from Merck . Ultrapure water was prepared using a Milli-Q Integral 5 water purification system .Genkwa Flos (50 g) was packed in a nonwoven bag  and transferred into a 1000-ml round-bottom flask. Water (500 ml) was added and then heated for 1 h under reflux. The filtrate of the Genkwa Flos solution was concentrated using a Rotavapor R-215 rotary evaporator  to a volume of 50 ml. The solution was then transferred to a separatory funnel and extracted with 50 ml of ethyl acetate three times. The ethyl acetate extract was lyophilized using a Martin Christ Beta 2–8 LD plus freeze dryer . The resulting Genkwa Flos extract powder (25 mg) was combined with 20 mg of glycyrrhizic acid in a 100-ml round-bottom flask. Water (10 ml) was added, and then extracted with reflux for 1 h to obtain a sample of Genkwa Flos/glycyrrhizic acid self-assembled complex. In addition, 25 mg of the ethyl acetate extracted Genkwa Flos powders was extracted separately as a parallel control for morphology characterization, UHPLC-Q-Orbitrap HRMS analysis and cell experiments. All samples prepared were stored in the dark at 4 °C.Genkwa Flos and the Genkwa Flos/glycyrrhizic acid self-assembled complex were characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The yuanhuacine/glycyrrhizic acid self-assembled complex was analyzed by 1H-NMR and NOESY 2D NMR.The morphology and size distribution of the 18 column  at a flow rate of 0.3 ml/min and maintained at 35°C. The mobile phase was composed of a water solution (A) and methanol (B). The elution conditions were set as follows: 0–3 min, 10–70% B; 3–10 min, 70–90% B; 10–15 min, 90% B. A sampling volume of 2 μl was injected for each run. The chromatograph was coupled with a Thermo Scientific Q Exactive quadrupole-Orbitrap mass spectrometer . Positive electrospray ionization (ESI+) mode was used, with a spray voltage of 3.5 kV. The capillary gas heater temperature was set to 320°C. The flow rates of sheath gas, auxiliary gas, and sweep gas were 40, 10, and 1 (in arbitrary units), respectively. Full-scan data within the range of m/z (mass-to-charge ratio) 50–750 were acquired at a mass resolution of 70,000 FWHM defined for m/z 200 at a scan rate of 3 Hz. Data-dependent MS/MS (dd-MS2) scan mode was set at a mass resolution of 17,500 FWHM. The stepped normalized collision energies (NCEs) were 20, 40, and 60 eV. The automatic gain control (AGC) target value was set at 1 × 105, with a maximum injection time of 50 ms. Data acquisition and processing were carried out using Xcalibur version 2.3 and TraceFinder version 4.1 software .UHPLC separation was performed on a Thermo Scientific UltiMate 3,000 liquid chromatographic system . Chromatographic separation was conducted on a Waters ACQUITY UPLC BEH C2 at 37°C. The Genkwa Flos and Genkwa Flos/glycyrrhizic acid self-assembled complex were dissolved in deionized water and added at required concentrations to the cell culture. Cells incubated without any additions served as the control.L-02  was obtained from the Chinese Academy of Medical Sciences and Peking Union Medical College. The cell lines were maintained in RPMI-1640 supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin  under a humidified atmosphere containing 5% COGenkwa Flos and Genkwa Flos/glycyrrhizic acid self-assembled complex were evaluated on L-02 cell lines in vitro using the MTT method. In short, cells growing in the logarithmic phase were seeded into 96-well plates at a density of 3 × 103 cells/well and incubated for 24 h. Then, each well was exposed to different concentrations of the tested samples of Genkwa Flos and Genkwa Flos/glycyrrhizic acid self-assembled complex (100 μl) and incubated for 48 and 72 h at 37°C with 5% CO2, respectively. Later, MTT solution  was added to each well and incubated for another 4 h. After removing the supernatant medium, 150 μl of dimethyl sulfoxide (DMSO) was added. The optical density (OD) values of the studied samples were measured at 490 nm with a plate reader . Wells without drugs were used as blanks. The cell viability (%) was calculated with the following equation:The cytotoxicities of % cell viability = (Sample group OD - Blank group OD)/(Control group OD - Blank group OD) × 100% 4 cells/well). After incubation for 24 h at 37°C with 5% CO2, 0.15625 mg/ml of Genkwa Flos and Genkwa Flos/glycyrrhizic acid self-assembled complex were added to each well, and the plate was incubated for another 72 h. After discarding the cell culture medium, they were washed three times with cold PBS, fixed with 4% paraformaldehyde for 10 min, washing three times with cold PBS again, then stained with 4’,6-diamidino-2-phenylindole  for 3 min in the dark. Cell nuclei were observed under a fluorescence microscope.L-02 cells in the logarithmic growth phase were seeded in 12-well plates  of three replicates. Statistical analysis was performed by SPSS software (version 20.0) to analyze variance. Independent samples Genkwa Flos and glycyrrhizic acid, we discovered that Genkwa Flos and glycyrrhizic acid could self-assemble into a hydrogel in an aqueous solution, which was confirmed by the inverted test tube method . The MassBank of North America (MoNA) was also used to identify the compounds in the Genkwa Flos/glycyrrhizic acid self-assembled complex. The total ion current chromatogram (TICC) of the Genkwa Flos/glycyrrhizic acid self-assembled complex in the ESI+ mode is shown in In this study, a total of 58 constituents were tentatively identified in the Genkwa Flos/glycyrrhizic acid self-assembled complex, including yuanhuacine, yuanhuadine, yuanhuafine, yuanhuatine, yuanhuapine, genkwadaphnin, daphne diterpene ester-7, and genkwanine H  and yuanhuadine (C32H42O10), respectively. A protonated molecule of yuanhuacine ([M+H]+) with a m/z of 649.3001 was detected in the positive ion mode. The protonated molecule lost a C17H22O4 moiety, forming a fragment ion [M+H–C17H22O4]+ at m/z 359.1476. By further losses of H2O and CO, product ions [M+H–C17H22O4–H2O]+ at m/z 341.1381 [M+H–C17H22O4–H2O–CO]+ at m/z 313.1431, [M+H–C17H22O4–2H2O]+ at m/z 323.1273 and [M+H–C17H22O4–2H2O–CO]+ at m/z 295.1325 were observed. As for yuanhuadine, a protonated molecule [M+H]+ at m/z 587.2848 was detected in the positive ion mode. By eliminating H2O moieties, fragment ions [M+H–H2O]+ at m/z 569.2275 and [M+H–2H2O]+ at m/z 551.2692 were generated. Yuanhuadine could also produce fragment ions at m/z 509.2544 [M+H–C2H2O–2H2O]+ and at m/z 491.2425 [M+H–C2H2O–H2O]+ by losing C2H2O and H2O. The protonated molecule lost C12H14O, H2O, and CO moieties, resulting in fragment ions at m/z 359.1486 ([M+H–C12H14O–3H2O]+), m/z 341.1381 ([M+H–C12H14O–4H2O]+), m/z 331.1556 ([M+H–C12H14O–3H2O–CO]+), m/z 323.1274 ([M+H–C12H14O–5H2O]+), m/z 313.1424 ([M+H–C12H14O–4H2O–CO]+), and m/z 295.1327 ([M+H–C12H14O–5H2O–CO]+). The possible fragments for