Hippophae rhamnoides ssp. sinensis (Rha) and H. tibetana (Tib), but studies on the types and contents of soluble sugars, organic acids, free phenolics, bound phenolics, vitamin C (VC), tocopherol (VE) and carotenoids of the two sea buckthorn berries from this region have not been reported. In this paper, we found that the soluble sugars in Rha and Tib were mainly glucose and fructose; Rha exhibited a higher content of total sugar and fructose compared to Tib. The organic acids were mainly quinic acid and malic acid; Rha exhibited a higher content of total acids and quinic acid, but lower tartaric acid and citric acid compared to Tib. Rha also possessed a higher total (free and bound) phenolic as well as total (free and bound) flavonoid content than those in Tib; twelve phenolic compounds were analyzed, among which flavonols were dominant. Catechin, isorhamnetin and quercetin were the main phenolic substances. VC and VE (γ-tocopherol (γ-VE) and δ-tocopherol (δ-VE)) were higher in Rha than Tib. The total carotenoid, lutein, β-carotene and lycopene content of Tib was remarkably higher than that in Rha. Moreover, both Rha and Tib showed good in vitro and cellular antioxidant activities, and Rha had a stronger antioxidant activity. Taken together, Rha had a higher antioxidant activity, which may be due to its higher content of phenolics, flavonoids, VC and VE.The Qinghai–Tibet Plateau is the main production area of  H. rhamnoides subsp. sinensis (Rha) is mainly distributed on the Inner Mongolia Plateau, the Loess Plateau and the Qinghai–Tibet Plateau, accounting for approximately 85% of all sea buckthorn in China. H. tibetana (Tib) is a sea buckthorn endemic to the Qinghai–Tibet Plateau [China is rich in sea buckthorn resources, accounting for over 90% of the world’s total production . H. rham Plateau . Sea buc Plateau ,4, such C) content of Rha was 5-to 10-fold higher than that in Hippophae rhamnoides L. and H. rhamnoides subsp. Mongolica [E) content in Rha was remarkably higher than that in H. rhamnoides subsp. Mongolica [β-carotene, zeaxanthin and β-cryptoxanthin were dominant [trans-β-carotene and β-cryptoxanthin [C, VE and carotenoid content, in addition to the cellular antioxidant activity of Rha and Tib berries from the Qinghai–Tibet Plateau.Large differences in soluble sugars, organic acids, antioxidant components and antioxidant activities have been reported among sea buckthorn. It is reported that the soluble sugars of sea buckthorn are dominated by glucose and fructose, and organic acids include quinic acid, malic acid, citric acid and oxalic acid . The totongolica . Tocopheongolica . Previoudominant . It has oxanthin . Meanwhioxanthin . What isoxanthin . The Qinoxanthin . AlthougC and VE; (4) determine the types and contents of carotenoids; (5) evaluate in vitro and Caco-2 cellular antioxidant activities.The objectives of the study were to: (1) measure the types and contents of soluble sugars and organic acids; (2) analyze the types and contents of phenolics and flavonoids; (3) examine the types and contents of VRha and Tib berries were collected from Luqu County, Gannan Tibetan Autonomous Prefecture, China, in September 2020 . Fruit branches measuring approximately 30 cm were cut from the plants, put into packaging boxes (2 kg/box) and transported to the laboratory.The human colorectal adenocarcinoma (Caco-2) cell line was purchased from Shanghai Bioleaf Biotech Co., Ltd. .The berries of Rha and Tib were frozen with liquid nitrogen, the branches were shaken to shake the fruits off and the frozen fruits were quickly ground into a powder using a mechanical grinder , and stored at −80 °C.g, 20 min). The extraction was repeated once and centrifuged, and the supernatant was collected and purified through a 0.22 μm filter. The analysis was performed using a high-performance liquid chromatograph  equipped with a differential refractive index detector from the same manufacturer. The separation was performed on a NH2 amino  column . Acetonitrile–water  at a 1 mL/min flow rate was used as the mobile phase at 30 °C. The standard calibration curve equations for glucose, fructose and sucrose were y = 203,913x − 9054.3 (R2 = 0.9992), y = 178,967x + 18,820 (R2 = 0.9992) and y = 204,997x − 8477.2 (R2 = 0.9993), respectively. The linear range of the calibration concentrations were 0.625, 1.25, 2.5, 5 and 10 mg/mL. Total sugars were obtained by summing the concentrations of the individual components. Soluble sugar content was expressed as mg/g FW (fresh weight).The soluble sugar content was determined by referring to the method of Petreikov et al.  with somg, 15 min, 4 °C) and purified through a 0.22 μm filter. A Waters ACQUITYArc system  equipped with a 2998 PDA photodiode matrix detector was used for the determination, and all separations were performed on a Hi-PiexH (300 × 7.7 mm) column (Agilent). The samples were analyzed with mobile phase 0.2 mM sodium dihydrogen phosphate (pH 2.7) at 210 nm and a flow rate 0.5 mL/min. The standard calibration curve equations were as follows: quinic acid: y = 578.57x + 6175.5 (R2 = 1); malic acid: y = 1871.7x + 20,660 (R2 = 0.9993); tartaric acid: y = 4540.1x − 13,373 (R2 = 0.9997); citric acid: y = 1550.3x − 7312.6 (R2 = 0.9991); and succinic acid: y = 153,773x − 225,757 (R2 = 0.9995). The linear range of the calibration concentrations was 30, 35, 40, 45 and 50 μg/mL. Total acids were obtained by summing the concentrations of the individual components. Organic acid content was expressed as mg/g FW.Organic acid content was determined by referring to the method of Coelho et al. . Frozen w/v) acetone for 10 min, and the supernatant was collected after centrifugation . The residue was re-extracted four times using the same procedures, and all supernatants were combined and evaporated at 45 °C using a rotary evaporator ; the free phenolic extracts obtained were dissolved in ultrapure water  for determination. The residue from the extraction of free phenolics was shaken at room temperature and digested with NaOH  for 1 h. The mixture was acidified to pH 2.0 with concentrated hydrochloric acid and extracted four times with ethyl acetate (30 mL), and the supernatant was obtained by centrifugation. Subsequently, the ethyl acetate fractions were pooled and evaporated at 45 °C using a rotary evaporator  to dryness, before being reconstituted in 20 mL of water and then stored at −80 °C until use. The phenolic and flavonoid results were mg GAE/100 g FW  and mg RE/100 g FW (rutin), respectively [Phenolic content was determined by referring to the method of Guo et al. . Specifiectively . Total pA Waters ACQUITYArc system (Waters) equipped with a 2998 PDA photodiode matrix detector was used for the determination, and all separations were performed on a ZORBAXSB-C18  column (Agilent). The samples were eluted with a gradient system consisting of solvent A (0.1% formic acid in water) and solvent B (acetonitrile), with a flow rate of 0.8 mL/min. The column temperature was maintained at 28 °C and the injection volume was 5 µL. The gradient elution program that followed was 0 min (10% B), 0–3.5 min (10–13% B), 3.5–4 min (13–15% B), 4–7.5 min (15–18% B), 7.5–8.3 min (18–20% B), 8.3–8.5 min (20–25% B), 8.5–9 min (25–30% B), 9–11 min (30–30% B), 9–11 min (30–30% B). 9–11 min (30–35% B), 11–12 min (35–40% B), 12–13 min (40–42% B), 13–15.5 min (42–44% B), 15.5–15.8 min (44–50% B), 15.8–16.1 min (50–55% B), 16.1–17 min (55–60% B), 17–18 min (60–65% B), 18–21 min (65–70% B), 21–25 min (70–90% B), 25–30 min (90–10% B) and 30–37 min (90–10% B). The phenolic compounds were identified via comparison with the retention time of the standard using PDA detection at 280 nm and 360 nm, respectively, and individual phenolic content was estimated on the basis of the peak area and the calibration curves of the corresponding standards. Phenolic content was expressed as mg/g FW.C content was determined by referring to the method of Sytařová et al. [w/v) oxalic acid; the supernatant was harvested after centrifugation  and was fixed with 1% oxalic acid to 1 mL and purified through a 0.22 µm nylon membrane. A Kromasil C18  column  was used for HPLC  analysis. HPLC conditions were as follows: mobile phases A was 0.1% oxalic acid and mobile phases B was methanol in the ratio of 95/5 (v/v). The flow rate was 0.8 mL/min, column temperature was 25 °C and the injection volume was 5 µL. The UV detection wavelength was 245 nm. VC was characterized by retention time with standard compounds and quantified by peak area and calibration curve. The concentration of VC in the sample was calculated with a standard curve. VC content was expressed as mg/100 g FW.Vá et al.  with a sE content was determined by referring to the method of Tkacz et al. [g, 10 min, 4 °C). N-hexane was then added to the centrifuged residue, and the supernatant was obtained by centrifugation. The process was repeated three times. Finally, all supernatants were combined and dried under a nitrogen stream; the residue was dissolved in 200 μL methanol and purified by passing through a 0.45 µm organic membrane. A YMC C30  column  was used for ultrahigh-performance liquid chromatography  equipped with a fluorescence detector from the same manufacturer for analysis. The HPLC conditions were as follows: ultrapure water (A) and acetonitrile (B) as the mobile phases; gradient elution at 0–15 min, 30% A and 70% B; at 15–25 min, 80% A and 20% B; at 25–30 min, 30% A and 70% B; and at 30 min, 100% A and 0% B. The flow rate was 1 mL/min, column temperature was 40 °C and the injection volume was 2 µL. The fluorescence detector was set at 294 nm for excitation and 328 nm for emission. VE was characterized by retention time with standard compounds , and quantified by peak area and calibration curve. The concentration of each tocopherol in the sample was calculated with a standard curve. VE content was expressed as mg/100 g FW.Vz et al. . Frozen v/v/v, containing 0.1% BHT) for 30 min in the dark; the supernatant was harvested after centrifugation . The supernatant was mixed with 10% ethanol and potassium hydroxide for 1 h, and washed twice with ultrapure water (saponification), which was the carotenoid extract. The extract was dried with a centrifugal vacuum concentrator  and dissolved in 0.2 mL of the acetone:methanol  mixture, and then purified by passing it through a 0.45 µm filter. A YMC C30  column (YMC-Europe GmbH) was used for ultrahigh-performance liquid chromatography  with the mobile phase comprising methanol/methyl-tert-butyl ether (MTBE)/water  (A)-methanol (B) at a 450 nm wavelength. The elution was performed as described below: at 0–15 min, 100% A and 0% B; at 15–25 min, 39% A and 61% B; at 25–30 min, 100% A and 0% B; and at 30 min, 100% A and 0% B. The injection volume was 2 µL, flow rate was 1.0 mL/min and column temperature was 40 °C. The total carotenoid was obtained by summing the concentrations of the individual components. Carotenoid content was expressed as mg/100 g FW.Carotenoid content was determined by referring to the method of Meléndez-Martínez et al. . Frozen g, 15 min, 4 °C) for determination.Antioxidant substances were extracted from the frozen powder of the berries (0.1 g) using ultrapure water (1 mL); the supernatant was harvested after centrifugation (8000× 2•−) radical scavenging capacity were analyzed according to the methods of Brand-Williams et al. [The DPPH, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and superoxide anion , 1% L-glutamine, 1% penicillin  and 1% streptomycin . Cells were cultured in a humidified atmosphere at 37 °C in 5% CO2 .4 cells/well) were sucked into each well of a 96-well plate and incubated at 37 °C and 5% CO2 for 24 h. Then, 20 μL solutions of various concentrations of sea buckthorn solution were added and placed under the same conditions for 24 h, with 20 μL of the serum-free cell culture medium used as the control group. Thereafter, the cell culture was removed and the wells were washed twice with DMEM; then, 10 μL of CCK-8 solution was added to each well and incubated for 4 h. The absorbance value was measured at 450 nm and cell viability was expressed as: cell viability (%) = (A1/A0) × 100%, where A0 represents the absorbance value of the control cells and A1 represents the absorbance value of the treated cells.Cytotoxicity was measured using a CCK-8 agent. In the presence of electronically coupled