This study was aimed at determining the antioxidant, anti-quorum sensing, and Auricularia auricula-judae, Termitomyces umkowaani, Trametes elegans, Trametes versicolor, and Microporus xanthopus were collected from Arabuko-Sokoke and Kakamega National Forests, in Kenya. Specimens were identified and extracted using chloroform (CHL), 70% ethanol (Eth), and hot water (HW) solvents. Antioxidant and cytotoxic activities of the extracts were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Vero cell lines, respectively, while anti-quorum sensing activities were tested against Chromobacterium violaceum. All data were compared using relevant descriptive and inferential statistics at a significance level of p ≤ 0.05.  Wild mushrooms of A. auricula-judae, T. umkowaani, T. elegans, and T. versicolor were observed in 70% Eth extract with the percentage values of 76.40 ± 0.12%, 68.40 ± 0.01%, 62.40 ± 0.07%, and 66.40 ± 0.04%, respectively, whereas the HW extract of Microporus xanthopus showed free radical scavenging activity at 65.90 ± 0.02%. None of the extracts, at the tested concentrations (up to 1000 µg/mL), had shown cytotoxic activity against the Vero cell line. The HW extract of T. umkowaani and the 70% Eth extract of T. versicolor showed a statistically significant difference in the inhibitory activity of violacein production against C. violaceum at the concentration of 200 µg/mL.  A total of 35 wild mushrooms were collected, identified, and classified into 14 genera. Among screened mycochemicals, fatty acids, flavonoids, polyphenols, and saponins were detected at higher concentrations. The highest free radical scavenging activities of  The antioxidant activity of wild mushrooms can help to tackle the diseases caused by free radicals. The anti-quorum sensing potential of wild mushrooms could also provide future alternatives to conventional drug therapies cost-effectively. Further detailed chemistry of the bioactive compounds and their possible mechanisms of action responsible for the observed antioxidant and anti-quorum sensing activities are needed. Mushrooms are primarily underutilized natural resources . Many acMicrobial cells use the signal transduction pathway known as quorum sensing (QS) to communicate with one another and within their own species. The QS transduction pathway is started once the signaling molecules have accumulated to a certain concentration and begun to bind to a protein detector. Finally, this route activates the genes that control pathogenicity, rotting, and biofilm formation –20. One Our bodies manufacture dangerous free radicals as a result of the respiration, digestion, and metabolism process . Free raTo the best of our knowledge, no research has been conducted on the detection of antioxidant and anti-quorum sensing activities of wild mushroom extracts obtained from Arabuko-Sokoke and Kakamega National Reserved Forests in Kenya. Therefore, the purpose of this work was to ascertain the antioxidant and anti-quorum sensing capabilities of bioactive compounds isolated from five indigenous Kenyan wild mushrooms.Arabuko-Sokoke and Kakamega National Forests are situated within 3°20′S and 39°55′E and 34°32′0″E and 0°10′15″S, respectively, as shown in An experimental and exploratory cross-sectional design was used. An experimental (to ensure that the right kind of data can provide a clear and effective response to the research questions) and exploratory cross-sectional design  was used.During the months of March and April 2018, random collections of wild mushrooms on either tree bark or other substrates  were made. To prevent breakage and preserve moisture content, specimens were wrapped in aluminum foil and put in an ice box. Molecular and morphological methods were used to identify the specimens, together with the assistance of mycology specialists and relevant literature. Finally, samples were preserved for additional analysis after being dried in an electric drying oven for 480 h at 30°C , 27.Chloroform (CHL), 70% ethanol (Eth), and hot water (HW) solvents were used for the extraction , 29. 100The extracts were subjected to a qualitative mycochemical screening analysis according to standard procedures . Distill3) test revealed the presence of polyphenols. A solution of 2 mL of distilled water, 3 drops of 10% FeCl3, and 3 drops of potassium ferrocyanide were mixed with 1 mL of each extract at a concentration of 10 mg/mL. Blue or green color formation shows the presence of polyphenols [A foam test was used to determine the presence of saponins. 