yuanhuacine and yuanhuadine are proposed in Diterpenoids were the major bioactive compounds identified in the wanine H . DiterpeGenkwa Flos/glycyrrhizic acid self-assembled complex, including genkwanin, hydroxygenkwanin, genkwanin-5-O-β-D-primeveroside, genkwanin-5-O-β-D-glucoside, etc.  originated from the loss of CH3. Fragment ions at m/z 167.0339 were produced by RDA fragmentation in the C-ring of formononetin, which further eliminated the CH3 and CO, resulting in a fragment ion at m/z 124.0156. Another example is genkwanin-5-O-β-D-glucoside, which exhibited a protonated molecule [M+H]+ at m/z 447.1280 in the positive ion mode. By losing the glucose, CH3, CO, and H2O moieties or undergoing the RDA reaction, a series of product ions could be produced such as m/z 285.0757, 271.0596, 242.0571, 127.0387 and 109.0280. The proposed fragmentation patterns of diosmetin, apigenin, oroxylin, wogonin, calycosin, etc are shown in Flavonoids are one of the largest classes of small molecular bioactive compounds in plants. A total of 35 flavonoids were identified in the de, etc. . The maiGenkwa Flos/glycyrrhizic acid self-assembled complex. The phenylpropanoids identified in this self-assembled complex included chlorogenic acids, neochlorogenic acid, cryptochlorogenic acid, 7-hydroxycoumarin, and isodaphnoretin. An investigation of their MS fragmentation patterns revealed common neutral losses of CO, CH3, and H2O. For example, isodaphnoretin exhibited a protonated molecule [M+H]+ at m/z 353.0652 in the positive ion mode. The protonated molecule lost a CH3 moiety, forming a fragment ion [M+H−CH3]+ at m/z 338.0417, which continued to lose a coumarin moiety to yield a fragment ion [M+H−CH3–C9H3O4]+ at m/z 163.0387. Due to consecutive losses of CO and OH, the ion at m/z 338.0417 produced more fragment ions at m/z 310.0467 ([M+H−CH3–CO]+), m/z 282.0517 ([M+H−CH3–2CO]+), and m/z 265.0488 ([M+H−CH3–2CO–OH]+). Likewise, the ion at m/z 321.0381 showed fragment ions at m/z of 177.0181, 161.0240, and 133.0285. Other fragments at m/z 208.0365 ([M+H−C9H5O2]+), 192.0417 ([M+H−C9H5O3]+), 146.0362 ([M+H−C10H7O5]+) and m/z 135.0441 ([M+H−CH3–C10H3O5]+) were also observed in the product ion spectrum. Some constituents belonging to the category of phenylpropanoids were also identified in the 10H13N5O5, had a protonated molecule [M+H]+ at m/z 284.0987 . The most intense product ion at m/z 152.0568 ([M+H–C5H8O4])+ was generated through the loss of a deoxyribofuranose, which was subjected to a further loss of NH3, producing a fragment ion at m/z 135.0302 ([M+H–C5H8O4–NH3]+). In addition, a fragment ion ([M+H–C5H8O4–CH2N2]+) at m/z 110.0350 was the product ion that resulted from the loss of imidazole from the ion m/z 152.0568. The proposed fragmentation patterns of adenine, guanosine, iso-guanosine, and adenosine are shown in Some nucleosides were identified in the positive ion mode, including adenine, guanosine, iso-guanosine, and adenosine. Guanosine, which has a formula of CGenkwa Flos/glycyrrhizic acid self-assembled complex. L-leucine, which has an elemental composition of C6H13NO2, displayed a protonated molecule [M+H]+ at m/z 132.1018 in the positive ion mode -3-ethyl-2-methylheptane. Another characteristic fragment ion [M+H–H2O–C10H20–C3H4]+ at m/z 231.1741, was produced by the loss of one C3H4 molecule from m/z 271.2055. Due to consecutive losses of CH2, the ion at m/z 231.1741 produced more fragment ions at m/z 217.1573 ([M+H–H2O–C10H20–C4H6]+), m/z 203.1431 ([M+H–H2O–C10H20–C5H8]+), and m/z 189.1277 ([M+H–H2O–C10H20–C6H10]+). The proposed fragmentation pattern of trigonelline is shown in Additional constituents were identified in the positive ion mode, including terpenoids, alkaloids, aliphatic aldehydes, and essential oils. As shown in Genkwa Flos/glycyrrhizic acid self-assembled complex, yuanhuacine, which is considered as both a toxic and an active compound [, as shown in δ = −0.01 ppm) and Glu H-1″  increased. Moreover, the chemical shifts of the H-20 in yuanhuacine moved low field from 3.50  of yuanhuacine correlated with the Glu-OH (δ 3.34 ppm) of GA; the hydrogens on H-20 (δ 3.55 ppm), and H-14 (δ 4.94 ppm) of yuanhuacine correlated with the Glu H-1′ (δ 4.48 ppm) of GA; and the hydrogen on Glu H-1″ (δ 4.38 ppm) of GA correlated with the H-7 (δ 3.58 ppm) of yuanhuacine. ChemBio 3D was used to predict the optimal conformation for the complex structure, as shown in In addition, NOESY 2D NMR spectra were used to ensure the conformation of the complex units as well as the self-assembly mechanism . In agrein vitro cytotoxicities of Genkwa Flos and the Genkwa Flos/glycyrrhizic acid self-assembled complex were evaluated on L-02 cells using the MTT assay. The cell viability of Genkwa Flos and the Genkwa Flos/glycyrrhizic acid self-assembled complex are shown in Genkwa Flos/glycyrrhizic acid self-assembled complex was more cytotoxic against L-02 cells than Genkwa Flos with increasing concentration at 48 and 72 h, which was consistent with the increase in the dissolution of yuanhuacine, a toxic component, detected in HPLC (The  in HPLC .Genkwa Flos and the Genkwa Flos/glycyrrhizic acid self-assembled complex for 72 h. As shown in Genkwa Flos and the Genkwa Flos/glycyrrhizic acid self-assembled complex groups were significantly less, especially with those exposed to the Genkwa Flos/glycyrrhizic acid self-assembled complex group. The contours of some cells became irregular and the nuclear fluorescence was weak. Thus, the results indicated that Genkwa Flos and the Genkwa Flos/glycyrrhizic acid self-assembled complex could induce apoptosis in L-02 cells, but that the Genkwa Flos/glycyrrhizic acid self-assembled complex induced cell apoptosis more significantly.To further study their mechanism of growth inhibition, DAPI staining was performed after treating L-02 cells with 0.15625 mg/ml of Genkwa Flos. The morphology and size distribution of the Genkwa Flos/glycyrrhizic acid self-assembled complex were characterized by SEM and DLS. In total, 58 compounds were identified within the supramolecular complex using accurate mass analysis, including eight diterpenoids, 35 flavonoids, five phenylpropanoids, four nucleosides, two amino acids, and four additional compounds. Their MS fragmentation pathways were further investigated. The formation mechanism of the yuanhuacine/glycyrrhizic acid self-assembled complex was fully investigated through 1H-NMR and NOESY 2D NMR analysis. This study provides fundamental scientific evidence of the presence of a self-assembled phytochemical supramolecular resulting from the combination of Genkwa Flos and glycyrrhizic acid, enabling better understanding of their clinical incompatibility and contraindication.UHPLC-Q-Orbitrap HRMS and supramolecular self-assembly were utilized to reveal the toxicity-enhancing essence of glycyrrhizic acid on