reagents, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium monosodium salt) was reduced by dehydrogenase in the mitochondria to produce a highly water-soluble orange–yellow formazan product, whose color depth was inversely proportional to cytotoxicity. Frozen powder (10 mg) was dissolved in a serum-free cell culture medium (1 mL) and evenly mixed. After sterile filtration, the dilution concentrations were 0.15625, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mg/mL. According to the Cell Counting Kit 8  method, Caco-2 cells  (25 μmol/L) and they were added in triplicate wells and incubated for 1 h. For the ‘PBS wash’ protocol, cells were washed with PBS; for the ‘no PBS wash’ protocol, cells were not washed prior to adding ABAP. The purpose of washing with PBS was to remove the interference of extracellular antioxidants. Finally, 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP)  was added into each well, the purpose of ABAP treatment of cells is that ABAP disperses in the cells and spontaneously decomposes to form peroxyl radicals, which attack the cell membrane to generate more free radicals or reactive oxygen species. The fluorescence value for 1 h was recorded every 10 min with the emission wavelength at 538 nm and excitation at 485 nm. The CAA value was expressed as µmol QE/100 g FW.CAA was measured following the method of Kellett et al. . CAA wasp < 0.05).All analyses within an experiment were conducted using three biological replicates. Excel 2021 was used to calculate the mean and standard error (±SE). OriginLab OriginPro 8.5  was used for drawing. Duncan’s multiple difference significance analysis at a 5% level was performed using IBM SPSS Statistics 24.0   were found in Rha and Tib berries. Among these, flavonols were dominant with 0.2069 ± 0.0375 mg/g FW, followed by flavanols, phenylpropanoids and benzoic acid derivatives  in Rha. Among the phenolics analyzed, the content of catechin in Rha was the highest (0.1345 ± 0.0085 mg/g FW), followed by isorhamnetin, quercetin and chlorogenic acid  in Rha. Tib showed the same trend as Rha. The gallic acid and protocatechuic acid content was lower than the results reported for four subspecies of sea buckthorn  from China. The contents of catechin, isorhamnetin and quercetin were in alignment with the results reported for four subspecies of sea buckthorn from China [In total, twelve phenolic compounds  were divom China . The genom China . The pheom China ,30. The om China . Furtherom China . TherebyC has a very high antioxidant effect [C content of Rha was 1080.30 mg/100 g, which was 836% higher than that of Tib  varieties [trans-β-carotene and β-cryptoxanthin were dominant [cis-epoxycarotenoid dioxygenase led to the difference in carotenoid levels among sea buckthorn berries [The total carotenoid content of Tib was 59.56 mg/100 g, which was 73% higher than that of Rha. Notably, Rha contained traces of otenoids . The cararieties . The dif berries . Thereby2•− free radical scavenging activity and FRAP are common indices to evaluate antioxidant capacity. Both Rha and Tib showed a good in vitro antioxidant activity, with the strongest scavenging activity being exhibited by ABTS, at 95.68 and 33.23 mg TE/g FW, followed by FRAP, DPPH and O2•−, in that order. The DPPH, ABTS and FRAP of Rha were 120%, 187% and 341% higher than those in Tib, respectively  and VE was weakly correlated with DPPH. Correspondingly, β-carotene and lycopene also scavenge ABTS free radicals [C and VE. DPPH, ABTS, Oectively , which iradicals . Tocopheradicals . Furtherradicals . It is rith DPPH , the synith DPPH ; VC afferadicals . These rCaco-2 cells are morphologically and structurally similar to small intestinal epithelial cells, suggesting that they may be a superior indicator of antioxidant activity in vivo . The cytC. In general, the total contribution of isorhamnetin, quercetin and kaempferol in the CAA was around 20% [C in Rha is an important reason for its high CAA value.The CAA method is a reasonable method for assessing antioxidant activity based on cell culture models, reflecting the absorption and metabolism of antioxidants, and the results have high biological relevance. Additionally, this method includes a ‘PBS wash’ method and a ‘no PBS wash’ method to evaluate the degree of uptake and membrane association of antioxidants . When thound 20% , since tound 20% . This phound 20% . The aboC and VE  of Rha were remarkably higher than those of Tib. The total carotenoid, lutein, β-carotene and lycopene contents of Tib were notably higher than that in Rha. Furthermore, both Rha and Tib showed good in vitro and cellular antioxidant activity, and Rha significantly outperformed Tib in terms of scavenging DPPH, ABTS free radicals, FRAP and its antioxidant capacity against Caco-2 cells. Thus, Rha berries could be considered for special medical or functional food processing due to their strong antioxidant activity.The total sugar, fructose, total acid, quinic acid, total (free and bound) phenolic, and total (free and bound) flavonoid contents of Rha were considerably higher than that in Tib. Twelve phenolic compounds were analyzed, among which flavonols were dominant. Catechin, isorhamnetin, quercetin and chlorogenic acid were the main phenolic substances. The V


Dracaena angustifolia Roxb has been used as an anti-inflammatory agent by the Li people in Hainan, China. In preliminary phytochemical studies conducted in our lab, its fractions were found to inhibit α-glucosidase in vitro, indicating a potential for alleviating glucose dysregulation.As a traditional herbal medicine, Through in vitro enzymatic assays, the abilities of the separated components to affect α-glucosidase and α-amylase were evaluated. By establishing concentration gradients and generating Lineweaver–Burk plots, the corresponding inhibition modes together with kinetic parameters were assessed. Following the evaluation of the outcomes of their combination with acarbose, computational docking and molecular dynamic simulations were carried out to analyse the interaction mechanisms and perform virtual screening against human enzymes.Compared with acarbose, 7 compounds, including flavonoid derivatives, amides and aromatic derivatives, with higher α-glucosidase inhibitory efficiencies were confirmed. It was found that those competitive/mixed candidates and acarbose interacted synergistically or additively on α-glucosidase. Moreover, 3 of them were able to inhibit α-amylase in mixed mode, and additive effects were observed in combination with acarbose. Through in silico docking, it was found that the active site residues as well as adjacent residues were involved in α-glucosidase and α-amylase binding, which were mainly achieved through hydrogen bonding. Among those dual-function flavonoids, Compound 9 was predicted to be a considerable inhibitor of human enzymes, as the formation of ligand–enzyme complexes was mediated by the residues responsible for substrate recognition and catalysis, the stabilities of which were reiterated by molecular dynamics simulations.Dracaena angustifolia Roxb in diabetes treatment.Despite their mild effects on α-amylase, considerable α-glucosidase inhibitory efficiencies and potential synergy with acarbose were exhibited by these natural candidates. Furthermore, a stable ligand, human α-glucosidase, was predicted by the performed simulations, which provided useful information for the application of The online version contains supplementary material available at 10.1186/s12906-022-03649-3. Type II diabetes mellitus T2DM) is a chronic disease that causes long-term body damage and mental issues. T2DM is diagnosed by persistent hyperglycaemia, which is caused by the insufficient secretion of insulin or insulin resistance . AlthougDM is a cRehmannia glutinosa, an essential constituent of Xiao Ke Wan  [Dracaena plants [Isolated compounds from pharmaceutical plants are considered to be alternatives. As indicated in clinical trials and laboratory studies, equivalent efficiency and improved safety could be ensured by monotherapy with those antidiabetic compounds or in combination with reduced acarbose , 11. Furiabetes) . Activita plants , 15.Dracaena genus, Dracaena angustifolia Roxb is an evergreen shrub widely distributed in tropical areas, and it is utilized by the Li people in China’s Hainan province as an herbal remedy [D. angustifolia Roxb [D. angustifolia Roxb in antidiabetic drug development, the IC50 values of each separated substance against α-glucosidase were initially evaluated. Compared to the reference acarbose, a total of seven compounds  with better inhibition efficiencies, ranging from 1.99 μM to 0.65 mM, were confirmed. Then, the corresponding standard curve and Lineweaver–Burke plots were constructed to investigate the corresponding inhibition models, and it was found that Compound 18 with the lowest IC50 and Compound 5 were an uncompetitive inhibitor and a noncompetitive inhibitor, respectively. Competitive or mixed-type inhibition was observed for Compounds 6, 8, 9, 22, and 24, all of which were able to exhibit synergistic effects with acarbose in the subsequent combination assay. Moreover, those five compounds were subjected to α-amylase activities assays, and only the flavonoids  showed weak inhibitory actives, with IC50 values two orders of magnitude higher than that of acarbose (10 μM). As revealed by kinetic and synergism analyses, the three compounds acting as mixed inhibitors were able to enhance the inhibition of acarbose against α-amylase. The docking technique was employed to assess the possible molecular mechanism. In addition to the active site residues and the adjacent residues, HIS112, GLU411, and ASP442 were thought to contribute to the interactions between those small ligands and α-glucosidase. For porcine α-amylase, GLN63 was important for the inhibitory activities of Compounds 6 and 9. Then, docking was repeated to assess the compounds that displayed inhibitory effects on α-glucosidase and α-amylase, in which homologous enzymes of humans were chosen. Compound 9 generated the best configuration, by which four sites for substrate binding  in human α-glucosidase, part of the catalytic sites and one residue for calcium binding  in human α-amylase were affected. With the information created by the molecular dynamics simulation, the stabilization of the human α-glucosidase–Compound 9 complex was confirmed. This study provides useful information for further in vivo research and antidiabetic drug discovery regarding D. angustifolia Roxb.As a no-resin-producing species of the l remedy . Accordilia Roxb , 18. Ourlia Roxb , and beiD. angustifolia Roxb were prepared as reported by Zhao et al. [Saccharomyces cerevisiae), and α-amylase (porcine pancreas) were purchased from Sigma–Aldrich. 4-Nitrophenol (pNP), 4-nitrophenol-α-D-glucopyranoside (pNPG), soluble starch from potato and other reagents were purchased from common commercial suppliers and used as received.Bioactive compounds from the stems of o et al. . 2S)-4′S-4′D. anD. angustifolia Roxb, which were diluted to 4 mM. After being kept at 37 °C for 10 min, 25 μL of α-glucosidase (0.5 U/mL) was added. Following another 10 min incubation at 37 °C, the reaction was stopped with 125 μL of 0.2 M Na2CO3, and the absorbance at 410 nm of each sample (converted pNP) was measured via a microplate reader . A standard curve was generated using serially diluted pNP, acarbose and assay buffer as positive and blank controls. The percent inhibition was calculated by the following formula: % inhibition = (1 - ASample / AControl) × 100%. For the compound with a greater inhibition rate (vs. the same amount of acarbose), IC50 values were determined by plotting the concentration gradient against the corresponding inhibition percentage.