5 mL of distilled water and 1 mL of each 10 mg/mL extract were added, and then the mixture was shaken vigorously. The presence of saponins is indicated by foam production. The ferric chloride  was used to treat a certain amount of the extracts, which resulted in the production of a cream-colored precipitate .2SO4 was used to determine flavonoids. A fraction of the extracts were treated with concentrated H2SO4 and the production of orange color was noticed [H noticed .Fatty acids were detected by placing 0.5 mL of extract onto filter paper. 0.5 mL of extract at 20 mg/mL was placed on a filter paper. The presence of fatty acids is indicated if the stain on the filter paper persists. A 2 mL of the extract at 20 mg/mL was dissolved in diethyl ether and evaporated to dryness. The presence of volatile oil is indicated by a pleasant odor .A. auricula-judae, M. xanthopus, T. umkowaani, T. elegans, and T. versicolor extracts in CHL, 70% Eth, and HW were evaluated using the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) method [The antioxidant properties of ) method , 29. In µg/mL) were used as a standard antioxidant (positive control). Free radical scavenging activity (FRS) of each extract was expressed as the percentage inhibition of the free radical by the extracts, and it was calculated by using the following formula:In a test tube, various extract concentrations  were made, and 1.00 mL of 400 M DPPH was added. When stable absorption values were attained, the mixture was rapidly mixed and left at room temperature for 1 hour in a dark area. As negative controls, 1.00 mL of distilled water, 70% Eth, CHL, and DPPH were made without extracts. A 1.00 mL sample solution was placed in a cuvette and the color changed from purple to yellow and optical density of the solution was measured using a visible spectrophotometer at 517 nm. Different concentrations of ascorbic acid  was calculated from the graph of RSA percentage against extract concentration [The extract concentration providing 50% inhibition  in a test tube), the number of viable cells was counted using an inverted phase-contrast microscope and the Trypan blue exclusion test on a hemocytometer slide. A 96-well plate was seeded with an aliquot of 2.0 104 cells/mL suspension and incubated in 5% CO2 at 37°C for 24 h. Then, in a nutshell, 150 mL of the medium was added to the wells in rows H along with 1,000 g/mL of extracts. From row H to row B, a serial dilution was performed. Row A served as the negative control, and the plates were incubated for 48 h at 37°C with 5% CO2. Cell viability was assessed using the 3--2,5-diphenyl tetrazolium bromide (MTT) test. The capacity of the living cells to convert the yellow MTT dye into a purple formazan product served as a measure of the cells' growth. 10 L of the MTT dye was added to the cells after 48 h, and they were then incubated for 4 h at 37°C with 5% CO2. After removing the entire media from the plates, formazan crystals formed by live cells were dissolved by adding 50 L of dimethyl sulfoxide and vigorously shaking the mixture. Using a microplate reader and a reference wavelength of 720 nm, the absorbance of each well was measured. The following formula was used to calculate cell viability percentage:50) values. If the IC50 is  > 90 µg/mL, the extract is classified as noncytotoxic. If the IC50 is between 20 and 89 µg/mL, the extract is classified as moderately cytotoxic. If the IC50 is <20 µg/mL, the extract is classified as cytotoxic [The Vero cell line (P171) was grown in T-75 flasks using minimal media that was supplemented with 10% fetal bovine serum (FBS) and 100 g/mL streptomycin (pH 7.25). For 72 h, the T-75 flasks were incubated at 5% COytotoxic , 34.Chromobacterium violaceum (CV026) was cultured in tryptic soy broth (Sigma) with shaking (180 rpm) and incubated at 30°C for 18 h [8 CFU/ml) using a spectrophotometer. A 100 μL of CV026 and 20 μL of the 100 μg/mL C6HSL (hexanoyl homoserine lactone) solution (dissolved in acidified ethyl acetate) were added onto a 5 ml of 1% warm molten tryptic soy agar. Then, the solution was gently mixed and poured immediately over the surface of a solidified tryptic soy agar plate as an overlay. Wells of 5 mm in diameter were made on each plate after the overlay was solidified. A 10 mg/mL extract was dissolved in DMSO and sterilized using a 0.45 µm filter membrane. Of the 10 mg/mL extract, 20 μL, 10 μL, and 5 μL were serially diluted and filled to each well. Another well was filled with 5 μL of 100 μg/mL of C6HSL and 45 μL of sterile tryptic soy broth as a negative control. Finally, assay plates were incubated at 30°C for 72 h [for 18 h . The ovefor 72 h . The lacC. violaceum (20 µL) was inoculated into four Erlenmeyer flasks containing 100 mL lauryl blue (LB) broth and incubated at 30°C for 18 h. The flasks were supplemented with 100 µL of 100 μg/mL C6HSL and 20 µL, 10 µL, and 5 µL of extracts from a stock solution (10 mg/mL). The flasks were incubated again at 30°C with shaking (150 rpm) for 20 h. A 1.00 mL of culture was taken from each flask to the test