Traditionally, the heterologous biosynthesis of (hydroxy)cinnamyl alcohols from (hydroxy)cinnamic acids involved CoA-dependent activation of the substrate. However, a recently explored alternative pathway involving carboxylic acid reductase (CAR) has proven efficient in generating the (hydroxy)cinnamyl aldehyde intermediate without the need for CoA activation. In this study, we investigated the application of the CAR pathway for whole-cell bioconversion of a range of (hydroxy)cinnamic acids into their corresponding (hydroxy)cinnamyl alcohols. Furthermore, we sought to extend the pathway to enable the production of a variety of allylphenols and allylbenzene.Segniliparus rugosus CAR (SrCAR) and Medicago sativa cinnamyl alcohol dehydrogenase (MsCAD2) as the most efficient enzymatic cascade for the two-step reduction of ferulic acid to coniferyl alcohol. To optimise the whole-cell bioconversion in Escherichia coli, we implemented a combinatorial approach to balance the gene expression levels of SrCAR and MsCAD2. This optimisation resulted in a coniferyl alcohol yield of almost 100%. Furthermore, we extended the pathway by incorporating coniferyl alcohol acyltransferase and eugenol synthase, which allowed for the production of eugenol with a titre of up to 1.61 mM (264 mg/L) from 3 mM ferulic acid. This improvement in titre surpasses previous achievements in the field employing a CoA-dependent coniferyl alcohol biosynthesis pathway. Our study not only demonstrated the successful utilisation of the CAR pathway for the biosynthesis of diverse (hydroxy)cinnamyl alcohols, such as p-coumaryl alcohol, caffeyl alcohol, cinnamyl alcohol, and sinapyl alcohol, from their corresponding (hydroxy)cinnamic acid precursors but also extended the pathway to produce allylphenols, including chavicol, hydroxychavicol, and methoxyeugenol. Notably, the microbial production of methoxyeugenol from sinapic acid represents a novel achievement.By screening the activity of several heterologously expressed enzymes in crude cell lysates, we identified the combination of The combination of SrCAR and MsCAD2 enzymes offers an efficient enzymatic cascade for the production of a wide array of (hydroxy)cinnamyl alcohols and, ultimately, allylphenols from their respective (hydroxy)cinnamic acids. This expands the range of value-added molecules that can be generated using microbial cell factories and creates new possibilities for applications in industries such as pharmaceuticals, flavours, and fragrances. These findings underscore the versatility of the CAR pathway, emphasising its potential in various biotechnological applications.The online version contains supplementary material available at 10.1186/s12934-023-02246-4. These allylphenols are important constituents of plant essential oils, contributing to the characteristic taste and aroma of numerous fruits, herbs, and spices [(Hydroxy)cinnamyl alcohols and allylphenols are two closely related groups of aromatic compounds commonly found in plants. The hydroxycinnamyl alcohols, namely coniferyl alcohol, tyrosine . These ttyrosine . Moreoved spices .Escherichia coli show a considerable range, varying from 4–91% [Nocardia iowensis CAR and Coptotermes gestroi aldo-keto reductase, a coniferyl alcohol yield of 97% was achieved under optimised growth conditions [The potential applications of (hydroxy)cinnamyl alcohols and allylphenols in the flavour and fragrance industries , as wellom 4–91% –11, 13. om 4–91% , 15. ThiPetunia hybrida coniferyl alcohol acyltransferase (PhCFAT) and Ocimum basilicum eugenol synthase (ObEGS) in combination with a CoA-dependent hydroxycinnamyl alcohol biosynthesis pathway to produce eugenol and chavicol from ferulic acid and p-coumaric acid, respectively [Petunia hybrida EGS in a bacterial co-culture system, resulting in the production of up to 244 mg/L eugenol from ferulic acid [E. coli expressing cinnamyl alcohol acyltransferase and propenylphenol synthase from Larrea tridentata and supplementing the bacterial culture with the respective hydroxycinnamyl alcohol precursors [Allylphenols can be produced from hydroxycinnamyl alcohols through two enzymatic steps Fig. . Firstlyectively . Howeverectively . Moreoveectively  was recelic acid . Other slic acid , or in Eecursors .Segniliparus rugosus CAR and Medicago sativa cinnamyl alcohol dehydrogenase as the best-performing enzyme candidates in vitro. Next, we optimise their expression levels through a combinatorial approach. This enabled the efficient in vivo biosynthesis of various (hydroxy)cinnamyl alcohols, achieving a nearly complete conversion of ferulic acid into coniferyl alcohol. Subsequently, by extending the pathway using PhCFAT and ObEGS, we demonstrate the production of eugenol, chavicol, hydroxychavicol, and methoxyeugenol from their respective hydroxycinnamic acid precursors in E. coli  and Segniliparus rugosus (SrCAR) as the top-performing enzyme candidates [Coptotermes gestroi AKR-1 (CgAKR-1), to catalyse both the in vitro and in vivo conversion of ferulic acid into coniferyl alcohol [Pseudomonas sp. strain HR199 coniferyl alcohol dehydrogenase  and Medicago sativa cinnamyl alcohol dehydrogenase (MsCAD2) for activity screening. These enzymes have previously been shown to effectively reduce various (hydroxy)cinnamyl aldehydes to their corresponding alcohols [In a recent study by Tramontina et al., the authors evaluated five CARs for their ability to reduce ferulic acid, and identified the CARs from ndidates . In thei alcohol . In thisalcohols , 20.Escherichia coli NEB5α expressing either NiCAR or SrCAR. The CARs were each coexpressed with Bacillus subtilis 4’-phosphopantetheinyl transferase (Sfp), which is essential for CAR activation [E. coli NEB5α whole-cell lysate [First, we monitored the conversion of ferulic acid to coniferyl aldehyde using whole-cell lysates of tivation . Among ttivation . It is il lysate . Among tNext, we investigated the double reduction of ferulic acid to coniferyl alcohol by combining the cell lysates containing the individual enzymes. The most efficient two-step reaction was achieved using SrCAR in conjunction with MsCAD2, resulting in a product yield of 50.3% coniferyl alcohol Table . The secE. coli NEB5α. To identify the optimum balance of gene expression levels, we employed a combinatorial pathway design approach, which has been successfully utilised in the microbial production of fine chemicals [trc and PlacUV5), as well as two different origins of replication  [E. coli NEB5α. Subsequently, the cells were supplemented with ferulic acid and the biosynthesis of coniferyl aldehyde and coniferyl alcohol was monitored for 48 h. Among the tested pathways, the top-performing design (SBC015866) yielded 2.99 mM coniferyl alcohol (538 mg/L) from 3 mM ferulic acid within 20 h  . Interge, ColE1) . These nE. coli for both coniferyl alcohol and caffeyl alcohol and was hypothesised to be due to either product consumption or volatility [E. coli NEB5α cells. In culture medium without cells, the level of coniferyl alcohol remained constant, while in cultures containing E. coli NEB5α, the titre decreased over time  and Ocimum basilicum eugenol synthase (ObEGS). E. coli DH5α ΔtyrR ΔpheLA carrying this pathway produced 0.17 mM eugenol when supplemented with 3 mM coniferyl alcohol [Eugenol is a volatile aromatic compound that is utilised extensively in the flavour and fragrance industries due to its spicy and clove-like aroma . It can  alcohol .E. coli NEB5α was transformed with the plasmid that contains the eugenol biosynthesis pathway (SBC009876) in combination with one of three selected plasmids that contain the coniferyl alcohol biosynthesis pathway , representing a slight increase over the eugenol titre of 0.49 mM (80 mg/L) attained after 24 h. Notably, since IPTG was added to the culture at the 24-hour time point to induce pathway expression, this suggests that the eugenol biosynthesis pathway had already been adequately expressed even in the absence of inducer, similar to the coniferyl alcohol production observed during the combinatorial plasmid library screen cinnamic acids and (hydroxy)cinnamyl aldehydes, respectively, the enzymes employed for the two-step conversion of coniferyl alcohol into eugenol also exhibit wide substrate tolerance. For example, PhCFAT acetylates a diverse range of (hydroxy)cinnamyl alcohols, including cinnamyl alcohol,  alcohol . Similar acetate  and in v acetate . Given tE. coli NEB5α strain was employed for the bioconversion of p-coumaric acid, caffeic acid, and sinapic acid, while the E. coli NEB5α ΔhcaE strain, unable to catabolise cinnamic acid [E. coli NEB5α or E. coli NEB5α ΔhcaE carrying the nine pathway variants were supplemented with 3 mM (hydroxy)cinnamic acids and the biosynthesis of (hydroxy)cinnamyl alcohols was monitored for up to 48 h.For each substrate, we initially performed a combinatorial plasmid library screen to determine the optimal pathway variant for the conversion of the (hydroxy)cinnamic acid into the respective (hydroxy)cinnamyl alcohol. Subsequently, two or three of these candidate pathways were combined with the pathway that converts (hydroxy)cinnamyl alcohols into allylphenols (SBC009876), as it was done for eugenol production. The mic acid , was usep-coumaryl alcohol, 0.87 mM caffeyl alcohol, 0.7 mM cinnamyl alcohol, and 2.33 mM sinapyl alcohol  to red  and brown  cinnamyl alcohol cinnamyl alcohol biosynthesis pathway by PhCFAT and ObEGS (encoded by plasmid SBC009876) enabled the production of chavicol, hydroxychavicol, and methoxyeugenol from their respective hydroxycinnamic acid precursors Table . The cha alcohol . Moreovewo steps . This fiE. coli NEB5α ΔhcaE carrying SBC009876 in combination with any of the three cinnamyl alcohol producing pathways  and when supplemented with cinnamic acid. However, in all three strains, we observed the considerable accumulation of a compound that matches cinnamyl acetate based on its GC-MS/MS fragmentation pattern cinnamyl alcohols to a significant extent, highlighting the need for an optimised chassis strain and an efficient allylphenol biosynthesis pathway to minimise the accumulation of potential by-products. We believe that the CAR-dependent pathway holds broad potential for the production of (hydroxy)cinnamyl alcohols and other related allylphenols.In this study, we developed an optimised pathway for the in vivo bioconversion of various (hydroxy)cinnamic acids into their respective (hydroxy)cinnamyl alcohols. By extending this pathway, we successfully produced the allylphenols eugenol, chavicol, hydroxychavicol, and methoxyeugenol. Our production of eugenol and chavicol exceeds previously reported titres achieved using a CoA-dependent pathway for (hydroxy)cinnamic acid activation, while the biosynthesis of methoxyeugenol from sinapic acid represents a novel accomplishment. Importantly, we observed that E. coli. In cases where no ideal enzyme candidates were listed, alternative options were suggested after conducting a manual literature search.RetroPath2.0  was usedE. coli was used for cloning, plasmid propagation, and the biosynthesis of coniferyl alcohol, eugenol, p-coumaryl alcohol, chavicol, caffeyl alcohol, hydroxychavicol, sinapyl alcohol, and methoxyeugenol. Strain