α-Glucosidase inhibitor screening was conducted according to a previously reported procedure with slight modifications . A measu2: 2% KI), and the optical density at 580 nm was directly proportional to its concentration. Their influences on α-amylase were calculated as percent inhibition according to the equation % inhibition = (APositive - ASample)/(APositive - ABlank) × 100%, where APositive and ABlank are the absorbance values of PBS buffer containing acarbose or without compounds from D. angustifolia Roxb. A starch calibration curve ranging from 0.0625 to 4 mg/mL and a plot of inhibition rate versus compound concentration were prepared to derive corresponding IC50 values.Changes in α-amylase activities were quantitatively analysed via a method based on starch-iodine colorimetric detection . Soluble50 value) was quantified to create Lineweaver–Burk plots. The equations were rearranged into double reciprocal forms:To acquire the inhibition type and related parameters of those inhibitors, kinetic assays were carried out, and the conditions of enzymatic reactions are detailed in the above section, in which the amount of enzymes was fixed. With an increasing amount of substrate, the amount of hydrolysed pNPG or starch in the presence of compounds  of acarbose and chosen compounds were combined using freely available software  [D)1 and (D)2 are the doses of sample and acarbose that inhibit a certain level of enzyme in the combination system, respectively, and (Dx)1 and (Dx)2 are the doses of a single inhibitor that produce the same level of inhibition. When the CI values were above 1.1, between 0.9 and 1.1, and below 0.9, the effects were defined as antagonistic, additive, and synergistic.Three concentrations  . A combiHomo sapiens (PDB IDs: 5NN8 and 2QV4) are available in the protein databank (www.rcsb.org/pdb). 3D structures with minimized energy of Compounds 6, 8, 9, 22, and 24 were sketched using Chem3D . Prior to docking, water molecule removal, Gasteiger charge addition, and the addition of hydrogen were carried out. Compounds were incorporated as ligands, whose bond rotations were set as default. The docking region was placed in the sites covering the active residues. For the α-glucosidase from Saccharomyces cerevisiae, a grid box, with a spacing of 30 Å, was centred at the coordinates of 20.159, − 6.004, and 22.986. For porcine pancreatic α-amylase, the X, Y, and Z centres were 36.436, 37.487, and − 2.166, respectively, with a spacing of 30 Å. For enzymes in human saliva and the pancreas, docking boxes with a spacing of 30 Å were generated, whose centres were defined as − 14.312, − 38.186, and 95.627 and 12.805, 46.042, and 25.979, respectively. Subsequently, the Broyden-Fletcher-Goldfarb-Shanno algorithm was chosen, and 50 independent calculations were conducted. Enzyme-compound complexes with the lowest binding energies were extracted, and the superimposed diagrams were analysed using PyMOL 2.6 [The mechanisms of action and possible interactions were predicted by AutoDock Vina . CrystalThe human enzyme–Compound 9 complexes were subjected to molecular dynamics simulations. After being separated, human α-glucosidase and α-amylase were prepared using Gromacs , in whicEach experiment was performed in triplicate, and the data were interpreted as the mean ± standard deviation (SD). Statistical analysis was carried out by one-way analysis of variance (ANOVA) using Origin 9.0 software.D. angustifolia Roxb were rendered for the preliminary assay, in which compounds with poor aqueous solubilities were excluded, and only 7 of them were found to have higher potency than acarbose (4.07 ± 0.516 mM). As shown in Fig. 50 values at low micromolar levels for Compounds 18 (1.99 ± 0.2 μM) and 22 (40 ± 4.0 μM), which are phenoic derivatives that have been briefly mentioned in the phytochemical screenings of Crataegus pinnatifida Bge and Polygonum aubertii Henry [50 values of 0.24 ± 0.03 mM, 0.37 ± 0.02 mM, 0.52 ± 0.01 mM, 0.65 ± 0.01 mM, and 0.78 ± 0.03 mM, respectively.Despite the lack of scientific evidence confirming the genetic susceptibility to T2DM in the Asian population, a robust association between inappropriate dietary habits and the disease incidence is undoubtable . Especiaii Henry , 30, werSince the mechanism underlying the postprandial blood glucose control of acarbose and other approved α-glucosidase inhibitors is the targeting of the enzyme in a competitive manner, the corresponding reversibility should be emphasized. Modes of their inhibition were distinguished based on kinetic analyses, and it was demonstrated that the type for Compound 18 showing the most effective inhibitory activity was uncompetitive Fig. A. As a r50 value.In addition to the verification of the competitive behaviour of Compound 8, others were found to be mixed inhibitors. For a further comparison, their kinetic constants were calculated. As listed in Table D. angustifolia Roxb on α-glucosidase activity were determined. As shown in Fig. To identify candidates that could be potential substitutes for acarbose or complementary, the impacts of the combination of acarbose with competitive or partly competitive inhibitors from 50 value of acarbose against α-amylase was found to be 10.25 ± 0.01 μM, while mild α-amylase inhibition was recorded; only flavonoid compounds from D. angustifolia Roxb  had IC50 values at the millimolar level   method usually used for the detection of converted maltose was found to be not acceptable . The int50, was supposed to be more probable  of α-glucosidase and all of the active site residues (ASP197 and GLU233) of α-amylase  and − 242,119.5 Kcal/mol (α-amylase–Compound 9). The details of the corresponding energy graphs, having minimal variations, are displayed in Fig. D. angustifolia Roxb have been verified, in which the functions of bioactive components are strongly related to their chemical structures. The previous phytochemical assay and the preliminary assessment of enzyme inhibition recalled the existence of substances that might interrupt carbohydrate digestion. In subsequent research, more efficient α-glucosidase inhibitors  were identified (with acarbose as a reference) and were proven to exhibit uncompetitive, competitive, noncompetitive, or mixed manners. While their abilities to inhibit α-amylase activities were unremarkable, only three of them  were considered to be bifunctional. As revealed in the computational docking, the mechanism of α-glucosidase inhibition was attributed to binding to residues HIS112, ARG213, GLU277, GLN279, ASP352, GLU411, and ARG442. Relatively weak hydrogen bonds with residues GLN63, ASP197, HIS299, and ASP300 were essential for α-amylase inhibition. Among compounds with dual inhibitory activities, Compound 9 was characterized by good redocking performance. Despite detecting a higher variation in RMSD values of human α-amylase–Compound 9, the possible human α-glucosidase inhibition of this compound isolated from D. angustifolia Roxb is worth being investigated in future research.As a representative of Li folk medicine, the bacteriostatic, antitumor, and anti-inflammatory features of Additional file 1.


Increased heart rate has been associated with stroke risk and outcomes. The purpose of this study was to explore the long-term prognostic value of initial in-hospital heart rate in patients with acute ischemic stroke (AIS).We analyzed data from 21,655 patients with AIS enrolled (January 2010–September 2018) in the Chang Gung Research Database. Mean initial in-hospital heart rates were averaged and categorized into 10-beat-per-minute (bpm) increments. The primary and secondary outcomes were all-cause mortality and cardiovascular death. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using multivariable adjusted Cox proportional hazard models, using the heart rate < 60 bpm subgroup as the reference.The adjusted HRs for all-cause mortality were 1.23 (95% CI 1.08–1.41) for heart rate 60–69 bpm, 1.74 (95% CI 1.53–1.97) for heart rate 70–79 bpm, 2.16 (95% CI 1.89–2.46) for heart rate 80–89 bpm, and 2.83 (95% CI 2.46–3.25) for heart rate ≥ 90 bpm compared with the reference group. Likewise, heart rate ≥ 60 bpm was also associated with an increased risk of cardiovascular death .High initial in-hospital heart rate is an independent predictor of all-cause mortality and cardiovascular death in patients with AIS.The online version contains supplementary material available at 10.1007/s00392-021-01953-5. Despite improvements in secondary prevention treatment, the incidence rates of mortality and recurrent cardiovascular events post-stroke remain high –3. This A slower heart rate is associated with greater longevity in many mammal species . Heart r2) = 186 × (serum creatinine)−1.154 × (age)−0.203 × 0.742  [2) [We conducted this retrospective cohort study using data from the Chang Gung Research Database , the lar female) . ChronicThe original cohort consisted of 41,241 patients aged ≥ 18 years. The exclusion criteria were patients: (1) hospitalized for less than 3 days; (2) admitted to a rehabilitation unit; (3) admitted through outpatient clinics; (4) with recurrent stroke during the study period; and (5) with less than one record of heart rate per day in the first 3 days of hospitalization. In addition, those without catastrophic illness cards were also excluded  . FinallyThe primary outcome was all-cause mortality, and the secondary outcome was cardiovascular death. We linked to the National Registry of Deaths Database provided by the Ministry of Health and Welfare in Taiwan from January 1, 2010 to December 31, 2018. The database includes death certificates coded using ICD-10 codes. Cardiovascular diseases were classified as ICD-10 codes I00–I99.Descriptive statistics are presented as number (percentage) for categorical data and mean (standard deviation) and median (interquartile range) for continuous data. The patients were classified into five subgroups according to mean heart rate . Differences between the groups were tested using the Kruskal–Wallis rank test for continuous data and the Chi-square test for categorical data. In addition to crude hazard ratios (HRs), adjusted HRs and 95% confidence intervals (CIs) were calculated with reference to the lowest risk group and estimated after adjusting for potential confounding factors in the Cox proportional hazard models. Model 1 included age, sex, and eNIHSS, and model 2 included age, sex, eNIHSS, history of hypertension (HTN), diabetes mellitus (DM), dyslipidemia, atrial fibrillation (AF), congestive heart failure (CHF), cancer before admission, smoking status, beta blocking agents, body mass index (BMI), total cholesterol, triglycerides (TGs), CKD stage, ALT, HbA1c, mean SBP, and mean DBP.Data analysis was conducted without imputing missing data. Variables with missing data were classified into a missing data category to minimize the effect of the missing data in the analysis.Interactions between mean heart rate and age, sex, eNIHSS, history of HTN, DM, dyslipidemia, AF, CHF, cancer before admission, smoking status, beta blocking agents, BMI, total cholesterol, TGs, CKD stage, ALT, HbA1c, mean SBP, and mean DBP at baseline were tested. Subgroup analyses were performed with heart rate as a continuous variable and when interactions were significant even after adjusting for the same variables as in the Cox proportional hazard model (model 2). HRs and 95% CIs for each subgroup were calculated for every one standard deviation increment in heart rate. A Cox model with restricted cubic spline smoothing technique was used to explore the overall trend of risks through the range of mean heart rate values. All analyses were performed with SAS  and R (version 4.0).A total of 21,655 adult patients with AIS were included in the analysis . The mean SBP and DBP were 151.04 ± 19.64 and 84.81 ± 11.17 mmHg, respectively, and the mean heart rate was 74.84 ± 11.27 bpm. There were 436,376 measurements of heart rate in this study. The median number of measurement per patient was 13 (interquartile range 9–18). The demographic data and baseline characteristics of the overall patient cohort and for each 10-bpm-increment heart rate subgroup are given in Table Crude and adjusted HRs for mean heart rate are given in Fig. Besides a high mean heart rate, age, male sex, eNIHSS, DM, CHF, history of cancer before admission, and CKD stages 2–5 were all positively associated with the risk of all-cause mortality. Conversely, a history of dyslipidemia, higher BMI, total cholesterol, ALT, and mean DBP levels showed a protective effect (Table Compared with the reference group (mean heart rate < 60 bpm), the adjusted HRs for cardiovascular death in model 2 were 1.18 (95% CI 0.95–1.46) for mean heart rate 60–69 bpm, 1.57 (95% CI 1.28–1.93) for mean heart rate 70–79 bpm, 1.98 (95% CI 1.60–2.45) for mean heart rate 80–89 bpm, and 2.36 (95% CI 1.89–2.95) for mean heart rate ≥ 90 bpm Below is the link to the electronic supplementary material.