tube and centrifuged at 8,000 rpm for 10 min to precipitate the insoluble violacein. The supernatant was discarded, and then 1.00 mL of DMSO was added to the test tube and shaken for 30 seconds to completely solubilize the precipitated violacein. The test tube was centrifuged at 8,000 rpm for 10 min to remove cells. Then, 200 µL of the supernatant was added to a 96-well plate in triplicates, and absorbance was read at 585 nm. The percentage of violacein inhibition is calculated asSimultaneously, the antibacterial effect of the extracts on the growth of the test bacteria was tested by culturing in the presence and absence of extracts and OD was read , 38.p ≤ 0.05 [The obtained results of each experiment were performed in triplicates and expressed as the mean ± SD values. Microsoft Excel Package was used to analyze quantitative data and draw graphs. All quantitative data were compared using relevant descriptive and inferential statistics at a significance level of p ≤ 0.05 .Agaricus, Auricularia, Boletus, Cantharellus, Daedaleopsis, Ganoderma, Lactarius, Leucocoprinus, Microporus, Pleurotus, Polyporus, Russula, Termitomyces, and Trametes. These genera were also classified into edible and inedible mushrooms. The edible mushrooms include Agaricus, Auricularia, Cantharellus, Ganoderma, Leucocoprinus, Pleurotus, Termitomyces, and Trametes. The inedible mushrooms are Daedaleopsis, Microporus, and Lactarius. The wild mushrooms which include both edible and inedible categories are Boletus, Polyporus, and Russula.A total of 35 wild mushrooms were collected from Arabuko-Sokoke National Forest (ASF) (23 specimens) and Kakamega National Forest (KF) (12 specimens). The collected wild mushrooms were classified into 14 genera, namely, Polyporus (ASF1) 70% Eth extract, Trametes (ASF3) HW and 70% Eth extracts, and Termitomyces (ASF14) HW extract. HW, 70% Eth, and CHL extracts of Termitomyces (ASF14) yielded moderate quantities of saponins, terpenoids, and fatty acids. Fatty acids and volatile oils were lacking in HW and 70% Eth extracts in all mushroom species.The extracts of the 23 wild mushrooms were qualitatively tested. Alkaloids, anthraquinones, fatty acids, flavonoids, polyphenols, saponins, tannins, terpenoids, and volatile oils were found in various amounts as shown in Different chemical constituents such as flavonoids, polyphenols, saponins, tannins, and terpenoids, among others, were obtained from all extracts as shown in Termitomyces spp. (KF2), whereas saponins, tannins, and anthraquinones were found in moderate concentrations. On the other hand, extracts of Termitomyces spp. (KF2) in 70% Eth included moderate amounts of tannins, alkaloids, flavonoids, and anthraquinones. Moderate fatty acid concentrations were found in the CHL extracts of Termitomyces (KF1), Termitomyces (KF3), and Auricularia (KF6). The majority of the chemicals lacking from the CHL extracts of most mushroom species were present in the HW and 70% Eth extracts of all mushrooms. All mushroom species' CHL extracts lacked alkaloids or anthraquinones. Similarly, none of the mushroom species' HW and 70% Eth extracts contained any fatty acids or volatile oils.tPolyphenols and flavonoids were found in the highest amounts in the HW extracts of The most prevalent substances in the HW and 70% Eth extracts of the majority of mushroom species were, in general, polyphenols, tannins, flavonoids, and terpenoids. Different amounts of the mycochemical components of the wild mushrooms were discovered, and they declined in polarity from more polar to less polar solvents. Most substances are soluble in HW and solvents containing 70% Eth, with the exception of fatty acids and volatile oils. The polyphenolic concentration of the aqueous and Eth extracts was higher than that of the CHL extracts.A. auricula-judae (KF6) exhibited dose-dependent free radical scavenging (FRS) activity, as shown in 50) values for 70% Eth, CHL, and HW extracts. The ascorbic acid showed the highest FRS activities despite the fact that all the extracts showed good FRS activities.Each extract of M. xanthopus (KF12) extracts improved FRS activities as extract concentration rose as shown in 50 values of CHL and HW extracts were determined to be between 50 and 60 g/mL.All T. umkowaani (KF2) extracts contained a significant amount of FRS activity as shown in 50 values of CHL extract, 70% Eth extract, and HW extract were 60–70 g/mL, 40–50 g/mL, and 50–60 g/mL, respectively.All T. elegans (ASF3) extracts in CHL, 70% Eth, and HW was 49.30 ± 0.03%, 62.40 ± 0.07%, and 55.90 ± 0.01%, respectively. When compared to all mushroom extracts, ascorbic acid (positive control) had the highest FRS activity as shown in 50 values for 70% Eth and HW extracts were 50 g/mL–60 g/mL and 60 g/mL–70 g/mL, respectively. However, the CHL extract