Castanopsis cuspidata var. sieboldii stems was selected as an experimental source for novel acetylcholinesterase (AChE) inhibitors. Five compounds were isolated from the extract by activity-guided screening, and their inhibitory activities against butyrylcholinesterase (BChE), monoamine oxidases (MAOs), and β-site amyloid precursor protein cleaving enzyme 1 (BACE-1) were also evaluated. Of these compounds, 4′-O-(α-l-rhamnopyranosyl)-3,3′,4-tri-O-methylellagic acid (3) and 3,3′,4-tri-O-methylellagic acid (4) effectively inhibited AChE with IC50 values of 10.1 and 10.7 µM, respectively. Ellagic acid (5) inhibited AChE (IC50 = 41.7 µM) less than 3 and 4. In addition, 3 effectively inhibited MAO-B (IC50 = 7.27 µM) followed by 5 (IC50 = 9.21 µM). All five compounds weakly inhibited BChE and BACE-1. Compounds 3, 4, and 5 reversibly and competitively inhibited AChE, and were slightly or non-toxic to MDCK cells. The binding energies of 3 and 4  for AChE were greater than that of 5 , and 3 and 4 formed a hydrogen bond with Tyr124 in AChE. These results suggest 3 is a dual-targeting inhibitor of AChE and MAO-B, and that these compounds should be viewed as potential therapeutics for the treatment of Alzheimer’s disease.Among 276 herbal extracts, a methanol extract of  The therapeutic efficacies of AChE inhibitors in AD have been shown to be due to augment synaptic ACh levels in the cerebral cortex and improve cholinergic transmissions3. In AD, levels of ACh are low and cause central nervous system (CNS) disorders, which are characterized by gradual declines in cognition, memory, and cognitive functions. Currently, AChE inhibitors approved by the FDA for the treatment of AD include donepezil, galantamine, and rivastigmine. Like AChE, butyrylcholinesterase (BChE) also importantly contributes to cholinergic neurotransmission. BChE is present in glial cells, hippocampus, and the temporal nerve cortex, and is involved in cognitive function. BChE has less substrate specificity than AChE, but both enzymes effectively hydrolyze ACh2.Cognitive dysfunctions, such as learning, memory, processing information speed, visual perception, mental flexibility, and persistent attention-deficit dysfunctions, are the primary symptoms of Alzheimer's disease (AD). Acetylcholine (ACh) regulates cognitive functions, especially learning and memory, via neurotransmission and is synthesized from choline and acetyl co-enzyme A in presynaptic neurons and then released into the synaptic gaps. Acetylcholinesterase (AChE) terminates the ACh-mediated neurotransmission and is mostly found in neurons4.Importantly, the formation and aggregation of β-amyloid peptide (Aβ) are associated with the hydrolysis of amyloid precursor protein (APP) by β-site amyloid precursor protein cleaving enzyme 1 , and since the anionic site of AChE is involved in Aβ aggregation, studies on dual AChE and BACE-1 inhibitors are being actively pursued5. Irreversible MAO-B inhibitors such as rasagiline and selegiline are used for the treatment of PD8, alongside levodopa, dopamine agonists, and catechol-O-methyltransferase inhibitors.On the other hand, monoamine oxidases (MAOs) are involved in the pathways leading to catecholamine and 5-hydroxytryptamine inactivation, and thus, MAOs are recognized targets in diseases associated with these pathways. Furthermore, selective MAO-A inhibitors have anti-depressant activity, and selective MAO-B inhibitors are recognized developmental targets for the treatment of AD and Parkinson's disease (PD)9.Recently, a multi-targeting treatment strategy was devised to target MAO-B and AChE, and it has been reported that MAO and AChE inhibitors can improve the cognitive functions and relieve symptoms in AD by increasing monoamine and choline ester levelsPrunus padus var. seoulensis, potently and selectively inhibits human MAO-A10, and that calycosin isolated from Maackia amurensis potently and selectively inhibits human MAO-B11. In this study, we screened Castanopsis cuspidata var. sieboldii as a potent AChE inhibiting herbal source, and isolated AChE inhibitors by activity-guided fractionation.During our on-going efforts to identify potent natural inhibitors of MAO-A, MAO-B, and AChE in a herbal extract library, we found that rhamnocitrin isolated from the leaves of C. cuspidata is an evergreen broad-leaved tree that is widely distributed in western Japan12, and has been reported to contain galloyl shikimic acid13, ellagitannins14, and dehydrodiallic acid, cretanin, chesnatin, chestanin15, galloyl ester triterpenoid, and hexahydorxydiphenic acid conjugated triterpenoid16. Furthermore, extract of C. cuspidata has antioxidant18, anticancer, and anti-inflammatory18, and anti-fungal19 properties.C. cuspidata was selected as an AChE inhibitor resource from a library of herbal extracts, five compounds were isolated and identified in an extract of the stems of the plant. Their inhibitory activities against AChE, BChE, MAO-A, MAO-B, and BACE-1 were evaluated, and kinetic and reversibility studies, cytotoxicity tests, in silico pharmacokinetics, and docking simulations were performed to identify novel candidate compounds for the treatment of AD and PD.In this study, 1H and 13C NMR spectroscopic data were recorded using JEOL ECZ500R and JEOL ECA600  instruments, respectively. HR-ESI-MS data were acquired using an ACQUITY UPLC I-Class/Vion IM-QTOF system  coupled with ACQUITY BEH C18 column . Preparative HPLC was performed using a YMC K-Prep LAB 300  equipped with a DAD-50-700S column (50.0 × 700 mm) packed with YMC ODS AQ HG resin  and a Gilson HPLC system  coupled with an Acclaim Polar Advantage II C18 column, 250 × 21.2 mm, 5 µm , respectively.C. cuspidata var. sieboldii was provided by the Korean Plant Extract Bank at the Korea Research Institute of Bioscience and Biotechnology  under agreement on Jan 4, 2020. The plant was identified by Dr. Gwanpil Song, and collected from Aewol-eup, Jeju-do, Korea in 2016. A voucher specimen (KRIB 0084147) was deposited at the KRIBB herbarium. All the experimental protocols adhered to the relevant ethical guidelines/regulations on the usage of plants. The extract (50 g) was separated by preparative reverse phase column chromatography eluting using a H2O/MeOH gradient  to obtain ten fractions (1–10) using the YMC K-Prep LAB300 unit . Of these fractions, two (CCS8 and CCS9) identified by bioactivity screening for AChE inhibition were purified by preparative HPLC. CCS8 (2.9 g) was subjected to reverse phase column chromatography using a YMC K-Prep LAB300 and a solvent gradient (0 → 100% MeOH over150 min at 100 mL/min) to yield ten sub-fractions (CCS8-1–CCS8-10). Sub-fraction CCS8-1 (179.6 mg) was purified by preparative HPLC  to obtain compound 1 (62.1 mg). Compound 2 (4.5 mg) was obtained from sub-fraction CCS8-5 (297.1 mg) by preparative HPLC  and compound 