Chelon ramada and the flathead grey mullet Mugil cephalus) have been stocked in the Sea of Galilee (Lake Kinneret) in order to increase fishermen’s income and lake water quality. These catadromous species do not reproduce in the lake, consequently, fingerlings have been introduced every year since 1958. Few additional mugilid species have been introduced unintentionally together with these two species, including C. labrosus. Following a survey of myxozoan infections in the Sea of Galilee, we described Myxobolus pupkoi n. sp. infecting the gill arches of C. labrosus, and reported Myxobolus exiguus from visceral peritoneum and gall bladder of C. ramada. Our study indicates that the parasites infecting C. ramada and C. labrosus belong to a lineage of myxozoans infecting mugilids. This result suggests that the infection took place in the Mediterranean Sea, where the fingerlings were caught, before their introduction into the Sea of Galilee. Since 2018 only farm-raised fingerlings have been introduced. We thus recommend to closely monitor the presence of these parasites in the future to determine if the presence of parasites disappear with the introduction of farm-raised fingerlings.Mullets (Mugilidae) are economically important fish in Israel. Two species of mugilids (i.e., the thinlip mullet  In Israel, two mullet species  have been artificially introduced in large numbers since 1958 in the Sea of Galilee (Lake Kinneret), the largest freshwater body in the Levant. The fish were introduced in order to lower the algal population and increase the annual fish yield3. Specifically, until 2018, mugilid fingerlings collected along the Mediterranean coast and in coastal streams were introduced into the Sea of Galilee after a 1–2 days stay in freshwater ponds for adaptation4. From 1960 to 2018, about one million fingerlings of M. cephalus and C. ramada were introduced every year into the Sea of Galilee5. Since 2018, the number of fish introduced annually has been reduced to only a few tens of thousands of M. cephalus fingerlings, originating from breeding farms .Mugilidae is a large fish family that includes 17 genera and 80 species. Mullets are present worldwide, mainly in coastal shallow marine and brackish waters of the tropical and temperate regions. They are an appreciated food resource with high commercial value8. Myxozoans form a clade of highly-reduced, microscopic, endoparasites Cnidaria. They have complex life-cycles involving two hosts, usually a fish (the intermediate host) and an annelid (the definite host)9. Over 90 myxozoan species have been reported to infect mullets11. The majority of myxozoans that parasitize mullets belong to the family Myxobolidae, including 50 species of the genus Myxobolus Butschli, 188213. Interestingly, while myxobolids are mainly freshwater species, both marine and freshwater myxobolids are known to infect mugilids10.Myxozoans are one of the main parasitic lineage infecting mulletsC. ramada specimen. A taxonomic and molecular study was performed to identify the parasites and determine whether they had a marine or freshwater origin. While Myxozoa are often host specific, a freshwater origin of the parasites could indicate a host switch. In contrast, a marine origin, would indicate that the fish and their parasites were co-introduced to the Sea of Galilee.During a survey of myxozoans infecting fish from the Sea of Galilee, we found myxozoan infections in the gall bladder, visceral peritoneum and gill arches of a few M. cephalus and twenty-three C. ramada specimens sampled in the Sea of Galilee on November 2020. While no myxozoan parasite was observed in M. cephalus, plasmodia were found in the gall bladder, in the visceral peritoneum lining the intestine and the gill arches of four C. ramada specimen. Each of the four fish was infected by a single parasite. No infection was found in other organs such as kidney, liver, eyes, brain, fins, muscles, and scales. The 18S rRNA sequences from the plasmodia from gall bladder and visceral peritoneum were identical to M. exiguus reported from Portugal (sequence accession number: MH236070)14, except for three insertions in the last 7 bp of the sequence, which most probably correspond to sequencing errors since most are located in the primer region. The parasite obtained from gill arches did not match any other myxosporean sequence available in the NCBI database.Myxozoan parasites were searched for in ten Plasmodia: Small plasmodia (about 0.5 mm), visible with naked eye, rounded, creamish white, freely floating in the bile or present in the visceral peritoneum lining the intestine, containing 300–500 myxospores per plasmodium.Myxospore: The morphological data are based on spores isolated from two plasmodia, one originating from the gall bladder and another from the visceral peritoneum. Few morphological differences were found between the spores of M. exiguus infecting the visceral peritoneum and the gall bladder of C. ramada.Mature gall bladder myxospores broadly oval to sub-spherical in valvular view and ellipsoidal in sutural view Fig. . They mePolar capsules pyriform and bottle-necked, equal in size and arranged side by side with two prominent pores at the anterior side of the myxospore measuring 3.79 ± 0.12 (3.54–3.97) µm in length and 2.54 ± 0.07 (2.41–2.67) µm in width ; 3.41 ± 0.11 (3.04–3.85) µm in length and 2.16 ± 0.05 (1.95–2.30) µm in width . Polar tubules form 3–4 coils while inside the capsule , vern. thinlip mullet, Family: Mugilidae.Locality: Sea of Galilee , Israel.Material deposited: Four slides with stained myxospores (SMNHTAU-AP-50-53) have been deposited in the parasite collection of the Steinhardt Museum of Natural History, Tel Aviv University, Israel.Infection site: Gall bladder bile and visceral peritoneum.Because of fish misidentification the prevalence of infection cannot be computed.Clinical signals: Whitish patches floating inside the gall bladder and in the intestine visceral peritoneum.Sequences deposited: Two identical sequences were deposited. GenBank accession numbers OM065835  and OL604467 .Remarks: The present observations on M. exiguus15 show that the spore size is smaller in the Israeli specimen than previously described14. Previous work reported M. exiguus15 from the visceral peritoneum of C. ramada, C. auratus, C. saliens, C. labrosus and M. cephalus from France, Tunisia and Portugal. A new locality—the Sea of Galilee, Israel—and a new tissue—the gall bladder—are here recorded for this parasite.Plasmodia: Small histozoic plasmodia, visible with naked eye, rounded, creamish white (about 1.0 mm in diameter), a single infected gill arch per fish with 2–4 plasmodia, 300–500 myxospores per plasmodium. Gills pale and mucous laden.Myxospore: The morphological data are based on the observation of myxospores isolated from a single plasmodium. Mature myxospores sub-spherical in valvular view and ellipsoidal in sutural view, measuring 5.84 ± 0.13 (5.67–6.14) µm in length and 5.41 ± 0.09 (5.30–5.53) µm in width (n = 12). Polar capsules pyriform, equal in size, and located at the anterior half of the myxospore measuring 2.44 ± 0.19 (2.25–2.73) µm in length and 1.34 ± 0.18 (1.14–1.80) µm wide (n = 12). Polar tubules making 4–5 coils while inside the capsule and measuring 7–10 µm when extruded; inter-capsular process absent; 8–9 sutural folds on the ring of the myxospore; sutural line thick, measuring 0.65 ± 0.03 µm and slightly curved; sporoplasm finely granular with two nuclei of similar size measuring 0.95 ± 0.04 µm, filling most of the extracapsular cavity , vern. thicklip grey mullet, Family: Mugilidae.Type locality: Sea of Galilee , Israel.Material: Two slides with stained myxospores (SMNHTAU-AP-48-49) have been deposited in the parasite collection of the Steinhardt Museum of Natural History, Tel Aviv University, Israel.Infection site: Gill arch.Because of fish misidentification the prevalence of infection cannot be computed.Clinical signals: Whitish plasmodia on the gill arch.Sequence: GenBank accession number OL605966 (2013 bp).Etymology: The specific epithet “pupkoi” has been given in honor of Prof. Tal Pupko, an expert in molecular evolution and bioinformatics, for his endless support, and encouragements.Remarks: For differential diagnosis, M. pupkoi n. sp. morphology, infection site and host have been compared with other Myxobolus species infecting members of the genus Chelon23 , M. cerveirensis (8.1 ± 0.2 × 6.8 ± 0.2 µm), M. labrosus (10.2 ± 0.2 × 8.1 ± 0.3 µm), M. muscularis (9.1 ± 0.6 × 7.0 ± 0.6 µm), M. parsi (9.1 × 8.1 µm), M. peritonaeum (8.1 ± 0.2 × 7.1 ± 0.2 µm), and M. pharyngobranchialis (9.3 ± 0.4 × 7.7 ± 0.4 µm), which have much larger spores. The new species is sub-spherical to spherical in shape, and hence differs from M. adeli, which is spindle shaped. The infection site of M. pupkoi n. sp. is the gill arch and hence it differs from M. episquamalis, which infects scales. It also differs from M. cerveirensis, M. exiguus, M. hepatobiliaris, and M. parvus, which infect various organs of the digestive track; and from M. renalis, which infects the kidney.The myxospores of M. parenzani, which has been described to have round myxospores (~ 5.4 × 5.4 µm). However, it differs from the present species in having larger polar capsules. The polar capsules of M. pupkoi n. sp. are 2.4 ± 0.20 µm long and occupy about half of the myxospores. Conversely, the myxozpores of M. parenzani have been described to be ~ 2 µm long, to be positioned along the membrane (rather than side by side) and to only occupy the upper third of the spore. Although M. pupkoi n. sp. and M. parenzani infect the same host the morphological differences mentioned above justify the definition of a novel species.The new species is very similar in size to 14.The phylogenetic analyses included 79 sequences of the 18S rRNA gene from the mugiliform-infecting lineage of myxobolid, as well as four outgroup sequences. The trees reconstructed based on maximum likelihood and Bayesian criteria only differ in the position of a few low-supported branches Fig. . These tMyxobolus ramadus and Myxobolus cochinensis . The second, which includes most species , is composed of three subclades. One subclade includes myxobolid lineages, infecting mainly fish hosts from the genus Chelon, and is where M. exiguus and the newly described species Myxobolus pupkoi n. sp. branch. The second subclade includes myxobolid lineages infecting mainly fish hosts from the genus Mugil. Both subclades include myxozoans from annelid hosts, for which the fish host is unknown .The phylogenetic tree divides the mugiliform-infecting lineage into two clades. The first harbors only M. pupkoi n. sp. and presents a novel tissue of infection, the gall bladder, for M. exiguus. It also reveals their presence in the Sea of Galilee. Morphologic and morphometric differences were observed between the myxospores of M. exiguus found in the visceral peritoneum and in the gall bladder, and their taxonomic identity could only be confirmed based on the 18S rRNA analysis. These data illustrate, once more, the importance of 18S rRNA sequences to complement the morphological identification of myxozoan species19, especially in the case of mugilids, which are infected with closely related myxobolid parasites10.The present study describes 27. Unfortunately, because of the small number of infected fish observed, the infected tissues were used for molecular identifications rather than histological preparation. Future