E. coli NEB5α ΔhcaE was employed to produce cinnamyl alcohol and allylbenzene. All strains used and generated in this study are listed in Additional file 1: Table New England Biolabs (NEB) 5α competent hcaE gene was performed using the OneTaq PCR Master Mix  in 25 µL reactions. All PCR-, HiFi-, and digestion reactions were set up following the manufacturer’s instructions. Chemically competent E. coli were prepared and transformed by heat shock [Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen). DNA for cloning was amplified by PCR in 50 µL reactions using the Q5 High-Fidelity 2X Master Mix from NEB. Gel-purified linearised DNA was extracted using the Zymoclean Gel DNA Recovery Kit (Zymo Research). NEBuilder HiFi DNA Assembly Master Mix and restriction enzymes were purchased from NEB. Screening of bacterial colonies to confirm the chromosomal deletion of the at shock .https://acs-registry.jbei.org) under the accession numbers ACS_000886–ACS_000894, respectively.Oligonucleotide primers were synthesised by Integrated DNA Technologies (IDT) and are listed in Additional file 1: Table E. coli NEB5α hcaE deletion strain was constructed using CRISPR-based gene editing as reported previously [hcaE gene was first screened for Cas12a protospacer adjacent motifs (PAMs) with the sequence TTTV. The CRISPR AsCpf1 insertion and deletion score web tool [hcaE gene [hcaE was confirmed by colony PCR, using primers EHseq049 and EHseq050 that flank both sides of the recombination site.The eviously . For theweb tool  was thencaE gene . The del2, 3 mM NADPH, and 3 mM ferulic acid in a 1-mL reaction volume. In the CAD/AKR assay, the reaction buffer contained 50 mM phosphate-buffered saline (pH 7.0), 0.2 mM NADPH, and 5 mM coniferyl aldehyde in a 1-mL reaction volume. The cascade assay buffer was the same as the CAR/AKR assay buffer, but with a higher NADPH concentration of 6 mM. All assays were incubated in 2-mL tubes at 30℃ with shaking at 250 rpm for 3 h before sampling and measuring the product titres using LC-MS/MS.The enzyme activity screening assays were performed following a previous protocol  with somTo assess the stability of different (hydroxy)cinnamyl alcohols in culture medium without cells, each alcohol was individually added to 1 mL of TBP medium in a 96-deepwell plate (DWP) to achieve a final concentration of 2 mM. The plate was then sealed with a breathable seal and incubated at 30℃ and 80% humidity with orbital shaking at 850 rpm. Samples were collected immediately after addition, as well as after 6 and 24 h.E. coli NEB5α were used to inoculate 1 mL of TBP medium supplemented with glycerol (0.4% w/v) in a 96-DWP. The plate was also sealed with a breathable seal and incubated at 30℃ and 80% humidity with orbital shaking at 850 rpm for 16–18 h to grow the seed cultures. The main cultures were set up by diluting the seed cultures to an OD600nm of 0.2 in 1 mL of fresh TBP medium supplemented with glycerol (0.4% w/v) and the respective (hydroxy)cinnamyl alcohol to a final concentration of 2 mM. Samples were collected immediately, as well as after 6 and 24 h. For both experiments, the (hydroxy)cinnamyl alcohol titres were quantified using either HPLC- or UPLC-DAD, depending on the specific target being analysed.To determine the stability of different (hydroxy)cinnamyl alcohols in culture medium containing cells, single colonies of E. coli NEB5α SBC015866 SBC009876 was performed in a 0.25 L bioreactor . The fermentation medium consisted of 17.5 g/kg glucose, 6 g/kg KH2PO4, 5 g/kg (NH4)2SO4, 2 g/kg NaCl, 3 g/kg sodium citrate, 6 g/kg MgSO4 × 7H2O, 0.03 g/kg CaCl2, 0.15 g/kg thiamine HCl, 0.0225 g/kg FeSO4 × 7H2O, 0.5 g/kg yeast extract, and 3 g/kg trace element solution [4OH, and the temperature was maintained at 37℃. The dissolved oxygen level was maintained at 40% through adjustment of the stirring rate, while a gas flow rate of 1 vvm was maintained. The bioreactor was inoculated with 2% biomass derived from a seed culture grown in shake flasks, and the initial glucose was consumed in a batch fermentation. Once the CO2 signal had dropped and the pH had increased to 7.15, a feed consisting of 55% (w/w) glucose was initiated. The feed rate was increased at an exponential rate of 0.1 h− 1 until an oxygen transfer rate of 180 mmol/kg/h was reached. Subsequently, a constant feed rate was maintained. The culture was induced by addition of IPTG to a final concentration of 0.1 mM, 24 h after inoculation.The fed-batch fermentation of solution . AdditioFor the biosynthesis of (hydroxy)cinnamyl alcohols, allylphenols, and allylbenzene, single colonies of freshly transformed cells were used to inoculate 1 mL of TBP medium supplemented with glycerol (0.4% w/v) and the appropriate antibiotics in 96-DWP, which were sealed with breathable seals. The seed cultures were incubated at 30℃ and 80% humidity with orbital shaking at 850 rpm for 16–18 h.600nm of 0.02 in 1 mL or 1.2 mL, respectively, of fresh TBP medium supplemented with glycerol (0.4% w/v) and the relevant antibiotics in 96-DWPs. These main cultures were returned to the shaker-incubator. For the biosynthesis of (hydroxy)cinnamyl alcohols, each seed culture was used to set up two main cultures: one remained uninduced, while the other was ultimately supplemented with isopropyl β-d-1-thiogalactopyranoside (IPTG).To produce (hydroxy)cinnamyl alcohols or allylbenzene, main cultures were set up by diluting the seed cultures to an OD600nm of 0.02 in 50 mL of fresh TBP medium supplemented with glycerol (0.4% w/v) and the relevant antibiotics in non-baffled 250-mL shake flasks. These cultures were incubated at 30℃ and 180 rpm.To produce eugenol, chavicol, hydroxychavicol and methoxyeugenol, main cultures were prepared by diluting the seed cultures to an OD600nm of 1.0–1.5, (hydroxy)cinnamic acids and optionally IPTG were added to final concentrations of 3 mM and 100 µM, respectively. Except for allylbenzene, the cultures were then returned to their respective shaker-incubator and grown for up to 72 h.To produce (hydroxy)cinnamyl alcohols, allylphenols, and allylbenzene, when the cultures reached an ODsec-butylbenzene (Sigma) to a final concentration of 0.005% (v/v). The vials were sealed with gas-tight screw caps and the cultures were incubated at 30℃ and 180 rpm for 24 h.For the biosynthesis of allylbenzene, after the addition of cinnamic acid and IPTG, 1 mL of culture was transferred to a 20-mL headspace vial. The culture was overlaid with 0.5 mL of 2,2,4-trimethylpentane (TMP) supplemented with g for 10 min. For HPLC-DAD and UPLC-DAD analysis, cell-free supernatants were diluted 10-fold, while for LC-MS/MS analysis, cell free-supernatants were diluted 1000-fold using methanol/water (10:90 v/v). To quantify allylbenzene using GC-MS/MS, the organic layer was transferred to a 2-mL Eppendorf tube and centrifuged at 16,000 g for 10 min. The organic layer was then diluted 5-fold using TMP, vortexed with anhydrous Na2SO4 to remove residual water and transferred to a GC vial for analysis. The TMP used for sample dilution was supplemented with 0.005% sec-butylbenzene, which served as an internal standard. Metabolite concentrations were determined using calibration curves generated from standards of known concentrations, prepared in the same manner as the samples. A summary of the quantification method and the instrument used for each target compound can be found in the Supplementary Methods in Additional file For the quantification of (hydroxy)cinnamyl alcohols, allylphenols, and allylbenzene, as well as their precursor and intermediate compounds, different methods and instruments were employed. For compound quantification using HPLC-DAD, UPLC-DAD, and LC-MS/MS, culture samples were quenched by adding an equal volume of 100% methanol, followed by vortexing and centrifugation at 16,000 Below is the link to the electronic supplementary material.Supplementary Material 1


PPC80, PPC82, and PPC84 inhibited pancreatic lipase (IC50 of 167–1023 µM) via competitive or mixed mechanisms. The activity of pancreatic α-amylase was suppressed by PPC80, PPC82, PPC84, PPC89, and PPC101 (IC50 of 162–519 µM), which acted as competitive or mixed inhibitors. Finally, PPC84, PPC89, and PPC101 also showed potent inhibitory effects on α-glucosidase (IC50 of 51–353 µM) as competitive inhibitors. The results suggest that these synthetic amino acid derivatives have inhibitory potential against digestive enzymes and may be used as therapeutic agents to control metabolic disorders.Over the last decades, the increased incidence of metabolic disorders, such as type two diabetes and obesity, has motivated researchers to investigate new enzyme inhibitors. In this study, the inhibitory effects of synthetic amino acid derivatives  on the activity of digestive enzymes were assessed using in vitro assays. The inhibitory effect was determined by the inhibition percentage and the 50% inhibitory concentration (IC The digestion of foods in the gastrointestinal tract of humans and animals is determined by the activity of enzymes that break down macronutrients into smaller molecules to be absorbed in the gut and used by the body . Some ofRegulation of nutrient absorption  through the inhibition of digestive enzymes is an effective manner to control metabolism. For example, acarbose can inhibit the activity of α-amylase and α-glucosidase enzymes by reducing glucose absorption and decreasing insulin secretion in postprandial glycemia, establishing a glycemic control mechanism associated with reduced glycosylated hemoglobin. This class of enzymatic inhibitors is indicated in patients with adequate fasting blood glucose and elevated postprandial blood glucose levels. In patients with impaired glucose tolerance, enzymatic inhibitors have been associated with a marked reduction in cardiovascular events and no risk of adverse side effects, such as weight gain or hypoglycemia . TherefoObesity is a complex disease that involves an abnormal or excessive accumulation of fat in the body and constitutes a public health problem worldwide . The conIn this context, the preparation of novel amino acid derivatives obtained from organic synthesis processes is a promising area that has been subjected to numerous biological studies. In addition to the functionalization of carboxylic and amine groups attached to the stereogenic center, the coupling of carbon side chains may also result in functional amino acid derivative drugs synthesized by conventional chemical reactions  . These dIn the present study, the inhibitory effects of synthetic amino acid derivatives on digestive enzymes were assessed using in vitro assays. This exploratory study may have predictive value for developing new therapeutic agents against metabolic disorders such as type two diabetes mellitus and obesity.The evaluated as amino acid derivatives, compounds PPC80 (342.52 g/mol), PPC82 (314.47 g/mol), PPC84 (287.40 g/mol), PPC89 (370.58 g/mol), and PPC101 (469.76 g/mol), were synthesized according to our previous report in the literature .p-nitrophenol palmitate, Triton-X 100, orlistat, porcine pancreatic α-amylase, 50 mmol/L Tris-HCl (pH 7.0), α-glucosidase, 100 mmol/L citrate-phosphate buffer (pH 7.0), acarbose, and