3 (9.3 mg) was isolated from sub-fraction CCS8-6 (30.9 mg) by preparative HPLC . CCS-9 (3.6 g) was separated by preparative HPLC, YMC K-Prep LAB300  to produce ten sub-fractions (CCS9-1–CCS9-10). Compound 5 (6.8 mg) was obtained from sub-fraction CC-9–1-2 (64.7 mg) by preparative HPLC  and compound 4 (4.2 mg) was purified from sub-fraction CC-9–1-4 (50.7 mg) by preparative HPLC .A methanol extract of the stems of Electrophorus electricus), BChE (equine serum), acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), tacrine, donepezil, and the BACE-1 activity detection kit were purchased from Sigma-Aldrich . Clorgyline and pargyline were obtained from BioAssay Systems .Recombinant human MAO-A and MAO-B, kynuramine, benzylamine, toloxatone, lazabemide, AChE , Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin solution were purchased from Hyclone Laboratories . The cell counting kit-8 (CCK-8) and dimethyl sulfoxide (DMSO) were obtained from Dojindo Laboratories  and Sigma-Aldrich, respectively.20, with slight modification22. Reactions were performed using ~ 0.2 U/mL of enzyme in the presence of 0.5 mM substrate and 0.5 mM of DTNB in 0.5 mL reaction mixtures, which were continuously monitored for 15 min at 412 nm. MAO-A and MAO-B activities were determined using a continuous spectrophotometric method, as described previously24. The Km values of MAO-A for kynuramine and of MAO-B for benzylamine were 0.040 and 0.17 mM, respectively. The concentrations of kynuramine (0.06 mM) and benzylamine (0.3 mM) used were 1.5 times and 1.8 times Km values, respectively. Reaction rates are expressed as changes in absorbance per min. BACE-1 assays were performed using a β-secretase (BACE-1) activity detection kit at excitation and emission wavelengths of 320 and 405 nm, respectively, using a fluorescence spectrometer , after reaction for 2 h at 37 °C with 7-methoxycoumarin-4-acetyl--amyloid β/A4 protein fragment 667–676-Lys-Arg-Arg amide trifluoroacetate as substrate25.AChE and BChE inhibitory activities were measured after preincubating enzymes with inhibitors for 15 min before adding substrates  and DTNB. AChE activities were assayed as previously described3, 4, and 5 were investigated by dialysis at concentrations of ~ 2 × IC50 values; tacrine and donepezil were used as reference AChE and BChE inhibitors26, and lazabemide and pargyline were used as reference reversible and irreversible MAO-B inhibitors, respectively21. After preincubating three compounds or reference inhibitors with enzymes for 15 min, residual activities of undialyzed (AU) and dialyzed (AD) samples were expressed as percentages of those of non-inhibitor treated controls. Reversibilities were assessed using AU and AD values and compared with those of reference compounds.The reversibilities of AChE and MAO-B inhibitions by 1 to 5 were initially investigated at a concentration of 10 µM28. IC50 values of the five compounds and the reference compounds  were determined. Kinetic parameters, inhibition types, and Ki values of the most potent AChE inhibitors (3 and 4) and MAO-B inhibitors (3 and 5) were analyzed. Kinetics of AChE inhibitions by 3, 4, and 5 and of MAO-B inhibitions by 3 and 5 were investigated at five different substrate concentrations  in the absence or presence of each inhibitor at concentrations of ~ 0.5 × , 1.0 × , and 2.0 × their IC50 values. Inhibitory patterns and Ki values were determined using Lineweaver–Burk (LB) plots and secondary plots of LB slopes.Inhibitions of AChE, BChE, MAO-A, MAO-B, and BACE-1 by compounds 2, and media were changed every two days.Madin-Darby canine kidney (MDCK) cells and human acute promyelocytic leukemia (HL-60) cells were obtained from the Korean Cell Line Bank . MDCK cells were cultured in DMEM, and HL-60 cells in RPMI-1640 medium containing 10% FBS, 1% penicillin/streptomycin, and 0.1% 2-mercaptoethanol. Cultures were maintained at 37 °C under 5% CO29. Briefly, MDCK cells or HL-60 cells were resuspended at 1 × 105 or 3 × 105 cells/mL and suspensions (100 µL) were added to wells of a 96-well plate and incubated in 5% CO2 atmosphere at 37 °C for 24 h. After incubation, 100 µL of each medium was treated with compounds at 1, 3, 10, 30, or 50 µM and incubated in 5% CO2 at 37 °C for 24 h. CCK-8 (10 µL/well) was then added to 100 µL aliquots from each cell and incubated 2–4 h. Absorbances were measured at 450 nm using a microplate reader .Cell viabilities were determined using the CCK-8 assay3, 4, and 5 with AChE and MAO-B, we used AutoDock Vina30, which has an automated docking facility. To define enzyme binding pockets, we used active sites defined by a complex of AChE with 4-carbamoyl-1-(3-{2-[(E)-(hydroxyimino)methyl]-1H-imidazol-1-yl}propyl)pyridin-1-ium (LND) (PDB ID: 6O5V) and a complex of MAO-B with (R)-rosiglitazone (RGZ) (PDB ID: 4A7A). To prepare 3, 4, and 5 for docking simulations, we performed the following steps: (1) created 2D structures of the three compounds, (2) converted these 2D structures into 3D structures, and (3) performed energy minimization using the ChemOffice program (http://www.cambridgesoft.com). Docking simulations of AChE or MAO-B with 3, 4, and 5 were performed using AutoDock Vina 1.1.231. Based on the docking results, we checked for possible hydrogen bonding interactions with bonding relaxation constraints of 0.4 Å and 10.0° using Chimera 1.15 program32.To simulate dockings of 3, 4, and 5 using the SwissADME web tool at http://www.swissadme.ch/33.Pharmacokinetic analyses for drug-like properties were performed in silico on C. cuspidata stems was selected based on its AChE inhibitory activity, novelty of the plant, and availability of raw material. During preparative HPLC of the extract, eluents were divided into ten fractions. AChE inhibitory analysis showed that fractions 8 (CCS-8) and 9 (CCS-9) had the lowest residual activities of 30.2 and 21.9%, respectively, at 50 µg/mL , and HRESIMS data with literature values. 1H, 13C, and MS data are provided in Supplementary Materials 34, 4′-O-(β-d-glucopyranosyl)-3,3′,4-tri-O-methylellagic acid (2)35, 4′-O-(α-l-rhamnopyranosyl)-3,3′,4-tri-O-methylellagic acid (3)36, 3,3′,4-tri-O-methylellagic acid (4)37, and ellagic acid (5)37. The purities of 1–5 were 95.2%, 99.1%, 99.1%, 92.0%, and 95.1% as determined by UPLC-PDA analysis  was previously isolated from the leaves of C. cuspidata and its structure elucidated, however, compound 5, that was ellagic acid, and compounds 2–4, which were identified as ellagic acid derivatives, were first isolated from C. cuspidata in this study. Ellagic acid, and ellagic acid derivatives may be considered secondary metabolites of the biosynthesis of gallic acid38.Chestanin (1 to 5) were assayed for inhibitory activities against five enzymes, namely, AChE, BChE, MAO-A, MAO-B, and BACE-1. 