work should determine, which tissue of the gill arch is the site of M. pupkoi infection , following Molnár et al.27. Myxobolus exiguus is a histozoic parasite described from the visceral peritoneum14. Its presence in the gall bladder bile, which is a coelozoic localization, is thus surprising. However, it is possible that the plasmodia found in the bile originated from the gall bladder wall or the hepatic bile ducts and thus is of a histozoic origin28.Tissue specificity is a characteristic of many histozoic myxozoans and an important taxonomical characterM. exiguus and M. pupkoi n. sp. belong to a well-defined clade of mugilid infecting myxozoans14. Members of this clade have also been found in marine, brackish and freshwater annelids present in estuaries29. Since mugilids are not native species from the Sea of Galilee and Jordan Valley system, it is most likely that these myxozoans were introduced to the Sea of Galilee with their mugilid hosts during stocking30, being the infection originated in the Mediterranean or the coastal plain estuaries of Israel from where the fingerlings originated before their introduction to the lake.Both 11. It was further suggested that the introduction of wild caught mugilid to new growing area may lead to the spread of their myxozoan parasites11. We here show that this prediction likely reflects the course of events that occurred in Israel where the transfer of infected fingerlings from the coastal environment led the presence of alien parasites in the Sea of Galilee.The fact that mugilid fingerlings are hosting numerous myxozoan parasites, sometimes even within a single individual, has been noted by Sharon et al.Myxozoans need two hosts to complete their life cycle, it may thus seem that myxozoans are not a serious threat to the Sea of Galilee since the annelid hosts were not transferred with the fish hosts. However, the establishment of a transmission cycle in that environment using local annelids species cannot be ruled out.Psammoryctides albicola as well as members of the genus Potamothrix (previously identified as Euilyodrilus)30. These freshwater annelids are known to harbor myxozoans closely related to the ones described in this work10  with a length of 18–20 cm, and Chelon ramada specimens (n = 23) with a length of 15–20 cm freshly collected from the Sea of Galilee, were obtained from a local fisherman and immediately transported to the lab. The fish sampling was performed under the authorization of the Fisheries and Aquaculture Department of the Israeli Ministry of Agriculture and Rural Development (authorization provided on 11.11.2020). The fish specimens were carefully examined externally for the presence of plasmodia followed by a dissection of each fish sample. Gills, scales, brain, kidney, visceral peritoneum, and other body parts were removed and examined under a stereo-microscope. When plasmodia were identified, fresh myxospores were photographed under a compound microscope with a DS-Ri2 Nikon photographic unit. The myxospores were stained with freshly prepared Ziehl–Neelsen and Giemsa solutions31. Spore measurements were done using a calibrated ocular micrometer.34 (Table M. exiguus and 34.7 ng/μL of M. pupkoi), 2.5 μL of 10X Taq buffer , 0.2 μL of Ex Taq polymerase , 2 μL of 20 µM dNTP mix , 2.5 μL of 5 pmol/μL of each primer , 0.2 μL of DMSO , 5 μL of 5 M Betaine , and 9.1 μL of molecular grade water. Amplification was performed using the following protocol: 1—initial denaturation at 94 °C for 5 min; 2—35 cycles of denaturation at 94 °C for 45 s, annealing of primers at 58 °C for 45 s, and extension at 72 °C for 2 min; 3—a final extension at 72 °C for 10 min. The obtained PCR products were purified using the ExoSAP method35.The samples for molecular work were fixed in absolute alcohol and stored at −20 °C. The extraction of genomic DNA was performed using the Qiagen DNeasy Blood and Tissue Kit according to the manufacturer’s instructions. PCR amplification of the 18S rRNA was performed using both universal and myxosporean specific primersSequencing was performed using the external primers ER1B1 and ER1B10 together with six additional primers Table  at the Dhttps://blast.ncbi.nlm.nih.gov/) on 05.09.2021. This allowed us to determine that both sequences belong to a distinct lineage of myxobolid infecting mugilids in the Histozoic III lineage sensus36. We downloaded all sequence hits from this clade that were longer than 800 bp. The sequences of Henneguya shaharini (EU643630), H. rhinogobii (AB447992), H. cynoscioni (JN017203), and Myxobolus khaliji (KC711053) were used as outgroups.To reconstruct the phylogenetic relationships of the Israeli species, we first blasted the obtained 18S rRNA sequences, against the NCBI nr databased  was used to align the edited sequences and to remove ambiguously aligned positions. Specifically, the sequences were aligned with the MAFFT algorithm under the options “Max-Iterate: 1000” and “Pairwise Alignment Method: localpair”. Positions with a score below 0.93 were removed as well as positions with more than 50% of missing data. Finally, we also removed positions corresponding to the annealing regions of the primers 18Se (5′-CTGGTTGATCCTGCCAGT-3′)38 and 18Sr (5′-CTACGGAAACCTTGTTACG-3′)39, the primers used to amplify most sequences in our sequence alignment40. The final dataset included 83 taxa and 1781 positions . The model selected based on the BIC criterion was the TVM + F + R3 model. Bayesian reconstructions were performed with PhyloBayes MPI version 1.842 under the CAT + GTR + GAMMA 4 model. Four chains ran for 55,000 points. The first 5000 trees from each chain were discarded as burn-in, and the consensus was built from trees sampled every 10 points . At the end of the run, the bpcomp maxdiff value was 0.051, indicating a proper convergence. We also verified that, for all parameters, the tracecomp eff size values were above 5000 and the rel_diff below 0.05, as recommended by the PhyloBayes manual.Phylogenetic relationships were reconstructed both under the maximum likelihood and the Bayesian criteria. Maximum likelihood analyses were performed with the program IQ-TREE version 1.6.12Supplementary Information 1.Supplementary Information 2.


In the dark or ambient light, LAKI is inactive. However, alternating transdermal illumination at 365 nm and 480 nm reversibly blocks and unblocks TREK/TRESK current in nociceptors, enabling rapid control of pain and nociception in intact and freely moving mice and nematode. These results demonstrate, in vivo, the subcellular localization of TREK/TRESK at the nociceptor free nerve endings in which their acute inhibition is sufficient to induce pain, showing LAKI potential as a valuable tool for TREK/TRESK channel studies. More importantly, LAKI gives the ability to reversibly remote-control pain in a non-invasive and physiological manner in naive animals, which has utility in basic and translational pain research but also in in vivo analgesic drug screening and validation, without the need of genetic manipulations or viral infection.By endowing light control of neuronal activity, optogenetics and photopharmacology are powerful methods notably used to probe the transmission of pain signals. However, costs, animal handling and ethical issues have reduced their dissemination and routine use. Here we report LAKI (Light Activated K Research on pain often relies on animals, and there is always a need for more precise and more ethical tools. Here, authors present a light-activatable molecule that induces pain in freely moving animal models in a reversible, non-invasive, and spatiotemporally defined way. Because of the low spatiotemporal resolution of the existing electrical, pharmacological, and genetic tools, the in vivo dissection of pain pathways has been limited2. Furthermore, the stimuli used are invasive, which is inherent to the study of nociception, and the effects are variable. Optogenetic and photopharmacological tools enable the control of action potential firing, in vitro and in vivo, through either the expression of exogenous opsin-proteins4 or the action of photopharmacological compounds, respectively6. Activation of these tools allows remote control of sensory neuron excitability non-invasively by transdermal illumination9 to modulate pain behavior, enabling control and test evaluations on the same animal with high spatiotemporal resolution. Although strongly facilitating the determination of cell and circuit function while reducing variability3, these approaches are neither widely nor routinely used due to various burdensome requirements for their use. The ideal tool would specifically modulate the nociceptor’s activity by controlling endogenous pain-related ion channels11 without the need for (i) genetic manipulation, (ii) viral infection, (iii) surgery, and (iv) extensive animal housing. In addition, this compound (i) would be in its inactivated state in the dark, enabling long-term experiments, (ii) would be ethically relevant by being non-invasive, (iii) would allow rapid, reproducible, and reversible control of pain in a physiological and pathological manner, and (iv) would be amenable to be used in different species. To obtain such a compound, we first need to identify a family of ion channels expressed in nociceptors that regulate their activity. Secondly, the photoswitch has to be designed and to be characterized for its properties and specificity. Ultimately, it needs to be tested in vivo on routinely used pain assays.Pain is an unpleasant sensory and emotional experience caused by noxious and/or potentially damaging stimuli2P) channels TREK1, TREK2, and TRESK, LAKI endows non-invasively a reversible and reproducible light-control of nociception in different freely moving animal models with a spatiotemporal resolution, for several days, without the need for transfection, infection, genetic manipulation or surgery procedures.In this work, we develop a light-activated antagonist of pain-related potassium channels (LAKI). By controlling the Two-Pore-Domain Potassium (K2P) channels TREK1, TREK2 and TRESK are highly expressed in nociceptors from the dorsal root ganglia (DRG) and trigeminal ganglia (TG)13. By being active at rest, TREK1, TREK2, and TRESK regulate nociceptor excitability by maintaining the membrane potential. Dysfunctional mutations inhibiting these channels in humans14 generate hyperexcitability of TG and DRG neurons underlying allodynia and migraine15, indicating their importance in pain induction. These properties make TREK1, TREK2 and TRESK channels suitable targets to regulate nociception.The two-pore-domain potassium   that is ~70% cis-LAKI. The thermal half-life of the cis-LAKI is 0.7 h at 21.5 °C and did not show photodegradation over ten cycles  Fig. . The ini2P channels, we tested whether it endows light sensitivity to TREK1, TREK2 and TRESK currents by expressing these channels in HEK293T cells. When we applied LAKI intracellularly, we did not observe any photomodulation of TREK and TRESK currents  it is inactive in the dark or ambient light, (ii) a brief pulse of light at 365 nm induces an efficient photoblock, (iii) the block is stable for long periods in the dark or ambient light, and (iv) a brief pulse of light at 480 nm rapidly removes the photoblock.15 but also acute TREK closing is sufficient to generate nociceptor hyperexcitability. Furthermore, as primary nociceptors are the first neurons involved in the complex processing system that evokes normal