did not reach the IC50 value in the concentrations tested in this study.The maximal percentage of FRS activity of T. versicolor (KF10) extracts rose as the extract concentration increased from 10 g/mL to 70 g/mL 50 value of 70% Eth was detected at concentrations ranging from 50 g/mL to 60 g/mL; however, the CHL and HW extracts did not achieve the IC50 values at the doses examined in this investigation.The percentage of FRS activity of A. auricula-judae (KF6) extracts was investigated as shown in p ≤ 0.05). HW, 70% Eth, and CHL extracts all had IC50 values that ranged from 250 to 500 g/mL, 125 to 250 g/mL, and 250 to 500 g/mL, respectively.Vero cell line cytotoxic activity of M. xanthopus (KF12) extracts showed no significant effect against the Vero cell line as shown in µg/mL. The IC50 values of CHL, 70% Eth, and HW extracts were observed at 125 µg/mL–250 µg/mL, 62.5 µg/m–125 µg/mL, and 250 µg/mL–500 µg/mL, respectively.The cytotoxic activity of T. umkowaani (KF2) demonstrated a dose-dependent cytotoxic effect on the Vero cell line as shown in µg/mL. The IC50 values of CHL, 70% Eth, and HW extracts were determined at 125 µg/mL–250 µg/mL, 500 µg/mL–1000 µg/mL, and 250 µg/mL–500 µg/mL, respectively.The extracts of T. elegans (ASF3) was detected in a very low effect against the Vero cell line as shown in 50 values of CHL, 70% Eth, and HW extracts showed within the range of 125 µg/mL–250 µg/mL, 500 µg/mL–1000 µg/mL, and 250 µg/mL–500 µg/mL, respectively. The maximum percentage of cell viability at 15.625 µg/mL was observed in HW extract (98.65 ± 0.02%) followed by a CHL extract (91.53 ± 0.03%) and 70% Eth extract (90.68 ± 0.01%). The pattern indicates that as the concentration of the extracts increases, the viability of the cells decreases.The cytotoxic activity of the three extracts of T. versicolor (KF10) extracts showed a very minimal cytotoxic effect against the Vero cell line as shown in µg/mL. The IC50 values of CHL, 70% Eth, and HW extracts were detected at 125 µg/mL–250 µg/mL, 125 µg/mL–250 µg/mL, and 250 µg/mL–500 µg/mL, respectively.The three All three extracts screened for anti-QS activity inhibited violacein production in a concentration-dependent pattern as shown in T. umkowaani (KF2) showed a maximum zone of inhibition (14.00 ± 0.04 mm) against the CV026 at the concentration of 200 µg/mL as shown in T. elegans (ASF3) and 70% Eth extract of T. versicolor (KF10), at a concentration of 200 mg/mL, also reduced the production of violacein 11.00 ± 0.05 mm and 11.00 ± 0.01 mm, respectively.The HW extract of T. elegans (75 ± 0.01%), HW extract of T. umkowaani (80 ± 0.02%), and 70% Eth extract of T. versicolor (65 ± 0.03%) at 200 µg/mL. The violacein production was reduced as the concentrations of extracts increased. It was observed that the HW extract of T. umkowaani and the 70% Eth extract of T. versicolor showed a statistically significant difference in the inhibition of violacein production against CV026  at 200 µg/mL.All three extracts showed anti-QS activity at sub-MIC concentrations as shown in All of the mushroom extracts in the current investigation had significant FRS activities in doses ranging from 10 g/mL to 70 g/mL. On the other hand, a prior investigation found that the FRS activity of mushroom extracts was 2.11 mg/mL . The exi50 values of the HW extracts of A. auricula-judae, M. xanthopus, T. umkowaani, and T. elegans were observed within the concentration range of 50 g/mL–70 g/mL, with the exception of T. versicolor. In contrast to the results of earlier investigations, the current results' IC50 values were observed at relatively low extract concentrations. For example, many researchers have observed IC50 values of mushroom extracts at 8.68 mg/mL [µg/mL [The results of this study show that the FRS activities among the five mushroom extracts varied widely. In this regard, the IC68 mg/mL , 340 µg/L [µg/mL , and 20.L [µg/mL . These sL [µg/mL . In addiL [µg/mL , 50.T. versicolor 70% Eth extract. This may be because the extract in question contains a variety of powerful antioxidant chemicals, which may work in concert to increase the FRS activity. According to one study report, different solvents may have different effects on the quality, strength, and quantity of antioxidant components [The most active FRS was found in the mponents . The premponents –55.50 values larger than 20 g/mL are regarded as noncytotoxic, according to the American National Cancer Institute (ANCI) recommendations [50 values of all five wild mushroom extracts were found to be within the range of noncytotoxic activity. The inhibitory concentrations (IC50) of the three extracts from each wild mushroom species differed against the Vero cell line. The discrepancies in cytotoxic activity among mushroom extracts may be attributed in part to changes