3 and 4 inhibited AChE by about 50% at 10 µM, whereas the other three compounds inhibited it by less than 30%  to 86.3% (AD), from 35.3% to 88.5%, and from 35.3% to 92.3%, respectively, and these values were similar to those observed for the reversible inhibitor tacrine (34.5 to 96.5%) , but not to those of the irreversible inhibitor pargyline (34.3 to 35.2%)  or HL-60 (cancer cell line) cells at 50 µM .In silico pharmacokinetic studies predicted the gastrointestinal (GI) absorptions of Glycin max Merrill39 and decursin from Angelica gigas Nakai40, and the MAO-B inhibitors, maackiain from Sophora flavescens21 and calycosin from Maackia amurensis11. In the present study, of the 276 herbal extracts examined, the methanol extract of the stems of C. cuspidata was selected as a potential source of AChE inhibitors and five compounds were subsequently isolated using a bioassay-guided method and identified.In our previous studies, we isolated the MAO-A inhibitors, hispidol from 3 and 4 effectively inhibited AChE, and 3 and 5 effectively inhibited MAO-B. Our results suggest that 3 is a dual-acting inhibitor of AChE and MAO-B, 4 is an AChE inhibitor, and 5 is a MAO-B inhibitor. Little information is available on natural dual AChE/MAO-B inhibitors, though, recently, macelignan was identified as a dual AChE/MAO-B inhibitor with IC50 values of 4.16 and 7.42 µM, respectively22.Of the five compounds isolated, 5, is a natural polyphenol with anti-proliferative, anti-oxidant, anti-diabetic, anticancer, and apoptosis-inducing activities42. Recently, ellagic acid was reported to have neuroprotective and cognition-enhancing effects in sporadic AD based on behavioral investigations, however, its IC50 for AChE was 132.92 μM, which was 3.2 times higher than our result (IC50 = 41.7 μM)43. Furthermore, the previously reported IC50 of ellagic acid for MAO-B was 0.412 μM using rat brain mitochondrial fractions, which was much lower than our result (IC50 = 9.21 μM)44. The inhibitory activities of the ellagic acid derivatives 3 and 4 on AChE and MAO-B have not been previously described.Ellagic acid, 50 values, i.e., high potency, IC50 < 15 μM; moderate potency, 15 < IC50 < 50 μM; and low potency, 50 < IC50 < 1000 μM45. Based on this classification, 3 (IC50 = 10.1 µM) and 4 (IC50 = 10.7 µM) are highly potent AChE inhibitors. The potencies of 3 and 4 were ~ 5 times lower or ~ 2 times higher than that of galantamine [IC50 values = 2.16 µM46 or 21.1 µM (6.07 µg/ml)47], which is a natural compound and used for the treatment of AD. Polyphenols that target AChE have attracted research interest as potential therapeutics for AD48. The AChE inhibitory potencies of 3 and 4 were higher than those previously reported for other natural polyphenol, e.g., C-glucosylflavone, isovitexin-7-O-methyl ether (swertisin)  from Anthocleista vogelii49, the flavonoids tiliroside (IC50 = 23.5 µM) and quercetin (IC50 = 19.8 µM) from Agrimonia pilosa50, curcumin (IC50 = 23.5 µM) from Curcuma longa51, and epigallocatechin gallate from green tea (IC50 = 14.8 µM)52, but lower than that of a xanthonoid α-mangostin (IC50 = 2.4853 or 6.3 µM54), a hydroxycinnamoylated catechin (IC50 = 2.49 µM) from Camellia sinensis var. assamica55, and two resveratrol oligomers vitisin A (IC50 = 1.04 µM) and heyneanol A (IC50 = 1.66 µM) from Vitis amurensis56. The potencies of 3 and 4 were similar to those of two khellactone derivatives (IC50 = 9.28 and 10.0 μM) from Peucedanum japonicum Thunberg26.Natural AChE inhibitors have been classified into three groups according to their IC3, 4, and 5 inhibited AChE in a competitive but partially mixed manner, whereas 3 and 5 inhibited MAO-B in a competitive manner. The majority of AChE inhibitors described to date are mixed or partially mixed inhibitors57, whereas the MAO-B inhibitors described are usually competitive type60.O-methyl group of 4 (IC50 = 10.7 μM) and the α-L-rhamnopyranosyl-3,3′,4-tri-O-methyl groups of 3 (IC50 = 10.1 μM) increased AChE inhibitory activity as compared with the 5 parent (IC50 = 41.7 μM). However, the β-D-glucopyranosyl group of 2 (IC50 = 46.4 μM), which replaced the α-L-rhamnopyranosyl group of 3 decreased AChE inhibitory activity to the level of 5. As regards MAO-B inhibitory activities, the α-L-rhamnopyranosyl of 3 (IC50 = 7.27 μM) and 5 (IC50 = 9.21 μM) increased the activity as compared with the 3,3′,4-tri-O-methyl group of 4 and the β-d-glucopyranosyl group of 2 .Regarding structure–activity relationships (SARs), the 3,3′,4-tri-3  and 4  for AChE were higher than that of 5 , largely due to hydrogen bond formation, which was not predicted for 5, and these results concur with their IC50 values as determined by inhibition assays, . Regarding docking to MAO-B, 3 and 5 both formed a hydrogen bond, whereas 4 did not, and the binding energies of 3  and 5  for MAO-B were higher than that of 4 , in line with their IC50 values . We attribute these differences to a combination of hydrogen bond formation, electrostatic bonding, van der Waals forces, dissolvent effects61, and the structural flexibilities of the compounds as determined by AutoDock program62.Docking analysis showed that the binding energies of 3, 4 and 5 are absorbed well in the GI tract, are not substrates for P-gp, which causes drug efflux to gut lumen, and inhibit some cytochrome P450, but do not cross the BBB. In terms of cytotoxicity, 3, 4, and 5 were non-toxic or slightly toxic to the normal and cancer cells at 50 μM. Ellagic acid and its derivatives are able to react with a polycyclic aromatic hydrocarbon metabolite, the ultimate carcinogen, and prevent its covalent binding to DNA63. However, some of them exhibited DNA-damaging activity in DNA repair-deficient yeast64. Therefore, when compounds 3, 4, and 5 are used, careful doses should be needed, though the compounds were non- or slightly toxic.Our in silico pharmacokinetic studies also predicted that 3 and 4 from Castanopsis cuspidata var. sieboldii, and show that these compounds have potential use for the treatment of neurological diseases like AD.In summary, we isolated the highly potent, reversible, selective AChE inhibitors Castanopsis cuspidata var. sieboldii by activity-guided screening for AChE inhibitors. Of these, 3 and 4 effectively and selectively inhibited AChE . In addition, 3 effectively inhibited monoamine MAO-B (IC50 = 7.27 µM) followed by 5 (IC50 = 9.21 µM). Binding energies of 3 and 4  for AChE were higher than that of 5  due to hydrogen bonding. In addition, compounds 3, 4, and 5 were slightly or non-toxic to MDCK cells. All five compounds weakly inhibited BChE and BACE-1. Our results show that 3 is a dual-targeting AChE and MAO-B inhibitor, 4 is an AChE inhibitor, and 5 is a MAO-B inhibitor, and suggest the potential use of these compounds for the treatment of AD.Five compounds were isolated from the methanol extract of the stems of Supplementary Figures.