and pathological pain, LAKI represents an attractive tool to remote-control pain signaling with high spatiotemporal precision but without delay or the need for genetic, viral, or surgical manipulations.We next addressed the functional effect on neuronal excitability of the light-dependent block and showed that alternating illumination between 365 and 480 nm increased and decreased the firing rate properties of nociceptors Fig. . This re19, making this system suitable for photopharmacological experiments on pain20. Topical application of vehicle solution or LAKI to the corneal surface of the mouse eye   Fig. . Further21) Fig. , validatAs TREK-TRESK acute closing in the sensory terminals triggers spontaneous pain, we wondered if we could use LAKI to specifically target TREK-TRESK in the most commonly used stimulus-evoked pain tests, which mimic studies of enhanced pain in humans. Notably, we looked at the ability of LAKI to trigger an enhanced response to noxious stimuli  or a nociceptive response to innocuous stimuli .22. After LAKI injection, mice were allowed to freely explore a chamber with a transparent floor. In the absence of light, LAKI did not modify the paw withdrawal latency compared to mice having received saline solution (P > 0.39), showing that LAKI is not active at rest and does not disrupt the normal physiology of mouse thermal perception  before the thermal stimulus induced a decrease of the paw withdrawal latency   Fig. .P > 0.74) due to the relaxation of LAKI to the trans-state in the dark. LAKI can be reactivated for further cycles following a new pulse of light at 365 nm without any loss of effect   but following a transdermal 20 s UV light pulse, we observed a ~6-fold decrease of the paw withdrawal threshold  , further supporting TREK channel involvement  Fig. . Similar98) Fig. . This UV27. To determine their activity at the single fiber level in vivo, we used the same injected mice used for the Von Frey test to investigate the effect of LAKI activation on the response of the spinal dorsal horn neurons following electrical stimulation of their receptive field. The evoked response, i.e. the number of delayed C-spikes induced by the stimulation, was increased by LAKI when activated by transdermal illumination before stimulation 31.Despite the relative simplicity of their nervous system, invertebrates share with vertebrates common genetic mechanisms regulating acute and chronic nociception32  induced an approximately four fold increase in the Omega turn numbers compared to control C. elegans (29.79 ± 4.35 vs 7.50 ± 1.32)   Fig. . Third, 12) Fig. . This be12) Fig. , support12) Fig. . This siAKI Fig. . This anAKI Fig. .Fig. 5LAC. elegans. This behavior can be considered nociception-related because UV light is deadly for worms and the response is reduced by analgesics. This demonstrates the conservation of the function of K2P channels in pain signaling within the animal kingdom as observed for other ion channels such as TRP channels33. More importantly, we found that the LAKI sensitization of worms to UV light-induced Omega turns can be used as an easy read-out for the development of a fast, inexpensive, and robust in vivo HTS method for analgesic drug discovery.Taken together, these results demonstrate that TREK/TRESK channel orthologs may be involved in the escape behavior of C. elegans are transducers in conserved pain perception pathways. More importantly, thanks to the localization and function of TREK-TRESK and LAKI light sensitivity, LAKI can be used to remotely control pain with many advantages. With only a simple injection or incubation, LAKI endows a reversible and reproducible light-control of nociception in different freely moving animal models, for several days, without the need for transfection, infection, genetic manipulation, or surgery procedures, and is adaptable to several species. Moreover, being inert in the dark and stable LAKI is not invasive, making it, with its stability in vivo for at least one week and bi-stability upon UV light illumination, compatible with long-term experiments35. These features make LAKI an appropriate tool in accordance with animal welfare rules by reducing animal housing and distress caused by existing methods for pain induction, and by improving the repeatability and reducing variability .To summarize, we synthesized LAKI, an easy, stable, and specific light-activated inhibitor of TREK1, TREK2 and TRESK channels. This photopharmacological tool enabled us to functionally map TREK/TRESK channels at the free nerve endings of both TG and DRG nociceptors. Importantly, we found that local acute channel closing in nerve endings generates instantaneous pain as well as hyperalgesia or allodynia, demonstrating the key role of TREK/TRESK in pain. Furthermore, LAKI enabled us to demonstrate that TREK/TRESK orthologs present in other species such as Finally, LAKI is ideal for basic and translational pain research, providing straightforward and reproducible control of pain with spatiotemporal resolution in freely moving animals, and for in vivo analgesic high throughput drug screening in worms before validation in mammals.3) and acetonitrile (CD3CN) with Varian (Innova) or Bruker (Neo) spectrometers, at 400 or 500 MHz for 1H spectra and 101 or 151 MHz for 13C spectra. Spectra were calibrated to residual solvent peaks as reported by Fulmer et al.36. UV-Visible absorption spectroscopy was performed using a Vernier UV-VIS Spectrophotometer and the spectra were analyzed using Logger Lite 1.9.4. HR-MS was obtained in ESI mode from the Mass Spectrometry Lab operated by the School of Chemical Science at the University of Illinois Urbana-Champaign.Reactions were performed using chemicals obtained from Alfa Aesar, Sigma-Aldrich, VWR, and Thermo Scientific. All dry reactions were performed in Extra Dry Solvents obtained from Acros Organics under a nitrogen atmosphere and in oven-dried glassware. Reactions were monitored using thin-layer chromatography on silica plates 60Å (250 1 was dissolved in 22 ml of dichloromethane (DCM), and to that, a solution of 4.45 g (7.24 mmol) of Oxone® (monopersulfate compound) in 20 ml of water was slowly added under continuous stirring. The reaction mixture was stirred at room temperature for 12 h until completion, at which point it was diluted with 50 ml of DCM and washed with sodium bicarbonate (50 ml × 3), brine (50 ml × 2), dried over sodium sulfate, and concentrated under vacuum to yield a green nitroso product (2). The concentrated product was used directly without further purification.About 500 mg (6.62 mmol) of 3-nitroaniline 2 was dissolved in 30 ml of ethyl acetate with 10 ml of glacial acetic acid and stirred at room temperature for 10 min under an N2 atmosphere. To this 0.44 ml (3.96 mmol) of m-toluidine 3 was added dropwise over 5 min. The reaction was stirred at room temperature for 22 h till reaction completion. The reaction was diluted with 30 ml of ethyl acetate and quenched with 25 ml of 1 M NaOH, followed by washing with sodium bicarbonate (50 ml × 3), brine (50 ml × 2), and dried over sodium sulfate. The product was purified via column chromatography using hexanes: ethyl acetate = 5:0.1 to yield 601.3 mg (71.9%) of a red solid 4.About 602 mg (3.96 mmol) of nitroso Rf = 0.47 (hexanes: ethyl acetate = 5:0.1)HR-MS (ESI): m/z calculated [MH]+ for C13 H12 N3 O2 is 242.0930, found [MH]+ as 242.0925.1H NMR : δ = 8.46 , 8.34 , 8.27 , 7.83 , 7.71 , 7.47 , 7.41 , 2.39 .13C NMR : δ = 152.1, 151.6, 148.6, 139.1, 133.1, 131.1, 129.8, 129.3, 125.3, 122.9, 120.7, 115.4, 20.8.4 and 1.84 g (7.66 mmol) of Na2S were added to the reaction vessel and flushed with N2. To this, 8 ml of Dioxane was added under constant stirring and then heated to 90 °C for 4 h followed by 12 h at rt. The reaction did not go to completion, so another 1.84 g of Na2S was added and the reaction was reheated for 4 h at 90 °C, followed by stirring at room temperature for another 12 h. The reaction was diluted with 30 ml of ethyl acetate followed by washing with Brine (50 ml × 4) and drying over sodium sulfate. The product was purified via column chromatography using hexanes: ethyl acetate: DCM = 3:1:1 to yield 449.8 mg (83%) of red oil 5.About 539 mg (2.55 mmol) of Rf = 0.69 (hexanes: ethyl acetate: DCM = 3:1:1)HR-MS (ESI): m/z calculated [MH]+ for C13 H14 N3 is 212.1188, found [MH]+ as 212.1185.1H NMR : δ = 8.72 , 8.30 , 8.23 , 7.78 , 7.70 , 7.44 , 7.36 , 2.48 .13C NMR : δ = 153.1, 152.3, 149.1, 139.3, 133.1, 130.0, 129.3, 129.2, 124.9, 123.5, 121.0, 117.1, 21.4.5 was dissolved in 12 ml of THF under an N2 atmosphere and cooled to 0 °C. To this 0.7 ml (4.02 mmol) of diisopropylethylamine was added and stirred at 0 °C for 5 min. 0.24 ml (1.60 mmol) of o-anisoyl chloride 6 was added dropwise over 10 min. The reaction was stirred at 0 °C for 4 h until reaction completion, at which point the reaction was diluted with 50 ml of ethyl acetate followed by washing with sodium bicarbonate (40 ml × 5), brine (50 ml × 2), and dried over sodium sulfate. The product was purified via column chromatography using hexanes: ethyl acetate = 6:1 to yield 405.2 mg (86.3%) of red solid LAKI.About 283 mg (1.34 mmol) of Rf = 0.37 (hexanes: ethyl acetate = 3:0.5)HR-MS (ESI): m/z calculated [MH]+ for C21 H20 N3 O2 is 346.1556, found [MH]+ as 346.1554.1H NMR : δ = 9.90 , 8.35 , 8.10 , 7.78 , 7.75 , 7.71 , 7.66 , 7.56 , 7.46 , 7.38 , 7.19 , 7.13 , 4.05 , 2.46 .13C NMR : δ = 164.7, 158.5, 154.0, 153.6, 140.7, 140.4, 134.4, 133.0, 132.5, 130.6, 130.1, 124.0, 123.8, 123.0, 122.1, 120.9, 119.7, 114.7, 113.0, 57.0, 21.3.3) and sealed. The prepared sample was kept in the dark to relax for 5 days. A 1H-NMR was taken in the dark in a Bruker (Neo) 500 MHz NMR to record the thermally relaxed photostationary state. Alternating NMRs were taken after irradiation with LED lamps of UV365nm (8.7 mW/m2) and Blue460nm (12.6 mW/m2) light from a distance of 1 cm for 15 min each.A solution of LAKI (440 µM) was prepared in a quartz NMR tube with Chloroform (CDCl365 nm (8.7 mW/m2) and Blue460nm (12.6 mW/m2) light for 5 min each from a distance of 1 cm. UV-Visible absorbance was measured with a Vernier UV-Vis spectrophotometer.A solution of LAKI (5 µM) was prepared in a quartz cuvette by diluting a 5 mM solution in DMSO with HEPES (10 mM) and sealed. The sample was irradiated alternatively with LED lamps of UVcis to trans isomerization) of a solution of LAKI (5 µM in HEPES solution containing 0.1% DMSO), the solution was irradiated with UV365nm light to achieve the PSS365. LAKI was allowed to thermally relax at 21.5 °C and was monitored at 330 nm for 20 h. Absorbance was plotted vs time and thermal half-life was calculated using −1), t is time (h), AbsBI is absorbance before illumination, and Abs365 nm is the absorbance at PSS365. The variables AbsBI, Abs365nm, and k were used as free parameters in the fit.To measure the thermal relaxation  293 T cells were purchased from ATCC (CRL-11268) and maintained at 37 °C in 5% CO2Ps and Kv channels was cloned in the pIRES2EGFP vector. HEK293T cells were maintained in DMEM with 10% FBS on plastic dishes. Cells were transiently transfected using calcium phosphate with 3.6 µg of DNA. When two genes were co-expressed, a ratio of 1:1 DNA was used. TG neurons were transfected with 0.75 µg of DNA using JetPRIME.DNA encoding Kl-glutamine, 10% fetal bovine serum, 100 ng/ml neural growth factor, and penicillin/streptomycin.Trigeminal ganglion tissues were collected from postnatal day 1–10 