p-nitrophenyl-α-D-glycopyranoside , while dimethylsulfoxide and starch . Unless noted, all chemicals utilized in the synthetic protocol were acquired from Sigma-Aldrich® Co., St. Louis, MO, USA, and used as received.The drugs and reagents used in this study were as follows: porcine pancreatic lipase, 50 mmol/L Tris-HCl buffer (pH 8.0), 2 and 25 mM NaCl. The p-nitrophenol palmitate substrate (8 mM) was dissolved in 0.5% w/v Triton-X 100. PPC80, while PPC82, PPC84, PPC89, and PPC101 amino acid derivatives and orlistat were solubilized in dimethylsulfoxide (DMSO) prepared at increasing concentrations ranging from 0.5 to 1.392 mM. A total of 100 µL of enzyme solution, 50 µL of p-nitrophenol palmitate substrate, and 50 µL of the amino acid derivative sample or orlistat were added to the microplate wells. Next, microplates were incubated at four different time intervals  in a water bath at 37 °C, and the reaction was stopped in an ice bath. All reactions were carried out in triplicate. The absorbance of the products was measured at 405 nm using a microplate reader .The pancreatic lipase inhibition assay was performed according to Santos et al.  with som2 and 1% starch. PPC80, PPC82, PPC84, PPC89, and PPC101 amino acid derivatives and acarbose were solubilized in DMSO prepared at increasing concentrations ranging from 0.15 to 1.590 mM. A total of 50 µL of enzyme solution, 50 µL of substrate, and 50 µL of amino acid derivative sample or acarbose were added to the microplate wells. Afterward, microplates were pre-incubated for 10 min in a water bath at 37 °C. A total of 100 µL of substrate was added to each well, and microplates were incubated at four different time intervals  in a water bath at 37 °C. The reaction was stopped using an ice bath. All reactions were carried out in triplicate. The absorbance of the products was measured at 405 nm using a microplate reader .The pancreatic α-amylase inhibition assay was carried out according to Freitas et al.  with som®, TP-Reader, Wuxi, China).The inhibitory effect against α-glucosidase was carried out according to Chelladurai and Chinnachamy  with somI%) was determined using “absorbance versus time” graphs. By means of linear regression, using the method of least-squares, the equations of the straight lines and the angular coefficients were obtained to determine the inhibition (I%) of the enzymatic activities by the equation:A is the angular coefficient of the straight-line equation (enzyme + substrate), a is the angular coefficient of the equation of the line (substrate), B is the angular coefficient of the straight-line equation (enzyme + substrate + sample), and b is the value of the angular coefficient of the straight-line equation (enzyme + sample).The percentage of inhibition (50) were determined through “response versus concentration” plots using the linear least-squares regression model.The 50% inhibitory concentrations  as a function of time (60 s). The absorbance values were converted into product concentration (µmol/L) using standard curves of glucose (α-amylase) and p-nitrophenol (pancreatic lipase and α-glucosidase). The value of the initial velocity (v0) of enzymatic reactions was estimated to create the “v0 versus substrate concentration” graph. Kinetic constants (mK and maxV) and slope were calculated, and the inhibition model was verified using Lineweaver–Burk plots ..Vmax equPhaseolus vulgaris) that inhibited pancreatic lipase in the range of 23–87%. Polylysine is a synthetic peptide that also acts as a lipase inhibitor, showing a remarkable inhibition (80%) on the activity of porcine pancreatic lipase at a concentration of 100 mg/mL [50 values below 50 µM.These findings are consistent with those of previous studies assessing the effects of amino acid and peptide derivatives on pancreatic lipase activity. Ngoh and Gan  identifi00 mg/mL . Further00 mg/mL  and hydr00 mg/mL  inhibiteThe results of the present study showed that PPC80 and PPC82 have inhibitory potential on the activity of pancreatic lipase, which may promote a reduction in intestinal fat absorption and potentially affect body weight . Therefo50 values  with IC50 values of 2.58 ± 0.08 and 2.62 ± 0.05 mM for GGSK and ELS, respectively [Ocimum basilicum) seeds that showed 36% inhibition on α-amylase [Glycine max) protein capable of inhibiting pancreatic α-amylase activity at IC50 values ranging from 0.16 to 8.30 mg/mL.An increasing number of studies have shown that synthetic compounds derived from amino acids and peptides exhibit an inhibitory action on α-amylase ,32,33. Tectively . Another50 = 51.00 ± 1.73 µM) was about 12-fold more potent than acarbose (IC50 = 639.00 ± 4.62 µM) against this enzyme . Furthermore, three peptides isolated from quinoa (Chenopodium quinoa) showed similar inhibitory activities against α-glucosidase [Acacia nilotica that exhibited potent inhibitory effects against α-glucosidase at low IC50 values (3.75 µg/mL).Several studies have reported the promising potential of amino acid and peptide derivatives as α-glucosidase inhibitors ,38,39. Fcosidase . Singh aThe human α-glucosidase is an enzyme found in the epithelium of the small intestine that catalyzes starch breakdown and the consequent release of glucose. Therefore, inhibition of this enzyme constitutes a promising strategy for reducing serum glucose levels in metabolic diseases, including type two diabetes . Our res50 values as low as 167 µM via competitive or mixed mechanisms. The activity of pancreatic α-amylase was suppressed by PPC80, PPC82, PPC84, PPC89, and PPC101, with IC50 values in a range of 162–519 µM, which acted as competitive or mixed inhibitors. In addition, PPC84, PPC89, and PPC101 also presented an inhibitory effect on α-glucosidase, with IC50 values as low as 51 µM acting as competitive inhibitors. The present study supports that amino acid derivatives are promising therapeutic agents for metabolic disorders, including type II diabetes and obesity. However, further pharmacological and toxicological investigations are needed to ensure their safe use as medicines.In summary, the results showed that PPC80, PPC82, PPC84, PPC89, and PPC101 amino acid derivatives are potential inhibitors of lipase, α-amylase, and α-glucosidase enzymes. For instance, PPC80, PPC82, and PPC84 inhibited pancreatic lipase with IC


We investigate the origin of the magnetite nanoparticle aggregates (MNAs) from the Peña Colorada iron-ore mining district (Mexico) to devise a nature inspired synthesis process. Three types of samples were used: natural MNAs recovered from the mine, concentrated magnetite microparticles as reference material, and thin berthierine films used to synthesize MNAs. The chemical, mineralogical, crystallographic and rock magnetic properties were determined by polarized microscopy, high-resolution transmission electron microscopy, electron microprobe, X-ray diffraction, Mössbauer spectroscopy, and thermomagnetic and hysteresis measurements. MNAs were synthesized in the lab with the following steps. We start with berthierine thin films, which are heated to temperatures between 495 and 510 °C leading to formation of numerous stable magnetite nanocrystals. They grow at a temperature above 650 °C. Space restrictions lead to the formation of dense MNAs. Smaller MNAs, <200 nm, with a Curie temperature of 650 °C, shows superparamagnetic behavior. While larger MNAs, >7 μm, show Curie temperature of 578 °C and ferromagnetic behavior. Based on present observations, we suggest that MNAs in the Peña Colorada iron-ore formed in a marine environment, where berthierine formation was accelerated by Fe-rich hydrothermal springs that supplied iron and increased the temperature. Most notably, our laboratory experiments mimicked natural conditions and were able to successfully nucleate and grow magnetite nanoparticles which developed into MNAs. These MNAs were similar to those recovered from the Peña Colorada iron-ore deposit. This study, thus, provides a nature inspired method for synthesis of magnetite nanoparticles and its aggregates. We investigate the origin of the magnetite nanoparticle aggregates (MNAs) from the Peña Colorada iron-ore mining district (Mexico) to devise a nature inspired synthesis process. For example, magnetite nanoparticles have the ability to remediate numerous pollutants, such as heavy metals , methylene blue, methyl orange, malachite green, and total nitrogen and phosphorus from wastewater, and have antifungal and antibacterial properties.4–6Magnetite nanoparticles and their larger counterparts have contrasting chemical, thermal, optical, physical and electrical properties. While these properties of larger counterparts are well defined, the nanoparticles are still poorly understood.3,7,8 To the best of our knowledge such methods for preparing magnetic nanoparticle aggregates (MNAs) have not yet been well established. Our study, thus, investigates the natural origin and genesis of MNAs found in the Peña Colorada iron-ore deposit. This iron-ore mine is located at the Pacific continental margin in southern Mexico 64O10(OH)8, and in minor proportion by calcite, quartz, apatite, cryptocrystalline and colloidal silica, hematite, siderite, feldspar, pyrite, chalcopyrite, pyrrhotite, marmatite, galena, covellite, native gold and argentite.10,11Well established methods of synthesizing nanoparticles require hazardous chemicals and physical conditions such as extremely high temperature and pressure. Thus, nature inspired synthesis procedures need to be established and developed.n Mexico . It is t12,13 In these MNAs, magnetite nanoparticles are embedded in the berthierine matrix , differential thermal and gravimetric analysis (DTA-GTA), polarized light microscopy, X-ray diffraction (XRD) and high-resolution transmission electron microscopy.2 This instrument classifies and separates the particles as a function of the size through centrifugal force under controlled conditions . MNA fractions were separated into size ranges: 56–30 μm, 30–22 μm, 22–15 μm, 15–10 μm, 10–7 μm and 7–0.1 μm. A few smaller MNAs, 2 to 15 nm in size, were also obtained. Then the effects of temperature on MNAs were analyzed using optical microscopy, differential thermal and gravimetric analysis (DTA-GTA), Xray diffractometry (XRD), electron microprobe (EPMA), and high-resolution transmission electron microscopy (HRTEM). Magnetic characterization was done by analysing the changes in magnetic susceptibility with frequency (χFD%) and high-temperature (thermomagnetic), and by investigating the changes in magnetisation with applied field (hysteresis).To study the changes in MNAs with variation in grain size and temperature we used mechanical and magnetic separations. For grain size analysis MNAs were separated into different sizes through a granulometric classification using a Warman-M8 cyclosizer equipment, sub-sieve sizer.