Z configuration about the C=C bond. The crystal structure features C—H⋯O and C—H⋯N hydrogen bonds together with C—H⋯π contacts and π–π stacking interactions. The crystal packing was further investigated by Hirshfeld surface analysis and the included surface areas from the title compound and an isomeric form were also investigated.The asymmetric unit of the title compound contains one molecule and the molecule adopts a  17H13NO3, adopts a Z configuration about the C=C bond. The isoxazole and methoxybenzylidene rings are almost coplanar with a dihedral angle of 9.63 (7)° between them. In contrast, the phenyl substituent is twisted significantly out of the plane of the oxazole ring, with the two rings inclined to each other by 46.22 (4)°. The crystal structure features C—H⋯O, C—H⋯N and C—H⋯π hydrogen bonds and π–π contacts. An analysis of the Hirshfeld surfaces points to the importance of H⋯H, H⋯C/C⋯H and H⋯O/O⋯H contacts. The included surface areas of the title compound were compared to those of the isomeric structure (Z)-4-(4-methoxybenzylidene)-3-phenylisoxazol-5(4H)-one .The title compound, C al. 2015. CrystEn This conformation is supported by the formation of an intramolecular C12—H12⋯O1 hydrogen bond -4-benzylidene-2-phenyl-1,3-oxazol5(4H)-one  and 3.9200 (9) Å] are found between the centroids of the isoxazolone ring and the methoxy-substituted benzene ring, which stack adjacent molecules in an obverse fashion along b.In the crystal, molecules stack along the on Fig. 2. Molecu1 were obtained using Hirshfeld surface analysis -4-benzylidene-3-phenylsoxazol-5(4H)-one yielded seventeen hits. Importantly, one of these, i.e. (Z)-4-(4-methoxybenzylidene)-3-phenylisoxazol-5(4H)-one  of the title compound with the methoxy substituent in the 4-position of the benzene ring. Another paper -4-(4-[dimethylamino)benzylidene]-3-phenylisoxazol-5(4H)-one (IDIBEE) together with two other related compounds, IDIBII and IDIBOO, that exhibit large second harmonic generation effects. The search also revealed four other structures in which the configuration about the C=C bond is Z, namely 4-(2-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one -4-benzylidene-2-phenyl-1,3-oxazol-5(4H)-one -4-benzylidene-3-methylisoxazol-5(4H)-one [MBYIOZ -4-(4-hydroxybenzylidene)-3-methylisoxazol-5(4H)-one  were mixed in a 25 ml flask equipped with a magnetic stirrer. The mixture was refluxed in 5 ml of water for 2.5 h (the reaction was monitored by TLC). On completion of the reaction, the mixture was gradually poured into ice-cold water. Stirring was maintained for a few minutes and the resulting solid was filtered and purified by crystallization from ethanol.2-Methoxybenzaldehyde (1 mmol), hydroxyamine hydrochloride (1 mmol), ethyl benzoylacetate (1 mmol) and KUiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021004308/ex2043sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021004308/ex2043Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021004308/ex2043Isup3.cmlSupporting information file. DOI: 2079211CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


X-ray analysis unveiled that PCAL and PCAC are nearly totally shielded in the β-CD wall. Their aromatic rings are vertically aligned in the β-CD cavity such that the functional groups on the opposite side of the ring  and 1-CHO/1-COOH groups) are placed nearby the O6–H and O2–H/O3–H rims, respectively. The preferred inclusion modes in 1 and 2 help to establish crystal contacts of OH⋅⋅⋅O H-bonds with the adjacent β-CD OH groups and water molecules. By contrast, the DFT-optimized structures of both complexes in the gas phase are thermodynamically stable via the four newly formed host–guest OH⋯O H-bonds. The intermolecular OH⋅⋅⋅O H-bonds between PCAL/PCAC 3,4-di(OH) and β-CD O6–H groups, and the shielding of OH groups in the β-CD wall help to stabilize these antioxidants in the β-CD cavity, as observed in our earlier studies. Moreover, PCAL and PCAC in distinct lattice environments are compared for insights into their structural flexibility.Protocatechuic aldehyde (PCAL) and protocatechuic acid (PCAC) are catechol derivatives and have broad therapeutic effects associated with their antiradical activity. Their pharmacological and physicochemical properties have been improved via the cyclodextrin (CD) encapsulation. Because the characteristics of β-CD inclusion complexes with PCAL ( Phellinus linteus : monoclinic, space group P21 (no. 4), a = 15.4213(11) Å, b = 10.0440(7) Å, c = 21.2795(15) Å, β = 110.281(2)°, V = 3091.7(4) Å3, Z = 2, T = 296(2) K, μ(MoKα) = 0.133 mm–1, Dcalc = 1.484 g cm–3, 60310 reflections measured (1.41° ≤ Θ ≤ 30.67°), 18989 unique  which were used in all calculations. The final R1 was 0.0620 (I > 2σ(I)) and wR2 was 0.1527 .Crystal data for 2 : monoclinic, space group P21 (no. 4), a = 12.5165(7) Å, b = 19.0287(12) Å, c = 14.0626(8) Å, β = 109.656(2)°, V = 3154.2(3) Å3, Z = 2, T = 296(2) K, μ(MoKα) = 0.132 mm–1, Dcalc = 1.471 g cm–3, 55384 reflections measured (1.54° ≤ Θ ≤ 30.61°), 19,288 unique , which were used in all calculations. The final R1 was 0.0602 (I > 2σ(I)) and wR2 was 0.1134 .Crystal data for Meaningful, reliable structures and thermodynamic data of CD inclusion complexes can be deduced from complete-geometry optimization by the DFT method. This has been successfully demonstrated in our previous works on the β-CD encapsulation of polyphenolic plants extracts, including tea epicatechins , olive o1 and 2, excluding the hydration water molecules. Before the calculation, all the underestimated X-ray-derived C–H and O–H distances were normalized to neutron hydrogen distances, 1.083 and 0.983 Å [Starting atomic coordinates of the 1:1 β-CD–PCAL and β-CD–PCAC inclusion complexes were taken from the converged crystal structure refinements of  0.983 Å . The two 0.983 Å  on a DELRegarding the protonated form of PCAC that was used in the DFT full-geometry optimization of the β-CD–PCAC inclusion complex, PCAC with pKa ~4.5 in a neutral pH solution containing β-CD was expected to exist in an anionic form. However, it turned out that PCAC and β-CD co-crystallized to yield a thermodynamically stable adduct in the single-crystalline state. Similarly, benzoic acid with pKa = 4.2 complexed with β-CD in solution  and the Estb and ΔEint) were calculated straightforwardly. For example, ΔEstb was obtained by subtracting the molecular energies of β-CD and PCAL/PCAC from the energy of the β-CD–PCAL/PCAC complex from the full-geometry optimization. Similarly, ΔEint considered the corresponding single-point energies in the complexed states. The DFT-derived inclusion structures of the two complexes together with their O−H⋯O hydrogen bonds, ΔEstb and ΔEint are given in The stabilization energy and interaction energy of the complex (ΔEstb and ΔΔEint) are sufficient to interpret the relative thermodynamic stabilities in relation to host–guest interactions and antioxidant properties.Note that no basis set superposition error (BSSE) correction is applied to the DFT-derived energies of the β-CD–catechol antioxidant complexes listed in 1) and PCAC (2) by a combined study of single-crystal X-ray diffraction and density functional theory (DFT) full-geometry optimization.Protocatechuic aldehyde (PCAL) and protocatechuic acid (PCAC) are polyphenol extracts from plants. They contain a catechol moiety and thus having wide therapeutic effects including antioxidant, anti-inflammatory, and anticancer activities. Their physicochemical and pharmacological properties have been improved via cyclodextrin (CD) inclusion complexation, of which their characteristics are still controversial. To address at the atomic-level of the inclusion complexation, we investigate the β-CD encapsulation of PCAL  and 1-CHO/1-COOH groups) are placed nearby the O6–H and O2–H/O3–H rims, respectively. The preferred inclusion modes in  and OLE . By cont


Scientific Reports 10.1038/s41598-020-70271-z, published online 06 August 2020Correction to: The original version of this Article contained errors. In Table 1, some of the values for “Concentration (% w/v)” and “Amount Given (mg/day)” were incorrectly reported.In Table 1, in the ‘Concentration’ column, for Levodopa/Spiperone/Lens (line 13) and Levodopa/Spiperone/Diffuser (line 16),“0.596/0.002”now reads“0.296/0.002”.Additionally, in Table 1, in the ‘Amount Given (mg/day)’ column, for 45 mM levodopa (line 9)“0.900”now reads“0.090”.The original version of the Article has been corrected.