mice of either sex and treated with a mix of 1 mg/ml collagenase type II (Gibco) and BSA for ~45 min, followed by 5 mg/ml trypsin for 10 min. Neurons were dissociated by triturating with fire-polished glass pipettes and seeded on poly-lysine and Laminin coated coverslips. The DMEM/F12-based culture medium contained 2 mM 2, 10 HEPES, pH 7.3 with KOH. Cells were patch clamped using a MultiClamp 700B (Molecular Devices) amplifier, recorded using pCLAMP 11, in an extracellular solution containing (in mM): 150 NaCl, 5 KCl, 2 CaCl2, 10 HEPES, pH 7.4 with NaOH. Currents were elicited in voltage-clamp mode with voltage-ramps . Photocurrents were elicited in voltage-clamp mode either with voltage steps  or in gap-free at several holding potentials upon alternating illumination at 480 nm (blue) and 365 nm (magenta) .HEK293T cells were recorded 24–48 h after phosphate calcium transfection (with 3.6 µg DNA). For co-expression of KCNQ1 and KCNE1 a DNA ratio of 1:1 was used. Glass pipettes were pulled with a resistance <5 MΩ and filled with intracellular solution containing (in mM): 155 KCl, 5 EGTA, 3 MgClTrek1−/−;Trek2−/− TG neurons were transfected with 0.75 µg of the pIRES2EGFP vector containing the TRESK-MT1 insert, in which there is no N-terminal tag on the insert and EGFP is co-translated as a transfection marker, or the pIRES2EGFP control plasmid with JetPRIME. The extracellular solution contained (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose, pH 7.4 with NaOH. The intracellular solution used was the same used for HEK293T cells. Recording pipettes had <5 MΩ resistance. Series resistance (<20 MΩ) was not compensated. Signals were filtered at 10 kHz and digitized at 20 kHz. After establishing whole-cell access, membrane capacitance was determined with amplifier circuitry. The amplifier was then switched to current-clamp mode to measure resting membrane potential (Vrest). Neurons were excluded from analysis if the Vrest was higher than −40 mV. To assess photoswitched neuronal excitability, the depolarizing current was injected to reach the threshold for triggering action potentials then neuronal excitability was studied upon alternating illumination at 480 nm (blue) and 365 nm (magenta). The amplifier was then switched to voltage-clamp mode to assess neuronal photocurrent at several holding potentials upon the same alternating illuminations.Photomodulation of neuronal excitability and photocurrent were studied in small-diameter TG neurons. Concentrations of LAKI and all compounds used are indicated in the respective figure legends.Trek1 and Trek2 were generated as described in ref. 37. Null mutations were backcrossed against the C57BL/6 J inbred strain for 10+ generations prior to establishing the breeding cages to generate subjects for this study. Age- and sex-matched C57BL/6 J WT mice, aged 9–12 weeks, were obtained from Charles River Laboratories .Mice lacking All mouse experiments were conducted according to national and international guidelines and have been approved by the ethical committees . The C57BL/6 J breeders were maintained on a 12 h light/dark cycle with constant temperature (21–23 °C), humidity (45–50%), and food and water ad libitum at the animal facility of Valrose or the Medical School of the University of Barcelona.Two groups of mice were injected subcutaneously into the plantar surface of the hind paws either with 15 µl of LAKI (100 µM) or the vehicle (1% DMSO). Mice were placed individually in compartments of Plantar test Hargreaves Apparatus  for 10 min for habituation. Mice were trained for 7 days before the experiments. The thermal withdrawal latency of mice was determined by exposition to an infrared source (intensity of 50%) before and after UV exposition. The hind paws were exposed to UV by illumination with a 365 nm lamp for 20 s. Recovery of the thermal sensitivity was also evaluated 5 min after UV exposition.Mice were injected subcutaneously in the plantar surface of the hind paws with 15 µl of LAKI (100 µM) or the vehicle (1% DMSO) for the contralateral side. Mice were placed in the testing cage (Ugo Basile) for 15 min for habituation. The withdrawal threshold was determined with von Frey filaments  using the ascending method  before and after UV exposition. The hind paws were exposed to UV by illumination with a 365 nm laser for 20 s and the mechanical assessment was performed after a delay of 20 s. The animals were re-exposed to UV every 2 min until the end of the test. Recovery of the mechanical sensitivity was also evaluated 4 min after UV exposition. Each Von Frey filament was applied five successive times (with a delay of 30 s) on the plantar surface of the hind paw of the mouse standing on its four paws. The withdrawal threshold was considered when the mouse responded positively to three out of five applications.In vivo recordings were performed on mice injected with LAKI in the hind paw following the Von Frey test. Mice were anesthetized with urethane 20% (1.5 g/kg) and placed on a stereotaxic frame . A laminectomy was performed on lumbar vertebrae L1–L3 and segments L4–L5 of the spinal cord were exposed. Extracellular recordings of wide dynamic range dorsal horn neurons  were made with borosilicate glass capillaries  . The signal was amplified and high pass filtered using a DAM80 amplifier  connected to CED1401 . The acquisition was performed using spike 2 software . The criterion for the selection of a neuron was the presence of an A fiber-evoked response (0-80 ms) followed by a C fiber-evoked response (80 to 300 ms) to electrical stimulation of the corresponding receptive field of the ipsilateral paw with subcutaneously implanted bipolar electrodes connected to a stimulator . LAKI-injected hind paw was exposed to a UV laser for 20 s every 2 min until the end of the recording. In the same experiment, a period of at least 15 min without UV was respected for recovery between two neuronal recordings.To assess mice nocifensive ocular responses, 5 μL of vehicle (DMSO 0.1%) or LAKI (100 μM) in saline solution, with or without Capsaicin (100 µM), were topically applied to the anterior ocular surface of the eye. Mice were lightly restrained (by grasping the scruff between the thumb and forefinger) so that the solution remained on the corneal surface. With the animal restrained, 20 s of UV light stimulation to the ocular surface was applied to activate LAKI. The number of nocifensive behaviors such as wipes  and scratches  directed to the treated eye were counted over a 5-min period. In control experiments with no UV light stimulation, the procedure was the same but without applying the light source.C. elegans N2 wild-type reference strain. C. elegans were maintained on NGM agar plates  carrying a lawn of E. coli OP5039. Animals were grown at 20 °C unless indicated otherwise and wild-type strain was freshly thawed prior to experiments.Our analysis included the C. elegans were included in the analysis at the adult stage. C. elegans were transferred on 35 mm diameter dishes containing M9 buffer supplemented either with LAKI or equivalent DMSO for 30-min incubation in the dark. C. elegans were recorded while exposed to ambient light, UV light , and blue light  for 15 s each in duration. The movies of C. elegans were acquired using Micro-Manager 1.4 and analyzed using Fiji software. Omega turns were counted manually and were included in the analysis when C. elegans fully circled. Concentrations of LAKI used are indicated in the respective figure legends.Hermaphrodite t-test when comparing two paired conditions or two-way ANOVA RM and mixed-effects model RM when comparing more than two paired conditions; otherwise, data were analyzed using Wilcoxon signed-rank test. When more than two conditions were analyzed, the tests described above were followed by Bonferroni’s, Holm–Sidak’s, or Tukey’s post hoc test for all pairwise comparisons or by Dunnett’s post hoc test for multiple comparisons to a control group.Analysis of obtained currents in whole-cell recordings was performed using ClampFit. Data were treated and analyzed using Excel, SigmaPlot 11.0, GraphPad Prism 8, and R according to their probability distributions. QuasiBinomial GLM was used for current inhibitions while Poisson and QuasiPoisson GLMs were used for Omega turn and nocifensive behavior counting. Neuronal excitability-, photocurrent-, current density-, paw withdrawal latency, and paw withdrawal threshold-related data were assessed to verify the assumption of Normality either with the Shapiro test or with a Q-Q plot. Data verifying Normality were analyzed using either paired Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


In this study, pectin (PC), whey protein isolate (WPI), and chitosan (CS) were combined with purple corn cob anthocyanins (PCCA). Four complexes, PC−PCCA, WPI−PCCA, WPI−PC−PCCA, and CS−PC−PCCA were prepared to evaluate the improvement in the α-glucosidase inhibitory activity and digestive stability of PCCA. The encapsulation efficiency (EE), particle size, physical properties, and mode of action of the synthesized PCCA complexes were evaluated. Among them, CS−PC−PCCA had the highest EE (48.13 ± 2.73%) except for WPI−PC−PCCA; furthermore, it had a medium size (200−300 nm), the lowest hygroscopicity (10.23 ± 0.28%), lowest solubility (10.57 ± 1.26%), and highest zeta potential (28.20 ± 1.14). CS−PC−PCCA was multigranular and irregular in shape; x-ray diffraction showed that it was amorphous; and Fourier transform infrared spectroscopy confirmed that it was joined with PCCA through hydrogen bonds and electrostatic interactions. Compared with PCCA, the four complexes showed a higher α-glucosidase inhibition activity and digestive stability, except for WPI−PC−PCCA. Furthermore, CS−PC−PCCA exhibited the best α-glucosidase inhibition and simulated digestion stability. Anthocyanins are bioactive phytochemicals that have a variety of colours that are affected by different pH values and are widely found in black beans, purple corn, purple sweet potato, etc. . Purple Studies have shown that encapsulation technologies, such as microencapsulation, liposomes, lotion encapsulation, and nanoparticles, can stabilize their bioactivity . In receKhalifa et al.  found thIn this study, PC, WPI, and CS were combined with PCCA to stabilize PCCA. CS and PC have good delivery effects as wall materials to prepare polysaccharide, protein, and protein−polysaccharide anthocyanin steady-state complexes. The physical properties, the α-glucosidase inhibitory activity, and the in vitro digestive stability of the four encapsulated complexes, PC−PCCA, WPI−PCCA, WPI−PC−PCCA, and CS−PC−PCCA, were compared. Additionally, their combined action was explored. The results of this study can help accelerate the development of related products and further the use of PCCA in the industry.Dried PCC (Heinuo 5) were crushed using a multifunctional grinder  and filtered with a 40-mesh sieve. The obtained powder was placed in absolute ethanol and 0.2 mol/L of citric acid solution (7:3) for the PCCA extraction under conditions of the solid−liquid ratio of 1:63 (g/mL) and an ultrasonic temperature and power of 58 °C and 240 W, respectively, for 41 min. The extract was evaporated in a vacuum at 40 °C and freeze-dried. The dried extract was dissolved in a pH 4.0 citric acid buffer solution at a concentration of 0.03 mg/mL. D-101 resins  were wet-packed into a glass column (1.0 cm × 30 cm) with a resin bed of 200 mL. According to the adsorption capacity