λ = 1.5418 Å radiation and graphite monochromator. Diffplus B-S software and international crystal powder diffraction (ICPD) database. Electron probe X-ray micro-analyses was done using JEOL JXA 8900-R with dispersive energy spectrometers of X-ray wavelength (WDS) standards SPI#02753-AB. DTA-GTA analyses using a Shimatsu ATR at room conditions with 70 mg sample under temperature from 19 °C to 1100 °C and an interval of 1 °C per minute. HRTEM was done with a JEOL 2010 FEG FASTEM.Light microscopy was done on a Leica MZ 7.5 stereoscope microscope a Leica DMLP polarizing microscope. XRD was done with Bruker D-8 advance with Cu Kα and χFD% and thermomagnetic measurements were done using a Bartington MS2 with an MS2W sensor, coupled to a furnace MS2WFP. Low and high frequency of 470 Hz and 4700 Hz were used for χFD% measurements, which are especially useful to distinguish ultrafine magnetite grain with superparamagnetic (SP) behavior. During thermomagnetic measurements the samples were heated in air from room temperature to 700 °C and then cooled back. The magnetic susceptibility is measured during the entire process of heating and cooling. Hysteresis was determined using small core-chips measured in a Princeton Instrument AGFM 2900 (MicroMag) employing a maximum applied field of up to 1.5 T.19 The intergranular material around the nanoparticles contains ca. 65% nanoscopic magnetite, ca. 30% berthierine, and ca. <5% associated minerals, which include, in order of decreasing proportion, granular quartz, amorphous colloidal silica, calcite, siderite, pyrite, chalcopyrite, marmatite, pyrrhotite, feldspar, apatite, sphene, native gold, argentite and organic matter. Berthierine is silky and occurs in low structural order, between amorphous to fine crystalline. The MNAs are occasionally cut by calcite veinlets, up to 20 μm thick, with apatite inclusions. Some MNAs are wrapped by up to a 35 μm thick layer of sulfides.MNAs are found in the mineralized bodies of the Peña Colorada iron ores. SEM and HRTEM reveal that the MNAs are comprised of spherical magnetite nanoparticles, 2 nm to 15 nm in size  and 3 and = 3.00 Å, (311) d = 2.55 Å, (111) d = 4.90 and (222) with d = 2.43 Å. Berthierine peaks correspond to (001) with d = 7.12 Å and (002) with d = 3.55 Å. The magnetite peaks are irregularly shaped with wide base, indicating a significant contribution from nanometer-scale grains.2The XRD spectrum analysis  reveals 3O4 molecule is in two magnetic states: ferromagnetic state indicated by a sextuple spectrum, corresponding to the micrometric size magnetite aggregates >0.2 μm; and superparamagnetic state represented by a doublet spectrum corresponding to the MNAs of nanometer-scale <200 nm. The magnetic state of the MNAs depends strongly on their degree of compaction.The Mössbauer spectrum of the intergranular material around the nanoparticles , shows s3+ = 15.59 to 15.69 wt% and Fe2+ ions = 7.78 to 7.83 wt%, and elemental trace impurities (<0.2 wt%) of Mn, Ca, Mg, Ti, Al, V, Si, Na and K.The chemical properties of the MNAs obtained by WDS multi-elemental analysis in selected fields  and (222) magnetite peaks, with d value of 2.53 Å and 2.15 Å, respectively.Thin berthierine films were separated from the ore fragments using a stereomicroscope. The films were annealed at 360, 495, 510, 570, 650 and 750 °C , and theExothermic reactions at 360 °C and 495 °C  mark dehca. 650 °C is owed to formation of new magnetite nanoparticles and growth of pre-existent magnetite nanoparticles to over 10 nm. These larger nanoparticles saturate the colloid favoring their contact and subsequent aggregation  (220) and (311) planes, respectively  temperature to explore the repeatability of the procedure , (311) and (210) planes, respectively  magnetite peak at 2 compare . Newly fectively . MagnetiThe DTA and GTA analyses reveal marked differences between reference magnetite, 56–30 μm and natural MNAs recovered from the iron-ores. The reference magnetite presents two exothermic reactions at 375 and 615 °C. The first one corresponds to magnetite changing to maghemite due to oxidation and the second one represents maghemite transforming to hematite . The GTAIn contrast, the MNAs  reveal f2O3 → α-Fe2O3), while their core remains maghemite. The DTA curve shows wide amplitude in the interval 600 to 675 °C , respectively. This decrease in χFD% corresponds to the transformation of remaining fractions of magnetite nanoparticles to maghemite, which present little remanence and very low coercivity  and 750 °C. The initial ercivity . In descercivity . After aercivity . Beyond ercivity . The hysTc) is encountered at 350–380 °C indicating maghemite,22,28,29 which is produced from the transformation of magnetite nanoparticles.23 All MNA sample show moderate to strong Hopkinson peak at c. 580 °C. Hopkinson peak can be attributed to abundance of smaller magnetite grains. At temperatures above the magnetite Tc, the thermomagnetic curves are reversible with the colling branch presenting the same shape as the heating branch  nucleation and growth of numerous stable crystalline magnetite nanoparticles homogeneously distributed in a colloidal berthierine matrix starts at 635 °C. (2) With increasing temperature, to 650 °C, the nucleation accelerates, and the pre-existing nanoparticles grow. HRTEM confirms that these particles are magnetite less than 15 nm. (3) Constant temperatures of 650 °C lead to the continuous growth of magnetite nanoparticles. Space restrictions lead to the formation of dense MNAs less than 200 nm in size.31 could spark a series of reactions and physicochemical adjustments in composition to form the MNAs.In marine conditions, berthierine is formed by colloidal diagenetic processes. Supply of heat and iron from hydrothermal solutions coming out of marine chimneysThe properties of MNAs are size-dependent. Magnetite MNAs, which are larger than 7 μm, start to transform to maghemite in the range of 280 °C to 380 °C and then to hematite at 615 °C. In contrast, the smaller MNAs, <0.2 μm, are more resistant to temperature induced oxidation and transformation. They show changes to hematite at temperatures >750 °C. The larger MNAs are ferromagnetic, while the smaller ones are superparamagnetic.Most notably, our laboratory experiments that mimicked natural conditions and were able to successfully nucleate and grow magnetite nanoparticles which developed into MNAs. The MNAs were similar to those recovered from the Peña Colorada iron-ore deposit. This study provides a nature inspired method for synthesis of magnetite nanoparticles and its aggregates which do not require harmful chemicals and the starting material is also naturally derived.The physical and chemical settings of the experiments allow the prediction of the environmental conditions for the formation of the natural magnetite nanoparticle aggregates. It is suggested that the magnetic nanoparticle aggregates at the Peña Colorada iron mine developed in shallow to a deep marine environment with redox conditions. This was facilitated by Fe rich hydrothermal source such as marine chimneys. Because of this, we propose that the MNAs act as genetic indicators and reveal the geological conditions of their formation.Alva-Valdivia: conceptualization, formal analysis, funding acquisition, writing – original draft, supervision. Agarwal: methodology, data curation, writing – review&editing. Urrutia-Fucugauchi: writing – review&editing, Hernández-Cardona: writing – review & editing.There are no conflicts to declare.RA-013-D3RA04065H-s001


The aim of the study was to evaluate the presence of metal(loid)s in lubricating eye drops used in the treatment of dry eye disease. The concentrations of Al, As, Ba, Cd, Co, Cu, Cr, Pb, Fe, Mg, Mn, Mo, Ni, Se, V, and Zn were determined in 19 eye drop samples using inductively coupled plasma optical emission spectrometry (ICP OES). The limit of detection (LOD) and limit of quantification (LOQ) values for the quantified elements ranged from 0.0002–0.0363 (mg/L) and 0.0007–0.1211 (mg/L), respectively. High values of concentrations of Al (2.382 µg/g), As (0.204 µg/g), Ba (0.056 µg/g), Cd (0.051 µg/g), Co (1.085 µg/g), Cr (0.020 µg/g), Cu (0.023 µg/g), Fe (0.453 µg/g), Mg (24.284 µg/g), Mn (0.014 µg/g), Mo (0.046 µg/g), Ni (0.071 µg/g), Pb (0.049 µg/g), Se (0.365 µg/g), V (0.083 µg/g), and Zn (0.552 µg/g) were quantified in samples of eye drops with and without preservatives. The concentrations of As (5 samples) and Cd (3 samples) were higher than those allowed by the Brazilian Pharmacopoeia for impurities . The value of Co content (µg/g) in a sample was higher than the value established by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH Q3D (R2)) in the parenteral route. The daily eye drop instillation exposure (µg/day) was below the values from the parenteral-permitted daily exposure (PDE) set by the ICH Q3D guideline (R2). The presence of heavy metals in eye drops is an alert to regulatory agencies in several countries so that control and inspections can be carried out. According to the World Health Organization WHO, 2019), eye conditions that do not typically cause vision impairment, such as dry eye (DE), must not be overlooked 9, eye co,4, whichThe diagnosis of DE is made from the identification of the symptoms and clinical tests. However, this disease has multiple symptoms and signs that often do not correlate with one another. As a result, greater attention is being paid to diagnosing and imaging technologies to assess the type and severity of dry eye disease ,6,7. ConThe eye drops used in dry eye disease may contain preservatives in order to avoid contamination of bacteria in the vials after opening . AlthougPrevious studies have reported that metals and metalloids play an important role in the pathogenesis of several ophthalmological disorders, such as Cd, Cu, Fe, Hg, Ni, and Zn in glaucoma ,14, as wThe impurities present in eye drops can be organic, inorganic, and residual solvents. Most of these impurities occur due to manufacturing processes, degradation, storage conditions, excipients, or contamination ,29,30. ISince metals have various oxidation states, their toxicities are different . Cr , presents a process to assess and control elemental impurities in the drug product administered orally, parenterally, and inhaled; that is, it establishes the limits of permitted daily exposure (PDE) in µg/day for each element of toxicological concern and the concentration-permitted levels of impurities in drugs, drug substances, and excipients ,41,42. HMotivated by the results published in Refs. ,22,23,24The results were presented as follows: A spike was generated by adding a known amount (0.5 mg/L) of analyte to a sample, testing the spiked sample. The recovery results were as follows: Al 91.37%, As 90.86%, Ba 95.38%, Cd 96.12%, Co 90.57%, Cr 99.05%, Cu 91.69%, Fe 96.19%, Mg 97.13%, Mn 95.31%, Mo 90.23%, Ni 92.70%, Pb 95.85%, Se 97.10%, V 91,12%, and Zn 98.17%.2) for the detected elements were 0.0002–0.0363 (mg/L), 0.0007–0.1211 (mg/L), and 0.9968–0.9996, respectively. 