Chemical compositions were obtained from available bottle labels. National-based bottle waters ranged from -2.47‰ to -10.65‰ in δ18O and from -10.4‰ to -78.0‰ in δ2H, while d-excess varied from +4.2‰ up to +17.0‰. International bottle waters ranged between -2.21‰ and -11.03‰ in δ18O and from -11.3‰ up to -76.0‰ in δ2H, while d-excess varied from +5.0‰ up to +19.1‰. In Costa Rica, only 19% of the brands reported chemical parameters such as Na+, K+, Ca+2, Mg+2, F−, Cl−, NO3−, SO4−2, CO3−2, SiO2, dry residue, and pH; whereas 27% of the international products reported similar parameters. The absence of specific geographic coordinates or water source origin limited a spatial analysis to validate bottled water isotope compositions versus available isoscapes in Costa Rica Global bottled water consumption has largely increased   In general, potential source elevations ranged from 233 up to 3386 m asl, with a mean value of 2020 m asl, which in turn reflects the large dependency of high elevation recharge processes and spring discharges for the bottled water industry in Costa Rica . Deuterium excess values of imported bottled waters varied from + 5.0‰ to + 19.1‰, with mean of + 12.4‰. As expected, bottled waters from the mountainous regions of Europe exhibited lower δ18O compositions due to strong latitudinal and temperature effects  and imported (N = 31) bottled water samples were purchased between 2019–2020 across a broad range of commercial stores  in Costa Rica, Honduras, Panamá, Guatemala, and El Salvador. Samples were classified as national-based (Costa Rica), imported from USA, Europe, Oceania, and other countries of Central America. As this type of sampling is opportunistic, seasonal effects are not included in our analysis. All samples were sealed with parafilm and refrigerated at 5 °C until analysis at the Stable Isotopes Research Group, Universidad Nacional . Chemical compositions were extracted from available bottle labels, the parameters included: Na+, K+, Ca+2, Mg+2, Fe+2/+3, F−, Cl−, NO3−, SO4−2, CO3−2, SiO2, dry residue, and pH. National-based samples were coded from CR1 to CR45; while imported samples were coded from FW1 to FW31. All information is available at https://www.hydroshare.org/resource/86225d08252747d5a78477c1f74eb158/https://www.hydroshare.org/) is an online platform to share data, models, and code.National-based (2.22H and ± 0.1‰ for δ18O (1σ). Stable isotope compositions are expressed as δ18O or δ2H = (Rs/Rstd - 1)•1000, where R is the 18O/16O or 2H/1H ratio in a sample (s) or standard (std) and reported in the delta-notation (‰) relative to V-SMOW/SLAP scale. The instrument accuracy was assessed with a combination of in-house and primary international water standards (SMOW and SLAP). Deuterium excess was calculated as d-excess = δ2H - 8•δ18O Samples were analyzed at the Stable Isotopes Research Group laboratory at the Universidad Nacional  using an IWA-45EP water analyzer  with a precision of ± 0.5‰ for δRicardo Sánchez-Murillo: Conceptualization, Methodology, Formal analysis, Data curation, Writing – original draft; Germain Esquivel-Hernández: Writing – review & editing, Writing – original draft; Christian Birkel: Writing – review & editing, Writing – original draft; Lucía Ortega: Writing – review & editing, Writing – original draft.The authors declare that they have no known competing financial interests or personal relationships that have, or could be perceived to have, influenced the work reported in this article.


Scientific Reportshttps://doi.org/10.1038/s41598-021-04510-2, published online 12 January 2022Correction to: The original version of this Article contained errors.In Figure 2, panels (G), (H) and (I) were omitted. The original Figure Similarly, in Figure 4, panels (C)–(H) and in Figure 5, panels (C) and (D) were omitted. The original Figures Additionally, in Table 1, values in columns “Total samples N (%)”, “Nuclear expression of MAGE-A11” and “Cytoplasmic expression of MAGE-A11” were omitted. The original Table Finally, the data in Table 2 did not display correctly. The original Table The original Article has been corrected.


In an additional analysis of the TCGA RCC dataset, γδ T-cell enrichment scores correlated strongly with those for CTLs, Th1 cells, “exhausted” T cells, and M1 macrophages, suggesting active involvement of γδ T cells in anti-tumor rather than pro-tumor activity, and Vδ1 cells were more abundant than Vδ2 or Vδ3 cells in RCC tumor samples. Thus, we posit that Vγ9Vδ1 T cells may represent an excellent candidate for adoptive immunotherapy in RCC patients with a high risk of relapse after surgery.Due to the highly immunogenic nature of renal cell carcinoma (RCC), the tumor microenvironment (TME) is enriched with various innate and adaptive immune subsets. In particular, gamma-delta (γδ) T cells can act as potent attractive mediators of adoptive cell transfer immunotherapy because of their unique properties such as non-reliance on major histocompatibility complex expression, their ability to infiltrate human tumors and recognize tumor antigens, relative insensitivity to immune checkpoint molecules, and broad tumor cytotoxicity. Therefore, it is now critical to better characterize human γδ T-cell subsets and their mechanisms in RCCs, especially the stage of differentiation. In this study, we aimed to identify γδ T cells that might have adaptive responses against RCC progression. We characterized γδ T cells in peripheral blood and tumor-infiltrating lymphocytes (TILs) in freshly resected tumor specimens from 20 RCC patients. Furthermore, we performed a gene set enrichment analysis on RNA-sequencing data from The Cancer Genome Atlas (TCGA) derived from normal kidneys and RCC tumors to ascertain the association between γδ T-cell infiltration and anti-cancer immune activity. Notably, RCC-infiltrating CD3 Postoperative relapse develops in a significant proportion of patients with organ-confined renal cell carcinoma (RCC) after curative surgery, and they have a universally unfavorable prognosis due to treatment failure . SurgicaAdditional progress will depend on understanding the immunobiology of RCC, which is complex and different from that of other immune-sensitive tumor types ,4,5. RCCUnfortunately, recent clinical trials have shown that although applications of γδ T cells to advanced solid tumors, including RCCs, yield promising clinical benefits and safety, obvious limitations remain, with an average response ratio of only 21% and a low proportion of complete remissions ,8,11. ThWe evaluated treatment-naïve patients with histologically confirmed RCC after partial or radical nephrectomy at Samsung Medical Center  from 2018 to 2019. All analyses were approved by the appropriate Institutional Review Board (IRB) at SMC (IRB No: 2018-04-037). Written informed consent was obtained from all patients. Heparinized peripheral blood and sections of fresh tumor tissue  were collected during surgeries. TM  gradient method as described previously [Explanted fresh tumor tissue was rinsed with phosphate-buffered saline to remove traces of blood and necrosis. The material, diced using scalpels, was filtered through a fine (70 μm) mesh of cell strainer , and red blood cell (RBC) lysis buffer  was used to lyse RBCs with minimal effect on white blood cells after centrifugation at 2000 rpm for 4 min. RCC TILs were finally isolated by repeated centrifugation , and the cell suspension was immediately analyzed after assessing cell viability using trypan blue exclusion. Subsequently, PBLs were enriched from heparinized peripheral blood collected from matched patients using the Ficoll-Paqueeviously .5–107) were labelled without stimulation or expansion using a panel of monoclonal antibodies specific for differentiation, activation, exhaustion, and cytotoxic molecules: anti-human antibodies against CD3 (HIT3a), γδTCR (B1), CD28 (CD28.2), Fas Cell Surface Death Receptor (FAS) (DX2), Vγ9 (B3) Vδ2 (B6) Programmed cell death protein 1 (PD-1) (EH12.2H7), Inducible T-cell co-stimulator (ICOS) (C398.4A), CD69 (FN50), CD27 (M-T271), CD45RA (HI100), B- and T-lymphocyte attenuator (BTLA) (MIH26), Lymphocyte Activating 3 (LAG3) (11C3C65), T-cell immunoglobulin and mucin domain-3 (TIM-3) (F38-2E2), T-cell immunoreceptor with Ig and ITIM domains (TIGIT) (A15153G), NK group 2 member D (NKG2D) (1D11), Perforin (B-D48), Granzyme A (CB9), Granzyme B (GB11), and Lysosomal-associated membrane protein 1 (LAMP-1) (H4A3)  and Vδ1 (TS8.2) . Cells were stained with 0.25 μg/mL human BD Fc Block  to block Fcγ receptors, and concentration- and isotype-matched control antibodies were used to detect the nonspecific binding of antibodies. After a 30-min incubation on ice followed by two PBS washes, cells were analyzed using the BD FACS CantoIITM flow cytometer  and FlowJo software  was used for data acquisition and analysis.For phenotyping, freshly isolated TILs and PBLs (10Von Hippel–Lindau (VHL) gene wild-type, well-differentiated) and ACHN  (4 × 104 cells/well) were incubated overnight in 48-well plates. Isolated RCC infiltrating Vγ9Vδ1 T cells or peripheral Vγ9Vδ2 T cells (5 × 103~1 × 104 cells/well) were added to target the tumor cells per well with and without activation. RCC-infiltrating Vγ9Vδ1 T cells were stimulated with anti-human Vδ1 Ab. For the cytotoxicity assay with Vγ9Vδ2 T cells, RCC cell lines were treated with hydroxymethylbutenyl-4-diphosphate (HMB-PP) for 20 h prior to performing cytotoxicity assays. After incubation for 24 h at 37 °C, tumor cell death was measured by eBioscienceTM Fixable Viability Dye eFluorTM 780  staining as per manufacturer’s instructions and analyzed by flow cytometry  and FlowJo software software .For cytotoxicity assessments, human RCC cell lines Caki-1 . Statistical significance is indicated when p < 0.05; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 in the Figures.Statistical analysis was performed using GraphPad Prism software . Results are expressed as mean ± standard deviation (SD). The statistical significance of differences between groups was determined using a high, intra-tumoral γδ T cells could be classified as CD3low (the major population) or CD3high (the minor population), both largely CD4-CD8-  (EMRA) that occur in both the CD4+ and CD8+ compartments of αβ T cells. Notably, intra-tumoral CD3 T cells A. As expand TIM3 ,14, cons+ CD27−) B. They aEMRA capable of immediate cytokine production and cytotoxicity [In addition, up-regulation of both activation/exhaustion molecules suggested the potent anti-tumor cytotoxic activity of these Vγ9δ1 T cells A,B 7,8,,11,15,16toxicity . SupportThe bias of intra-tumoral Vγ9δ1 T cells toward cytotoxicity rather than cytokine production, as demonstrated by an inability to secrete cytokines in response to either phorbol myristate acetate or ionomycin , suggest+ T cells. To confirm the association between γδ T cell infiltration and cytotoxic T lymphocyte (CTLs) activity, we performed a GSEA on RNA-seq data from TCGA derived from normal kidneys (n = 72) and RCC tumors (n = 524)  A. Immunen = 524) . γδ T ceImportantly, within the TCGA dataset, both TCR Vγ9- and Vδ1-chain sequences were detectable in ~20% of RCC but in only ~10% of normal kidney samples B. ConsisRCC-infiltrating γδ T cells are diverse, comprising either prominent Vδ1, Vγ9Vδ2, or mixed Vδ subfamilies ,8,9,15 bhigh Vδ1 T cells and CD3high Vγ9δ2 T cells have been reported in RCC [In this study, we conducted the first comprehensive immunophenotypic analysis of γδ T cell subsets isolated from RCC tumor tissue and matched PBL and assessed their anti-tumor cytotoxicity to allogeneic human RCC tumor cell lines. TILs and PBLs isolated from patients with treatment-naïve RCCs were immediately assessed without any manipulation because activating and expanding γδ T cells ex vivo may induce changes in the phenotypic characterization and cellular function of these naturally occurring cells capable of recognizing RCC in vivo. The TCR in γδ T cells consists of TCRγδ and CD3 subunits , and γδ T cells have certain unique features in the TCR/CD3 complex and its downstream signaling pathways that dictate their maturation and effector function ,8,11,19.d in RCC ,11,19, Cd in RCC .low Vγ9δ1 T cells in only RCC tumor tissues, and most of them were of the FAS+CD28-CD45RA+ TEMRA cells. Moreover, this indicated that newly defined CD3low Vγ9δ1 T cells could be predominantly in situ anti-RCC memory effectors compared to conventional peripheral circulating CD3high Vγ9δ2 γδ T cells that have been generally utilized in ACT [low Vγ9δ1 T cells might have been activated by RCC-associated antigens still uncharacterized in the TME. Collectively, these data indicate that RCC-infiltrating CD3low Vγ9Vδ1 T cells with an effector memory phenotype could be emerging as important candidates for adaptive immunotherapies against advanced RCC.In contrast, an observation made in our study was an exclusive infiltration of CD3d in ACT ,11. Notad in ACT . CD3low low Vγ9 δ1 T cells, whereas peripheral circulating CD3high Vγ9 δ2 T cells only expressed high levels of granzyme A. Granzyme A is the first to become detectable during differentiation into memory cells, followed by granzyme B and later perforin [low Vγ9 δ1 T cells and peripheral circulating CD3high were TEM and TCM, respectively. In fact, T cell exhaustion describes a state of late-stage differentiation usually associated with active prevention of functionality via ligation of negative signaling receptors on the cell surface, which can be recovered by manipulating extrinsic regulatory pathways, for example, by immune checkpoint blockade [Upon target recognition, Vδ1 T cell-mediated killing occurs via perforin and granzymes using mechanisms similar to those of Vδ2 T cells . Notablyperforin , supportblockade . A stateblockade ,15,16. Flow Vγ9δ1 T cells isolated from patients with RCC, which are phenotypically and functionally distinct from Vγ9δ1 PBLs. Our study is the first to identify a distinct population of RCC-infiltrating CD3low Vγ9δ1 T cells largely absent from the blood, suggesting that RCC selectively retains these subsets that adopt a terminally differentiated effector memory phenotype and are in a state of exhaustion as a result of chronic stimulation by RCC tumor cells [low Vγ9δ1 T cells. For example, a large part of the Vδ2 compartment is reportedly made up of CD27low CD45RAhigh Vδ1+ T cells in liver tissue, which are predominantly clonally expanded [low Vγ9δ1 TIL for combating RCCs.Despite recent data suggesting that Vδ1 T cells exhibit a radical new adaptive immunobiology ,9,10,15,or cells ,14. It iexpanded . This stlow Vγ9δ1 T cells. In the context of cancer, Vδ1 γδ T cells recognize altered-self lipids presented by CD1d [low Vγ9δ1 T cells. A greater understanding of the relationship between different Vγ9Vδ1 TCR ligands, their mode of recognition, tissue expression, and regulation/dysregulation will undoubtedly provide novel therapeutic avenues and insights into RCC [Nevertheless, an important unanswered question in RCC is the nature of the antigens that are targeted by CD3 by CD1d . The abu by CD1d ,23,24,25into RCC .low Vγ9δ1 TILs with a high proliferative capacity, a more pronounced Th1 polarization, an increased cytotoxic capacity and secretion of cytokines, and combination with other immunotherapeutic strategies, such as immune checkpoint inhibitors, should also be considered, aiming to maximize their therapeutic potential.The results of our analysis of the RCC TILs and TCGA dataset support γδ T cell anti-tumor activity and confirm preferential infiltration of the RCC TME by the Vδ1 cell subset. Infiltrating Vγ9Vδ1 T cells likely undergo chronic stimulation within the RCC TME, producing a terminally differentiated effector CTL phenotype. Thus, Vγ9Vδ1 T cells may represent an excellent candidate for adoptive immunotherapy in high-risk patients with locally advanced RCC. Optimized techniques promoting clinical-grade expansion and purification of CD3