of the resin, eleven bed volumes (BVs) of the above solution were loaded onto D-101 resins at 1 min/mL. The loaded resin was washed with distilled water and 10% ethanol (pH = 2) to remove the impurities and it was subsequently eluted with 30% ethanol (pH = 2) to collect the eluent, which was freeze-dried  to obtain PCCA.With a slight modification, PC−PCCA, WPI−PCCA, WPI−PC−PCCA, and CS−PC−PCCA were prepared, according to the description of Koh et al. , Zang etg, 30 min), the sediment was collected and freeze-dried to obtain a complex.PC−PCCA: the PC solution (6 mg/mL) and PCCA solution (4 mg/mL) were prepared using a glacial acetic acid buffer (25 mM) at a pH of 4.0, mixed in equal volumes, and allowed to stand for 18 h. After centrifuging , the sediment was collected and freeze-dried to obtain a complex.WPI−PCCA: the WPI solution (1.5 mg/mL) was prepared with 0.1 M of citric acid buffer solution (pH 6.6) and was rehydrated overnight at 4 °C. The PCCA solution (2.5 mg/mL) was prepared using a pH 3.0 citric acid buffer solution with the same concentration, and both solutions were mixed in equal proportions. After centrifuging (8000× w/w) and PC solution  were prepared with deionized water, rehydrated overnight at 4 °C, subsequently mixed in equal proportions, and adjusted to a pH of 7 using NaOH (1 M). The resulting solution (40 mL) and 10 mL of the PCCA solution (0.2 g of PCCA dissolved in a pH 3.0 citric acid sodium citrate buffer) were mixed and stirred magnetically  for 2 h, and subsequently adjusted to a pH of 3.5 using hydrochloric acid (1 M). After centrifugation , the sediment was collected and freeze-dried to obtain a complex.WPI−PC−PCCA: the WPI solution , the sediment was collected and freeze-dried to obtain a complex.CS−PC−PCCA: the CS solution (1 mg/mL) was prepared with 1% acetic acid aqueous solution, followed by an ultrasonic treatment for 30 min and magnetic stirring at room temperature for 24 h. Thereafter, 10 mL of PCCA solution (0.3 g of PCCA dissolved in a pH 3.0 citric acid buffer) was mixed with 100 mL of CS solution and stirred magnetically for 40 min. Afterwards, 20 mL of PC solution  was slowly added to the solution above, and the pH was adjusted to 4.0 using NaOH (1 M) and homogenized  for 5 min. After centrifuging  and W1 is the concentration of PCCA in the supernatant (mg/mL).As described in g for 10 min, then the supernatant was collected and dried to a constant weight at 105 °C. The solubility is expressed as the percentage mass of the solid powder before and after drying.With a slight modification to the method by Shittu & Lawal , 0.1 g oWith slight modifications to the method by Kanha et al. , the comThe PCCA complex was dissolved in distilled water to a concentration of 1 mg/mL, and the zeta potential was measured using a ZS90 nanoparticle size and zeta potential analyzer .Using a Hitachi Regulus 8100 scanning electron microscope , the microstructures of the complexes were observed. The sample powder was placed on the stage, sprayed with gold using an MC1000 ion sputtering instrument, and observed with 10 k × power mirrors and 100 k × power mirrors.−1 range using the average true range (ATR) method.Using a Fourier transform infrared spectrometer , the infrared spectrum of the complex was scanned in the 4000−400 cmThe complex was analysed using XRD  in the step scanning mode. The scan rate was 4°/min, the range was 5−60° (2θ), and the step width was 0.02°.The inhibitory ability of PCCA on α-glucosidase was evaluated according to the description of Kumar et al. . The abs2 (pH = 7), 30 mL of 0.1 M HCl solution, and 40 mL 0.1M NaHCO3, respectively, to simulate the saliva, gastric juice, and intestinal fluid.An in vitro digestion was carried out with a slight modification of the description by Zang et al. . Totals Simulate mouth digestion: the sample (PCCA or the complex) was placed in a 20 mL NaCl solution (9 g/L) and mixed with 1 mL of simulated saliva at 37 °C, and shaken for 10 min. Simulate gastric digestion: the pH value was adjusted to 3.0, mixed with 2.5 mL of simulated gastric juice, and shaken for 2 h. Simulate intestinal digestion: the pH value was adjusted to 7.5, mixed with 5 mL of simulated intestinal fluid, and shaken for 2 h.The digestive juice for each part was freeze-dried, dissolved in 70% ethanol solution, and centrifuged. The resulting supernatant was used to determine the concentration of the total and individual anthocyanins.2 = 0.9993), y = 188.93x + 86,944 (R2 = 0.9992), y = 109.5x + 17,196 (R2 = 0.9995), y = 156.37x + 23,146 (R2 = 0.9992), and y = 1818.3x + 1,000,000 (R2 = 0.9995). The linear range was 0.01−1000 (μg/mL).Anthocyanins were identified and determined using HPLC-Q-TOF-MS systems (Agilent 1260 and Agilent 6530) equipped with an Agilent ORBAX SB-C18 column . The column temperature was set to 30 °C and the injected sample volume was 5 μL. The mobile phase was composed of 0.1% formic acid in water (A) and acetonitrile (B) at a flow rate of 0.5 mL/min. The Gradient elution program was: (1) 0−55 min, 95−0% A; (2) 55−60 min, 0% A. The MS was equipped with an electrospray ionization (ESI) source and operated in a positive ion scanning mode (scan range: 100−1000 Da). The parameters for the MS identification were the gas temperature and flow with the sheath gas temperature and flow, which were 360 °C, 7 L/min, 400 °C, and 11 L/min, respectively; fragmentor of 150 V; collision energy of 20/30/40 V. The MS parameters for the quantification were the gas temperature and flow with the sheath gas temperature and flow which were 350 °C, 8 L/min, 380 °C, and 11 L/min, respectively. The anthocyanin quantification was carried out using an external standard method. Cyanidin-, peonidin-, pelargonidin-, malvidin, petunidin-3-O-glucoside were used to make the standard curve and their equivalents were used to express the content of their derivatives. The standard curves were as follows: y = 602.7x + 34,288 (Rp < 0.05) was done using SPSS 26 software.All the experiments were repeated in triplicate. The results are expressed as the mean ± standard deviation (SD). The statistical analysis (p < 0.05), indicating that the combined material was more efficient for the encapsulation of PCCA. A low solubility can help delay the release of complexes, mainly due to a complicated coagulation [p < 0.05), indicating that CS−PC−PCCA has the best-sustained release among the four complexes.gulation . The solp < 0.05). The high hygroscopicity of the composite leads to agglomeration, reducing the retention of the active components and hindering their dispersion [The hygroscopic values for the four complexes ranged from 10.23 ± 0.28 (CS−PC−PCCA) to 20.93 ± 0.12 (WPI−PCCA) g/100 g, with significant differences (spersion . In contspersion .p < 0.05) but significantly higher than that for PC−PCCA (p < 0.05), suggesting that the stability of the CS−PC−PCCA solution was the best, while that of PC−PCCA was the worst.The greater the absolute value of the zeta potential, the better the stability, while the smaller the potential, the easier it is for the molecules to agglomerate . The abs2) [3), which may be caused by the surface shrinkage and automatic aggregation of PCCA, CS, and PC during the freeze-drying [The apparent structure of the complex after freeze-drying was observed using a scanning electron microscope. 2) . Moreovee-drying . In addie-drying .−1, which are due to the influence of O−H and C−H bond stretching vibrations, benzopyran ring (C=O), and C=C vibration [−1 and a C−H bending vibration peak at 844 cm−1 [PCCA has strong absorptions at 3268, 2977, 1723, and 1474 cmibration . In addi844 cm−1 . The fou−1 for PC, and a C=O vibration absorption peak in the 1713−1594 cm−1 range [−1 is caused by the symmetrical stretching vibration of the carboxylic acid functional groups, whereas the peaks near 1271 cm−1 and 1032 cm−1 are caused by the stretching vibration of the C−O bond in carboxylic acid [−1 is observed, indicating that there is a hydrogen bond in PC−PCCA [−1 shifted slightly and are significantly weakened, indicating that PC and PCCA are joined by hydrogen bonds [−1 range . In addilic acid . After a PC−PCCA . The absen bonds .−1 and after adding PCCA, the peak widens and shifts slightly, which may be due to the stretching vibration of O−H and intermolecular hydrogen bonds, indicating that there is a hydrogen bond between PCCA and WPI in WPI−PC [−1 to 1602 cm−1 (affected by the C=O tensile vibration), meaning that the α-helix content in WPI and the strength of the amide I band decreases. This indicates the interaction of the hydrogen bonds and the hydrophobic attraction between WPI and PCCA [n WPI−PC . Figure and PCCA .−1 is generated by the tensile vibration of the O−H and N−H groups. Compared with CS−PC, the absorption band shifts, indicating that CS−PC and PCCA are joined by hydrogen bonds [−1, respectively [−1 are slightly wider with significantly decreased intensities, indicating that the amino group in CS−PC has intermolecular electrostatic interactions with PCCA [en bonds . In addiectively . Howeverith PCCA .Crystallinity can be determined from the diffraction peaks in the spectrum. Sharp and narrow diffraction peaks indicate a high crystallinity, whereas wide diffraction peaks indicate an amorphous state . Figure 50 values were 1.221 ± 0.141, 0.815 ± 0.006, 0.869 ± 0.001, 5.504 ± 0.105, and 0.45 ± 0.002 mg/mL, respectively, indicating that combining PC, WPI, and CS−PC with PCCA can increase the hypoglycaemic activity of PCCA. Studies have shown that anthocyanins can bind to the active site of α-glucosidase and inhibit its catalytic activity through hydrogen bonding to exert a hypoglycaemic effect. The binding energy and the number of binding sites were the main factors affecting the inhibitory ability of α-glucosidase [The α-glucosidase inhibition rates of PCCA, PC−PCCA, WPI−PCCA, WPI−PC−PCCA, and CS−PC−PCCA significantly increased with the increasing concentrations . Their Icosidase .5 position, which reduces the nucleophilicity of the C6 and C8 positions, making PCCA less vulnerable to an electrophilic attack [Twenty-three individual anthocyanins were identified from PCCA . Based oc attack . The temc attack ,45. Aftec attack . HoweverFour types of PCCA complexes, PC−PCCA, WPI−PCCA, WPI−PC−PCCA, and CS−PC−PCCA, were prepared. The four complexes exhibited different shapes and sizes (100−400 nm), encapsulation efficiencies (41.80 ± 0.05−58.84 ± 2.82%), physical properties, crystallinity, and combination modes through hydrogen bonding, a hydrophobic interaction, and an electrostatic interaction with PCCA, thus affecting their protective ability. In general, PC−PCCA, WPI−PCCA, and CS−PC−PCCA significantly improved the inhibition of α-glucosidase, the total PCCA content, and the individual anthocyanins in the simulated digestion process. Among them, CS−PC−PCCA is multiparticulate and irregular in shape, owing to the combination of electrostatic interactions and hydrogen bonds. Moreover, CS−PC−PCCA (48.13 ± 2.73%) has an EE second only to WPI−PC−PCCA, a medium size (200−300 nm), the weakest hygroscopicity (10.23 ± 0.28), the lowest solubility (10.57 ± 1.26), and the highest zeta potential (28.20 ± 1.14). In addition, it exhibited the best α-glucosidase inhibition and simulated digestion stability. Therefore, loading PCCA into a complex containing CS and PC is of a great significance in stabilizing PCCA and improving its hypoglycaemic ability, which helps expand the application of PCCA.