2.The range values of the detection limits (LOD), quantification limits (LOQ), and correlation coefficients s in 19 commercial samples of lubricating eye drops used in the treatment of dry eye disease in Brazil were analyzed using optical emission spectrometry with inductively coupled plasma (ICP OES). A total of fourteen samples with preservatives (samples 1–14) and five without preservatives (samples 15–19) were analyzed. Exposure to metal(loid)s through the daily instillation of lubricating eye drops was obtained by multiplying the concentration of quantified elements in the eye drops  by the mSpike tests generated information about the recovery of the spiked samples. Since there are no certified reference metal and metalloids samples in eye drops, spike recovery tests must be used for quality control. The values of LOD and LOQ are displayed in eye drops 1 (with preservatives): Mg > Zn > Se > Fe > As > Al > Cd > Ni > Mo > Ba > Co > Cu > V > Mn;eye drops 2 (with preservatives): Se > Mg > As > Zn > Ni > Cd > Mo > Pb = Co > Ba > V > Mn > Cu;eye drops 3 (with preservatives): Se > Mg > Fe > As > Z > Cd > Ba > Mo > Ni > Co > Cu > V > Mn; eye drops 4 (with preservatives): Se > As > Zn > Cd > Mo > Ni > Ba > Co = V > Cu > Mn;eye drops 5 (with preservatives): Mg > Co > Se > As > Cd > Zn > V > Mo > Ni > Ba > Mn > Cu;eye drops 6 (with preservatives): Se > As > Zn > Cd > Mo > Ni > Co > Fe > Ba > V = Cu > Mn;eye drops 7 (with preservatives): Mg > Se > As > Zn > Ni = Cd >Mo > V > Co > Ba > Mn > Cu;eye drops 8 (with preservatives): Mg > Se > Zn > As > Ni > Cd > Mo > Co > Ba > Pb > V > Cu > Mn > Fe;eye drops 9 (with preservatives): Mg > Se > As > V > Ni > Zn > Cd > Pb > Mo > Co > Ba > Cu > Mn > Cr;eye drops 10 (with preservatives): Mg > Se > Zn > As > Ni > Cd > Mo > Pb > Co > Ba > V > Mn > Cu;eye drops 11 (with preservatives): Mg > Zn > Se > As > Ni > Cd > Mo > Co > Ba > V > Pb > Mn > Cr > Cu;eye drops 12 (with preservatives): Mg > Al > Fe > Se > As > Ni > V > Zn = Cd > Mo > Co = Ba > Pb > Mn > Cu;eye drops 13 (with preservatives): Al > Fe > Se > Mg > Zn > As > Ni > Cd > V > Mo > Ba > Co > Pb > Mn > Cu;eye drops 14 (with preservatives): Se > Mg > As > Al > Cd > Ni = Zn > Mo > Ba > Co > Mn > V > Cu;eye drops 15 (without preservatives): Se > Al > Fe > As > Cd > Co > Ni = Mo > Ba = Mg > Zn > V > Cu = Mn;eye drops 16 (without preservatives): Se > As > Cd >Ni > Ba > Mo > Zn > Co > V > Mn > Cu;eye drops 17 (without preservatives): Se > As > Ba > Cd > Mo = Ni > Zn > Cu > Co > V > Mn;eye drops 18 (without preservatives): Al > Fe > Se > Mg > Cd > Zn > As > Ni > Co > V > Mo > Ba > Cu = Mn;and eye drops 19 (without preservatives): Se > Zn > As > Fe > Cd > Co > Ba = Ni > Mo > Cr > V > Mn > Cu.The concentrations of Al, As, Ba, Cd, Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, Pb, Se, V, and Zn in lubricating eye drops from different manufacturers  decreaseValues of the concentrations of metal(loid)s quantified in lubricating eye drops with preservatives and without preservatives are shown in p < 0.05) ( < 0.05) . For allAccording to The concentration of As in the eye drop samples ranged from 0.036 µg/g to 0.204 µg/g . In eye In The concentration of Cd in the eye drop samples ranged from 0.044 µg/g to 0.051 µg . The Cd As shown in As shown in The contents of Cu in the eye drops ranged from 0.002 µg/g (eye drops 14 with preservative) to 0.023 µg/g (eye drops 17 without preservative), being below the concentration allowed by the BP (25 µg/g) and ICH Q3D guideline (R2) parenterally (30 µg/g) . ExposurIn The concentration of Mg ranged from 0.021 µg/g (eye drops 15 without preservatives) to 24.284 µg/g (eye drops 9 with preservatives). However, there are no Mg concentration values in drugs allowed by the BP and ICH Q3D guideline (R2) parenterally. According to Mn levels in eye drops 5 (0.006 µg/g), 8, and 9 (0.014 µg/g) with preservatives are below the concentration value established by the BP for impurities in parenteral drugs (25 µg/g). However, there are no values for Mn established by the ICH Q3D (R2) guideline for parenteral administration. The Mo content in eye drops ranged from 0.025 µg/g (eye drops 18 without preservatives) to 0.046 µg/g (eye drops 9 with preservatives); however, such values were below the permissible BP concentration (2.5 µg/g) and ICH Q3D guideline (R2) for the parenteral route (150 µg/g). Exposure to Mo considering the daily instillation of eye drops ranged from 0.005 µg/day (eye drops 17 without preservatives) to 0.026 µg/day (eye drops 10 with preservatives). Therefore, Mo exposure values  were fouThe concentration of Ni ranged from 0.023 µg/g to 0.071 µg/g, but these values were found to be below the concentration value of impurities in drugs established by the BP (2.5 µg/g) and ICH Q3D guideline (R2) for parenteral administration (2 µg/g) . ConsideIn As shown in The concentration values of V in the eye drops ranged from 0.008 µg/g (eye drops 14 with preservatives) to 0.083 µg/g (eye drops 9 with preservatives) . In addiThe concentration of Zn ranged from 0.019 µg/g (eye drops 15 without preservatives) to 0.552 µg/g (eye drops 11 with preservatives) . HoweverAs we observed in the results and discussion above, although the concentrations of several elements are below the limit values that have been established by regulatory agencies, such as the BP and ICH Q3D (R2), it does not mean that their use is safe. Metals and metalloids can be dangerous for human health as they have a long biological half-life and are not biodegradable, and some are toxic even at very low concentrations ,96,97.This study shows that there is a presence of toxic metals in eye drops used in the treatment of dry eye; therefore, regulatory agencies must establish a PDE level in micrograms per day (µg/day) through the instillation of lubricating eye drops in the eyes.In this study, a total of 95 eye drop samples from different manufacturers were purchased in December 2021 from pharmacies in the city of Campo Grande, state of Mato Grosso do Sul, Brazil. Only samples of lubricating eye drops with a volume of 10 mL and available in drops were selected, with 5 samples of manufacturers without preservatives (25 samples from each batch and same eye drop manufacturer) and 14 samples of manufacturers with preservatives (70 samples from each eye drop manufacturer). From each company, 5 copies of the same batch were purchased; however, samples from each batch and from the same company were mixed to obtain a representative sample for analysis.v/v) and nitric acid  for a minimum period of 24 h, following which they were rinsed in ultrapure water and dried in an oven at 42 °C.All materials used in the study, such as Falcon glassware and plastic tubes, underwent a chemical demineralization process before being used. The materials were placed in a solution of Extran , and 1.0 mL of H2O2 .Approximately 0.5 mL of each sample was placed in a glass test tube, followed with the additions of 2.0 mL of HNOThe samples were digested in an open digestion system for 40 min at a temperature of 80 °C. Subsequently, all samples were transferred from glass test tubes to Falcon tubes, where 2.5 mL of ultrapure water (conductivity 18.2 MΩcm (Millipore), Biocel, Germany) was added.All steps of the digestion analysis were performed in triplicate and the analytical blanks were prepared following the same procedure used in the samples.The contents of 16 elements  were quantified using ICP OES . Instrumental and operational parameters for ICP OES are shown in An analyte addition and recovery test (spike test) was performed, in which 0.15 mL of analytes were added to 0.5 mL of a sample of lubricating eye drops. Spike-and-recovery and linearity-of-dilution experiments are important methods for validating and assessing the accuracy of ICP OES. Spike and recovery are used to determine whether analyte detection is affected by differences in the standard curve diluent and organic sample matrix.Elementary standard stock solutions of 100 mg/L of Al, Co, Ca, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Se, and Zn  were utilized. In addition, calibration curves for all analytes were obtained using eleven different concentrations in the range from 0.001 ppm to 2 ppm.2 values were determined.The LOD and LOQ followed the analytical standards established by the IUPAC . For eacFor the maximum daily dose (mL/day) of lubricating eye drop instilled in the eyes of an adult person, it was considered that it could be obtained through multiplying the volume of 2 drops  by the maximum frequency of daily instillation (5 times a day) See .Upon instillation, part of the eye drops on the ocular surface is absorbed by the cornea, conjunctiva, and sclera, and most of it goes to the systemic circulation via the mucous membrane and rhinopharynx. That is, only a small percentage of the applied dose is released into the intraocular tissues, while about 50–100% of the administered dose may reach the systemic circulation via the conjunctiva and nasolacrimal duct ,99,100.Parenterally administered drugs also enter the systemic circulation directly and are not subjected to the first-pass effect or the gastrointestinal environment. Therefore, concentrations of metals in the eye drops, as well as the daily application, can be compared with the values of concentrations established by the Brazilian Pharmacopoeia (BP) and ICH Q3D guideline (R2) for the parenteral route.Since the elemental impurities (EIs) absorbed through the ophthalmic route are not considered in the pharmacopoeias and in the ICH Q3D (R2) guideline ,41,43, iThe PDE in micrograms per day (µg/day) give the maximum permitted quantity of each element that may be, in a medication, administered through the oral, inhaled, and parenteral routes ,41,43. HTherefore, in our study, the daily exposure to elemental impurities (EIs) through the instillation of lubricating eye drops in the eyes (µg/day) was compared to the PDEs for parenteral drugs defined in the ICH Q3D guideline (R2). In addition, the ICH Q3D (R2) guideline allows for the use of parenterally administered PDE values without modification ,41,42.p < 0.05.One-way analysis of variance (ANOVA) and Tukey’s post hoc multiple comparison test were used to test for significant differences in concentrations between eye drop brands and significant differences in values of daily instillation of lubricating eye drops with and without preservatives. All statistical analyzes were performed using the GraphPad Prism 9.0 statistical package . The significance level was set at In the present study, samples of 19 brands of Brazilian eye drops used to treat dry eye with and without preservatives showed different concentration values of As, Ba, Cd, Co, Cu, Mn, Mo, Ni, Se, V, Zn, Al, Cr, Fe, Mn, and Pb.2) for the parenteral route. However, the contents of As in the eye drops with preservatives (four different samples) and one eye drop without preservatives, as well as Cd in three eye drops with preservatives are all above the concentration value defined in the BP for parenteral use. One eye drop with preservatives had a Co concentration value greater than that established by the ICH Q3D guideline (R2) parenterally. On the other hand, the concentrations of Ba, Cr, Cu, Mn, Mo, and Ni in the eye drops were below the values established by the BP or set by the ICH Q3D (R2) parenteral guideline.There are no concentration limit values for Al, Se, Fe, Mg, Ba, Co, and Se in drugs established by the BP and Mn defined by the ICH Q3D guideline .In several countries, regulatory agencies must carry out the inspection of eye drops used in the treatment of ophthalmic diseases. Therefore, complementary studies are needed to investigate the possible risks of toxicity due to the daily instillation of lubricating eye drops containing heavy metals and metalloids.


Th. bessarabicum used in the study was indicated erroneously.In the published article, there was an error in the legend for The legend previously stated:“Ae. crassa, 4x (A–L) and 6x , and of Th. bessarabicum after FISH with different probe combinations; (A–C) the cell of 4x Ae. crassa AE 742 hybridized consecutively with CL244 + CL258 followed by CL170 + pTa-713 and pAs1 + pSc119.2; (D–F) - the cell of 4x Ae. crassa AE 742 hybridized consecutively with CL239 + CL232 followed by CL170 + pTa-713 and pAs1 + pSc119.2;  - the cell of 4x Ae. crassa AE 742 hybridized consecutively with CL131 + CL219 followed by CL170 + pTa-713; (I)- the cell of 4x Ae. crassa AE 742 hybridized with CL232 and CL257; (J–L) - the cell of 4x Ae. crassa AE 742 hybridized consecutively with CL209 + CL261 followed by CL170 + pTa-713 and pAs1 + pSc119.2;  – the cell of 6x Ae. crassa AE 131680 hybridized consecutively with CL228 + CL258 followed by pAs1 + pSc119.2;  – the cell of Th. bessarabicum PI 201890 hybridized with CL193 + CL241 followed by pTa-713. Probe combinations are given on the top of the respective images; probe color corresponds to signal color.”Metaphase cells of The corrected legend appears below:“Ae. crassa, 4x (A–L) and 6x , and of Th. bessarabicum after FISH with different probe combinations; (A–C) the cell of 4x Ae. crassa AE 742 hybridized consecutively with CL244 + CL258 followed by CL170 + pTa-713 and pAs1 + pSc119.2; (D–F) - the cell of 4x Ae. crassa AE 742 hybridized consecutively with CL239 + CL232 followed by CL170 + pTa-713 and pAs1 + pSc119.2;  - the cell of 4x Ae. crassa AE 742 hybridized consecutively with CL131 + CL219 followed by CL170 + pTa-713; (I)- the cell of 4x Ae. crassa AE 742 hybridized with CL232 and CL257; (J–L) - the cell of 4x Ae. crassa AE 742 hybridized consecutively with CL209 + CL261 followed by CL170 + pTa-713 and pAs1 + pSc119.2;  – the cell of 6x Ae. crassa AE 131680 hybridized consecutively with CL228 + CL258 followed by pAs1 + pSc119.2;  – the cell of Th. bessarabicum PI 531711 hybridized with CL193 + CL241 followed by pTa-713. Probe combinations are given on the top of the respective images; probe color corresponds to signal color.”Metaphase cells of 1 and D2 were mistyped, the accession number of Th. bessarabicum used in the study was indicated erroneously, and the source of Th. bessarabicum accessions was incorrect.In the published article, there was an error in The corrected Abstract, page one; the Introduction, paragraph two; in Materials and methods, Fluorescense in situ hybridization, paragraph two; in Results, Fluorescence in situ hybridization, paragraphs eight, ten, eleven, fifteen, sixteen, and eighteen; in Discussion,Novel markers for chromosome and genome identification, paragraph seven; in Discussion,Evolutionary changes of repetitive DNA families in Aegilops crassa genome, paragraph five.In the published article there were errors in the Aegilops crassa D1 and D2 were given incorrectly as D  and D , respectively.The designation of genomic formulas and chromosomes, the subgenomes of The corrections have been made throughout the text as follows.Abstract, page one, the sentence previously stated:In the n = 4x = 28, D1D  XcrXcr and 2n = 6x = 42, D1D  XcrXcrD2D , respectively) that are similar morphologically.”“It consists of tetraploid (4x) and hexaploid (6x) cytotypes  that are similar morphologically.”“It consists of tetraploid (4x) and hexaploid (6x) cytotypes  XcrXcr and 2n = 6x = 42, D1D  XcrXcrD  D , respectively] that are similar morphologically.”“The sentence has been corrected as follows:Ae. crassa consists of tetraploid and hexaploid cytotypes  that are similar morphologically.”“Introduction, paragraph two, the sentence previously stated:In the Ae. crassa subgenomes, designated D , is related to the D genome of Ae. tauschii , which also served as the cytoplasmic genome donor to this tetraploid species .”“One of the The sentence has been corrected as follows:Ae. crassa subgenomes, designated D1, is related to the D genome of Ae. tauschii , which also served as the cytoplasmic genome donor to this tetraploid species .”“One of the Materials and methods, Fluorescence in situ hybridization, paragraph two, the sentence previously stated:In Ae. crassa, IG 131680 carries a translocation T1D L:7D L (T10) with interstitial breakpoints in addition to two species-specific translocations, T1 (Acr:6Xcr) and T2 .”“At the same time, we have found earlier that hexaploid The sentence has been corrected as follows:Ae. crassa, IG 131680 carries a translocation T1D1L:7D1L (T10) with interstitial breakpoints in addition to two species-specific translocations, T1 (Acr:6Xcr) and T2 .”“At the same time, we have found earlier that hexaploid Results, Fluorescence in situ hybridization, paragraph eight, the sentence previously stated:In cr (middle of the short arm), 5Xcr , and on a distal part of 7D L arm , 5Xcr , and on a distal part of 7D1L arm L L and 6D L.”“Hexaploid The sentence has been corrected as follows:Ae. crassa has smaller signals of CL232 in a terminal part of 4D1S and distal quarters of 7D1L and 6D2L.”“Hexaploid Results, Fluorescence in situ hybridization, paragraph eleven, the sentence previously stated:In crL, CcrL, 5XcrL, 2D L, and 3D1), but tetraploid form contains two additional loci on 1D L and 2D1S.”“Among them, five sites are common , but tetraploid form contains two additional loci on 1D1L and 2D1S.”“Among them, five sites are common , and one or a pair of small signals are present in opposite arms of 2D1 and 7D1 , similarly to 3DL , and other intense sites are present in the proximal third of 2DL  and a distal part of 7DThe sentence has been corrected as follows:2L, similarly to 3D1L, and other intense sites are present in the proximal third of 2D2L and a distal part of 7D2S.”“The largest signal occurs on 3DResults, Fluorescence in situ hybridization, paragraph eighteen, the sentence previously stated “Most prominent sites appear on chromosome 2D1; subterminal signals on 3DL  and 3DL  are also large.”In The sentence has been corrected as follows:1; subterminal signals on 3D1L and 3D2L are also large.”“Most prominent sites appear on chromosome 2DDiscussion, Novel markers for chromosome and genome identification, paragraph seven, the sentence previously stated:In 1 and another one, overlapping with CL228, on 3DL .”“Thus, three prominent CL131 clusters were detected on 2DThe sentence has been corrected as follows:1 and another one, overlapping with CL228, on 3D1L.”“Thus, three prominent CL131 clusters were detected on 2DDiscussion, Evolutionary changes of repetitive DNA families in Aegilops crassa genome, paragraph five, the sentence previously stated:In 1S) and distal 7DL  chromosome regions.”“Genomic shock might cause massive amplification and spread of the repeat to other chromosomal sites, leading to the emergence of prominent CL219 clusters in proximal (7DThe sentence has been corrected as follows:1S) and distal (7D1L) chromosome regions.”“Genomic shock might cause massive amplification and spread of the repeat to other chromosomal sites, leading to the emergence of prominent CL219 clusters in proximal .”“The D subgenome is also present in two polyploid The sentence has been corrected as follows:Aegilops species and in hexaploid bread wheat Triticum aestivum (2n = 6x = BBAADD).”“The D subgenome is also present in two polyploid Introduction, paragraph two, a sentence previously stated:In the Ae. tauschii subsp. four exaploidy, while Ae. tauschii subsp. tauschii contributed the D subgenome to polyploid Aegilops species Ae. cylindrica and 6x Ae. crassa .”“According to molecular analysis of nuclear genome  and hybridization pattern of pAs1 probe , the D subgenome of polyploid wheat was inherited from The sentence has been corrected as follows:Ae. tauschii subsp. strangulata, while Ae. tauschii subsp. tauschii contributed the D subgenome to polyploid Aegilops species Ae. cylindrica and 6x Ae. crassa .”“According to molecular analysis of nuclear genome  and hybridization pattern of pAs1 probe , the D subgenome of polyploid wheat was inherited from Introduction, paragraph three, a sentence previously stated:In the Ae. comosa and suggested genomic formula DM for tetraploid and DDM for four hexaploid Ae. crassa.”“Kihara (1963) proposed that this subgenome could be inherited from The sentence has been corrected as follows:Ae. comosa and suggested genomic formula DM for tetraploid and DDM for hexaploid Ae. crassa.”“Kihara (1963) proposed that this subgenome could be inherited from Materials and methods, Real-time quantitative PCR, paragraph one, a sentence previously stated:In “Primers 2) were synthesized at Syntol Ltd. .”The sentence has been corrected as follows:“Primers were synthesized at Syntol Ltd. .”Materials and methods, DNA probes for FISH, paragraph one, a sentence previously stated:In Ae. crassa genome: CL3, CL8 (highly homologous to CL16 found in Ae. Tauschii subsp. nine trangulate), CL18, CL60, CL131 (highly homologous to CL149 found in Th. bessarabicum), CL170, CL193, CL209, CL219, CL228, CL232, CL239, CL241, CL244, CL257, CL258, CL261 (highly homologous to CL198 found in Th. bessarabicum); ii) derived from 6x Ae. crassa genome: CL27_232; iii) derived from Th. bessarabicum genome: CL2, CL148.”“The following novel repeats identified by means of low-coverage sequencing followed by bioinformatics analysis were used as FISH probes: (i) derived from 4× The sentence has been corrected as follows:Ae. crassa genome: CL3, CL8 (highly homologous to CL16 found in Ae. tauschii subsp. strangulata), CL18, CL60, CL131 (highly homologous to CL149 found in Th. bessarabicum), CL170, CL193, CL209, CL219, CL228, CL232, CL239, CL241, CL244, CL257, CL258, CL261 (highly homologous to CL198 found in Th. bessarabicum); ii) derived from 6x Ae. crassa genome: CL27_232; iii) derived from Th. bessarabicum genome: CL2, CL148.”“The following novel repeats identified by means of low-coverage sequencing followed by bioinformatics analysis were used as FISH probes: (i) derived from 4× Materials and methods, Fluorescence in situ hybridization, paragraph one, a sentence previously stated:In n and pTa-535 according to Komuro et al. (2013); chromosomes of Ae. Tauschii were classified as suggested by Badaeva et al.  and Zhao et al. (2018), Ae. crassa as in Abdolmalaki et al. (2019), and Badaeva et al. (2021).”“The A subgenome chromosomes of wheat were classified using additional probe combination GAAThe sentence has been corrected as follows:n and pTa-535 according to Komuro et al. (2013); chromosomes of Ae. tauschii were classified as suggested by Badaeva et al.  and Zhao et al. (2018), Ae. crassa as in Abdolmalaki et al. (2019), and Badaeva et al. (2021).”“The A subgenome chromosomes of wheat were classified using additional probe combination GAAMaterials and methods, Fluorescence in situ hybridization, paragraph two, the sentence previously stated:In Ae. crassa, 4x), K-112 (Ae. tauschii subsp. strangulata), PI 201890 (Th. bessarabicum), and Chinese Spring have normal karyotypes typical to the respective species .”“Our previous analyses showed that accessions K-2485 and AE 742 , K-112 (Ae. tauschii subsp. strangulata), PI 531711 (Th. bessarabicum), and Chinese Spring have normal karyotypes typical to the respective species .”“Our previous analyses showed that accessions K-2485 and AE 742 genomes.”“According to bioinformatics and qPCR the novel CL239 repeat is absent in The sentence has been corrected as follows:Ae. tauschii genome.”“According to bioinformatics and qPCR the novel CL239 repeat is absent in the Discussion, Evolutionary changes of repetitive DNA families in Aegilops crassa genome, paragraph eight, a sentence previously stated:In cr genome, but is abundant in the D (sub)genome of wheat and Ae. tauschii, Ae. crassa, and Th. bessarabicum.”“We can reach a similar conclusion considering that the CL170 repeat is absent from XThe sentence has been corrected as follows:cr genome, but is abundant in the D (sub)genome of wheat and Ae. tauschii, Ae. crassa, and Th. bessarabicum.”“We can reach a similar conclusion considering that the CL170 repeat is absent in XThe publisher apologizes for these errors. The original article has been updated.


Chrysanthemum indicum L., which exists in various shades of yellow and has pharmacologically active constituents. It is widely used for medicinal purposes in China, Japan, and South Korea to treat inflammatory diseases. Its external color is usually used to judge its internal quality in trade; however, the correlation between its color and chemical constituents is unknown. Here, we simultaneously determined five phenylpropanoids  and four flavonoids  of 70 CIF germplasms using a newly established UPLC method; furthermore, we measured their color parameters  using a spectrophotometer. Our results showed considerable variations in the bioactive constituent contents and color parameters of CIF. The content of the five phenylpropanoids and the relative correlation degree γi of the nine constituents were positively correlated with color parameters, which could be rapidly predicted based on L* and/or b*. Moreover, we screened out a high-quality germplasm with a high linarin content and bright colors using the hierarchical clustering method. Our results provide comprehensive insight into CIF’s quality evaluation process, particularly the methods for procuring high-quality medicinal materials and breeding by color.Chrysanthemi Indici Flos (CIF) is the inflorescence of  Chrysanthemum indicum L. , the inflorescence of dicum L. , has fevted TCMs .C. indicum, a polymorphic species with various morphological characteristics and pharmacologically active constituents, is mainly distributed in northeast, southwest, northern, eastern, and central China [Chinese Pharmacopoeia 2020, CIF is brownish yellow in color [al China . In accoin color . In CIF Medicinal material appearance characteristics, including shape, color and odor, are considerable indicators for quality evaluations, especially the colors that are widely used in trade ,9. HowevChinese Pharmacopoeia 2020, linarin content, the only chemical marker for CIF quality control, cannot be less than 0.8% [According to the han 0.8% ; howeverhan 0.8% . Despitehan 0.8% , the corL* a* b* system by the Commission International on Illumination (CIE); nine major bioactive constituents in CIF, including five phenylpropanoids  and four flavonoids , were determined simultaneously using a newly established ultra-performance liquid chromatography (UPLC) method; furthermore, grey relational analysis (GRA), a quantitative comparative analysis method that is widely used in the quality assessment of TCMs [In our study, we measured CIF color parameters using a spectrophotometer based on the  of TCMs , was perC. indicum germplasms were collected from 11 provinces in China, including Anhui, Henan and Hubei, the main CIF-producing areas  was transferred into a conical flask with 100 ml 70% methanol aqueous solution and extracted by refluxing for 3 hours. Then, we cooled the extraction to room temperature, compensated the loss weight with 70% methanol aqueous solution and filtered the supernatant using a 0.22 um millipore filter. We stored the subsequent filtrate at 4°C until UPLC analysis.Based on the methods of the -1 neochlorogenic acid, 21.19 μg·mL-1 chlorogenic acid, 22.44 μg·mL-1 isochlorogenic acid B, 22.81 μg·mL-1 isochlorogenic acid A, 20.17 μg·mL-1 isochlorogenic acid C, 45.90 μg·mL-1 linarin, 20.70 μg·mL-1 luteolin, 24.10 μg·mL-1 apigenin, and 21.81 μg·mL-1 acacetin in the prepared mixed reference solution.Reference standards  were weiTM system H-CLASS , which was equipped with a binary solvent delivery manager and a sample manager. We performed chromatographic separations using an Acquity UPLC HSS C18 column . The mobile phase was composed of solvents A (100% acetonitrile)  and B (0.05% formic acid aqueous solutions) . We maintained the column temperatures, flow rates, injection volumes of the reference standards and samples, and detection wave lengths at 25°C, 0.4 mL·min-1, 3.0 μL, and 326 nm, respectively. We optimized the gradient program as follows: 0–6 min, 10%-18% A; 6–7 min, 18%-20% A; 7–12 min, 20%-20% A; 12–19 min, 20%-23% A; 19–20 min, 23%-28%; 20–25 min, 28%-30% A; 25–38 min, 30%-40% A; 38–40 min, 40%-65% A; 40–42 min, 65%-90% A; 42–44 min, 90%-90% A; 44–44.1 min, 90%-10% A; 44.1–47 min, 10%-10% A.We conducted identification and quantification of the nine bioactive constituents on a Waters Acquity UPLCC. indicum germplasms into a glass cell  and measured the color parameters  using a YS3060 spectrophotometer  with a pulsed xenon arc lamp  and a 10° observer. We measured each CIF sample 3 times. Additionally, we calibrated the spectrophotometer by black and white correction plate before use. Moreover, we determined the standard color difference value E*ab using the formula:We transferred the CIF powder of the 70 γi of the nine bioactive constituents of CIF based on the method outlined by Hua et al. [H’ of the bioactive constituents, γi and the color parameters  of CIF were calculated using the PCH’ Diversity Index Calculation Tool designed by the researcher Beiluozhongyuan (https://pan.baidu.com/s/1jG3eWKI). We conducted Pearson correlation and multiple linear regression (MLR) analyses using SPSS v21  to elucidate the relationships between the nine bioactive constituents, γi and their color parameters. We performed cluster analysis on the γi and color parameters using TBtools v1.0983 [γi and color parameters among different cluster groups using SPSS v21 using one-way ANOVA followed by Fisher’s least significant difference (LSD) test.We used MS Excel 2019  to calculate the relative correlation degree a et al.  to evalu v1.0983  and SPSSAnalytical method validation. The CIF sample extractions and the mixed reference solutions were detected according to the above-mentioned UPLC conditions. The chromatographic resolution between component peaks was greater than 1.5, and the number of theoretical plates was more than 31,000. The nine components in the mixed reference solution and the CIF sample extractions showed high-resolution separation without interference, and their retention time was stable  six times, added reference solutions, extracted the bioactive constituents and detected constituents according to the above-mentioned UPLC conditions. The recovery and the RSD of the nine bioactive constituents was between 98.62% and 101.57%, and between 0.31% and 1.69%, respectively .γi and color parameters of C. indicum germplasms all exhibited abundant genetic variation (H’ > 3)  and the smallest H’ (3.456). In contrast, neochlorogenic acid had the highest H’ (4.106) and the smallest CV (54.938%). The values of L*, a* and b* were 53.938–71.312, -2.620–5.240, and 18.240–39.397, respectively. Therefore, the predominant CIF color was white, its subordinate color was yellow, and E*ab was mainly affected by L* and b*.The bioactive constituents, (H’ > 3) . The meaL*, a* and b* values  and b* . The a* value was significantly negatively correlated with chlorogenic acid (P = 0.016), but had no significant correlation with neochlorogenic acid (P = 0.260). Isochlorogenic acid A and γi were significantly positively correlated with L* value . Isochlorogenic acid C and acacetin were significantly positively (P = 0.049) and negatively (P = 0.027) correlated with b*, respectively. However, linarin, luteolin, and apigenin had no significant correlation with its color parameters , chlorogenic acid , isochlorogenic acid B , isochlorogenic acid A , isochlorogenic acid C , acacetin  and γi  had linear relationships with CIF’s color parameters , and they had the highest γi and L*, and the lowest a*; therefore, they had the highest quality and the most brightness compared with the others. As the γi, L*, b*, and linarin of AHTC2 were higher than those of HAHC, AHTC2 was more suitable for medicine or tea. Sixteen germplasms in Group Ⅳ had the highest b* and E*ab, although we observed no considerable differences in their comprehensive quality γi with Group Ⅱ.When the rescaled distance cluster combination was 15, the 70 ups Figs . The 40 γi, and the color of C. indicum germplasms, and that there were considerable correlations among them. We could quickly predict the γi and phenylpropanoid content of CIF based on its color. However, we found no significant correlation between the CIF color and its chemical maker linarin. Therefore, CIF’s external color can be used to evaluate its internal quality in the medicinal material trade and for breeding; however, we cannot assess whether its linarin content conforms to the standard of the Chinese Pharmacopoeia 2020 through its color.Medicinal materials have many kinds of pharmacology constituents that play considerable roles in physiological regulation through synergistic or antagonistic effects, and it is inappropriate to rely on only a few constituents to assess their quality ,18. GRA Chinese Pharmacopoeia 2020 standard. These CIF characteristics, including rich genetic diversity and low linarin content, have been reported in several studies. It has been reported that there was high genetic diversity among C. indicum geographic populations, and their genetic distances showed no considerable correlations with geographic distance [Chinese Pharmacopoeia 2020 [Our results showed that the bioactive constituent content and the color parameters of the 70 CIF germplasms were different compared with each other; these germplasms were clustered into different groups. For example, we clustered 16 germplasms from 13 prefecture-level cities of seven provinces of China into Group Ⅳ. Moreover, the linarin content of these germplasms, which comprised more than 58%, was not up to distance . Furthereia 2020 . Althougeia 2020 ,26. For eia 2020 .C. indicum is a cross-pollination plant with maternal inheritance, and its phenotypes, including morphological character and chemical components of its sexual offspring, are partially similar to its maternal parent [Chrysanthemum, our selected germplasm can be expanded by asexual propagation to realize popularization; however, the cost is high [C. indicum germplasm resources.l parent ,29. In o is high . Further is high . Breedinb* (yellow-blue chromaticity). However, b* was positive with phenolic acids . The correlation phenomenon between the yellow and phenolic acids of CIF, apart from linarin, luteolin and apigenin of CIF need to be further studied. Furthermore, although CIF drying methods such as drying in the sun or drying after steaming are recommended by the Chinese Pharmacopoeia 2020, other drying methods, including stoving, stoving after stir-fried blanching and stoving after steam blanching, are widely used in CIF production. Different processing methods will lead to different colors of CIF [Yellow is the main color of CIF, which is mainly composed of carotenoids, largely lutein . These cs of CIF . The infγi of CIF, were positively correlated with and could be estimated based on color parameters L* and/or b*. However, there were no significant correlations between the color parameters and CIF’s chemical marker—linarin. Our work largely contributes to quality evaluations of CIF by color to breed and procure high-quality medicinal materials.In our study, we established a new UPLC method to determine nine major bioactive constituents of CIF; furthermore, we revealed the correlations between the nine bioactive constituents and CIF color parameters. Our results showed that the concentrations of CIF’s neochlorogenic acid, chlorogenic acid, isochlorogenic acids A, B and C, and acacetin, as well as the comprehensive quality S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Impairment of atrial function was greater in Group 1 compared to Group 2. A PALS variation > 20.8% identified patients who were most likely to develop asymptomatic mild cardiotoxicity . Conclusions: PALS and LASi significantly change during chemotherapy in association with GLS. Atrial strain is an additional parameter that could be measured together with GLS to detect cardiotoxicity early.Left ventricular global longitudinal strain (GLS) has an important role in the diagnosis of cancer therapy-related cardiac dysfunction (CTRCD). Little is known about the role of atrial function in diagnosing CTRCD. The aim of our study was to assess the impact of anti-cancer drugs on atrial function measured by speckle-tracking echocardiography in breast cancer women. A prospective multicenter study was conducted enrolling 169 breast cancer women treated with anthracyclines. A cardiological evaluation including an electrocardiogram and echocardiogram with an analysis of GLS, left atrial (LA) strain, and LA stiffness (LASi) was performed at baseline (T0), 3 (T1), and 6 months (T2) after starting chemotherapy. The patients were divided into two groups: patients with asymptomatic mild cardiotoxicity at T1  and those without (Group 2). We did not find a significant change in left ventricular ejection fraction (LVEF) at T1 and T2; we found a significant change in GLS ( It is known that anti-cancer drugs can cause cardiovascular complications such as left ventricular dysfunction, heart failure, arrhythmias, myocardial ischemia, and arterial hypertension. The mechanisms underlying cardiotoxicity may involve direct and/or indirect mechanisms of cardiomyocytes. Anthracyclines are associated with a high incidence of heart failure as a consequence of direct damage to cardiomyocytes which, once their function is altered, could lead to cell death. Other mechanisms underlying cardiotoxicity may include vascular dysfunction and thromboembolic ischemia. Other adverse effects result from impaired signaling of cardioprotective factors for cardiomyocytes, such as Neuregulin-1 (NRG1), or for other cardiac cell populations, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) ,3,4. SevA prospective multicenter study was carried out enrolling 169 women with newly diagnosed breast cancer, scheduled to receive anthracyclines (ANT) therapy followed by paclitaxel plus trastuzumab. Eight different Italian centers were involved: University Hospital “Paolo Giaccone”—Palermo (64 patients), ASST Metropolitan Hospital “Niguarda”—Milan (28 patients), University Hospital “Ospedali Riuniti”—Foggia (26 patients), University Hospital “San Giovanni di Dio”—Cagliari (23 patients), University Hospital “Borgo Trento”—Verona (16 patients), University Hospital “Gaetano Martino”—Messina (12 patients), University Hospital of Pisa, and University Hospital of Siena. Inclusion criteria were newly diagnosed breast cancer, age between 18 and 80 years old, and treatment with anthracyclines for at least 4–6 cycles followed by trastuzumab. Exclusion criteria were prior chemotherapy or radiotherapy; ischemic heart disease , severe valve diseases, baseline LVEF < 50%, atrial fibrillation and sustained ventricular arrhythmias, life expectancy < 1 year, poor acoustic window, and unfeasible STE. After approval by the local ethics committee and after providing written informed consent, all the patients underwent cardiological evaluation at baseline (T0), 3 (T1), and 6 (T2) months after starting chemotherapy. Clinical and anamnestic data were collected. An electrocardiogram and an echocardiogram with STE analysis were performed at baseline and during follow-up. The diagnosis of CTRCD was made according to the 2022 ESC guidelines on cardio-oncology, based on the LVEF and/or GLS reduction from baseline. Patients were divided into 2 groups: patients with mild asymptomatic cardiotoxicity, defined as a relative reduction in GLS > 15% at T1 compared to baseline (Group 1), and those without (Group 2). Echocardiographic examination was performed according to EACVI/American Society of Echocardiography (ASE) recommendations for chamber quantification , using aSTE analysis was performed offline to assess GLS and LA strain using semiautomated 2D Strain GE software . The operators analyzing the images were not aware of the clinical characteristics of the patients. The GLS was obtained by recording apical 2-, 3-, and 4-chamber views with a frame rate between 60 and 80 fps and a stable electrocardiographic trace. The analysis was performed offline by two experienced and independent echocardiographers. Normal reference values of GLS were considered: −21.5 ± 2%, with a lower limit of normal of—18% ,23:○PALS: 42.5% (IQR 36.1–48.0%), LLN 26.1 ± 0.7%○LA conduit strain: −25.7% (IQR 20.4–31.8%), LLN—12.0 ± 0.5%○PACS: −16.3% (IQR 12.9–19.5%), LLN—7.7 ± 0.3%○0.12 (IQR 0.10–0.15) for ages 20–40, with LLN 0.22 ± 0.010.13–0.22) for ages 40–60, with LLN 0.42 ± 0.040.24 (0.18–0.29) for age > 60 years, with LLN 0.55 ± 0.09LASi:t-test or, when necessary, the Mann–Whitney test was used to compare the two groups. Categorical variables were evaluated based on percentages of the total population and compared using the χ2 test and Fisher’s exact test. A one-way ANOVA test was used to determine if there was a statistically significant difference between the means of three or more groups. After adjustment for lack of sphericity in repeated measures, the ANOVA was performed with the Greenhouse–Geisser correction. ROC analysis was performed to evaluate the predictive ability of the percentage reduction in LA strain value between T1 and T0 on the development of subclinical dysfunction, as assessed by a reduction in GLS > 15% from T0 to T1. Continuous variables were expressed as means and standard deviations or medians with interquartile ranges as appropriate. The Student’s p-value < 0.05 was considered statistically significant. Bonferroni’s correction was used to correct the significance of multiple tests. All the statistical analyses were performed using RStudio software (version 1.4.1103 2009–2021 RStudio).A two-tailed After collecting and analyzing data from the different centers involved, the “total study population” consisted of 169 patients  with newly diagnosed breast cancer, “HER2 receptor-positive”. According to the TNM classification, most patients were in stage IIA. More precisely, the anatomical classification according to TNM is shown in As for echocardiographic parameters at baseline, our cohort showed normal values of LVEF (60 ± 1.7%), GLS (−20.7 ± 2.1%), PALS (36 ± 8.9%), and normal values of LAVi and normal right ventricular function. Regarding the assessment of diastolic function in all the population at baseline, we found that at T0, 37% of patients had a normal diastolic function, 62% had grade I diastolic dysfunction, and only 1 patient had grade II diastolic dysfunction; no patients had grade 3 diastolic dysfunction. p-value > 0.05). Analyzing diastolic function, the percentages of diastolic dysfunction present at baseline (T0) remained unchanged at T1; at T2, only 1 patient had grade II diastolic dysfunction, 74% of the patients had grade I diastolic dysfunction, and 25% of the patients had no diastolic dysfunction. In addition, during the follow-up, the E/A ratio changed significantly , but the mean E/E’ ratio  and pulmonary systolic pressure did not change significantly ; see p-value < 0.0001) and in PALS . Furthermore, LASi worsened during follow-up ; see p-value = 0.03). We evaluated the development of CTRCD during follow-up, and we found that no patients developed symptomatic CTRCD or severe asymptomatic CTRCD. One patient developed moderate asymptomatic CTRCD at T1, and two patients developed moderate asymptomatic CTRCD at T2. A total of 28 patients  developed asymptomatic mild CTRCD.In the whole population after 3 (T1) and 6 (T2) months of anti-cancer therapy, we did not observe a significant reduction in LVEF ; a significant reduction in GLS over time ; a significant reduction in PALS ; and a significant increase in LASi . The LAVi and mean E/e’ ratio did not change significantly ; the E/A ratio changed significantly ; see Specifically, we found in Group 1 a non-significant reduction in LVEF during the follow-up , PALS , LASi , and mean E/e’ ratio , we did not find significant changes in LVEF. We also observed significant changes in GLS (p = 0.06, sensitivity 45%, specificity 69.5%].In particular, we found a 20% reduction in PALS in Group 1 at T1 compared to baseline . This reIn addition, when comparing the two groups at baseline, we found no significant differences in baseline PALS value or other echocardiographic parameters; only the baseline GLS and mean E/e’ ratio differed significantly but remained within normal values .The present multicenter study was one of the first to evaluate LA function by STE in cardio-oncology. Our study showed the cardiotoxic effects of anthracyclines and trastuzumab on atrial function evaluated by STE, and it identified a percent change in PALS able to detect patients at higher risk of CTRCD. In our study, GLS and PALS changed significantly in all the patients treated with anti-cancer drugs, LASi increased significantly, and LVEF did not change during follow-up. Thus, in agreement with the literature data, our study reconfirms the ability of deformation imaging parameters such as GLS to identify signs of cardiotoxicity. In fact, GLS is more sensitive than LVEF and other echocardiographic parameters such as TDI for detecting early cardiac damage . For a lOur multicenter study highlights the usefulness of LA function assessment using STE in women with breast cancer treated with chemotherapy because atrial function also changes significantly during anticancer treatment. Particularly in our study, the impairment of LA strain was significantly higher in patients who developed asymptomatic mild CTRCD. A cut-off of reduction in PALS of 20.8% was identified to detect patients at higher risk of cardiotoxicity. Thus, the assessment of PALS in addition to GLS could be considered to discover subclinical cardiotoxicity more accurately. In daily clinical practice, the use of this parameter could help in identifying cancer patients with a greater risk of cardiotoxicity to more precisely manage cardioprotective therapy and design tailored cardiological follow-ups.