in compound polarity and solubility, bioactive component modes of action, and the presence of various cytotoxic chemicals in each extract [Significant cytotoxic activity against the Vero cell line was shown by all five mushroom species' extracts. Crude extracts having ICndations . The IC5 extract . In cont extract . This vaT. versicolor polysaccharides, particularly β-glucans, were found to be the most effective antitumor agents [The results demonstrate that the CHL and 70% Eth extracts have higher cytotoxic activity than the HW extract. The existence of numerous and similar chemicals inside the extracts could explain why the CHL and 70% Eth extracts performed better. r agents . Water-ir agents , 61.T. umkowaani HW extract, the T. elegans CHL extract, and the T. versicolor 70% Eth extract was qualitatively evaluated against CV026. There are no prior publications to compare this work with, as it is the first to demonstrate the anti-QS efficacy of the mentioned mushroom extracts against CV026.The anti-QS activity of the The anti-QS effect of the extracts may result from their ability to limit the production of acyl-homoserine lactones (AHLs), to degrade AHLs, or to block the actions of the autoinducers, despite the fact that their mode of action has not been thoroughly explored. In addition, the CV026's QS system may be interfered with and the genes involved in the quorum sensing process may be downregulated as a result of the synergistic effects of the bioactive substances found in the extracts. According to earlier research, biological quorum quenchers typically disrupt the bacterial quorum sensing circuits by inhibiting the production, transmission, and reception of AHLs signals as well as their receptors with antagonist molecules that are structurally related to AHLs competitive inhibitors , 63.C. violaceum was significantly reduced as a result of the mushroom extracts' anti-quorum sensing abilities. The development of novel alternatives for quorum sensing disruption is attracted by the extracts' ability to quench the quorum sensing activity of the bacterium. Untapped mushrooms' anti-quorum sensing capabilities may add a new dimension to research into the creation of inventive anti-quorum sensing agents that could one day offer cost-effective alternatives to the current pharmacological regimens. The bioactive ingredients that are responsible for the antioxidant and anti-quorum sensing actions need to be identified through additional research. The bioactive substances' precise chemical makeup and any potential mechanisms underlying the observed antioxidant and anti-quorum sensing properties also require a further research.Fatty acids, flavonoids, volatile oils, polyphenols, and saponins, which have potential uses in nutrition and medicine, were found in considerable concentrations among the mycochemicals that underwent qualitative screening. According to the present findings, mushroom extracts could be good candidates for anti-quorum sensing and antioxidant agents. The mushroom extracts demonstrated very little cytotoxic activity against the Vero cell line indicating that the mushrooms are safe for use in future downstream processes for drug development. The synthesis of the pigment violacein in


Curcumae Rhizoma that possesses high biological activity and extensive pharmacological effects. As a traditional Chinese medicine, Curcumae Rhizoma can inhibit the development of many types of cancer, especially colorectal cancer. However, the anti-colorectal mechanism of its monomer curdione remains unclear.Curdione is a sesquiterpene isolated from Colorectal cancer (CRC) cells were treated with curdione at doses of 12.5 μM, 25 μM, and 50 μM, and then the cells’ activity was measured with methyl thiazolyl tetrazolium (MTT). Nude mice were administered different doses of curdione subcutaneously and oxaliplatin by tail vein injection, and then hematoxylin–eosin (HE) staining was adopted to examine tumor histology. Moreover, flow cytometry was applied to detect reactive oxygen species in cells and tissues. Kits were employed to detect the levels of iron ions, malondialdehyde, lipid hydroperoxide, and glutathione. Polymerase chain reaction (PCR) and Western blotting were adopted to detect ferroptosis and m6A modification-related factors. A methylation spot hybridization assay was performed to measure changes in overall methylation. SLC7A11 and HOXA13 were measured by MeRIP-qPCR. The shRNA-METTL14 plasmid was constructed to verify the inhibitory effect of curdione on CRC.2+, and lowered the activity of glutathione. According to the qPCR and Western blot results, curdione promoted the expression of METTL14 and YTHDF2 in CRC cells and tissues, respectively, and decreased the expression of SLC7A11, SLC3A2, HOXA13, and glutathione peroxidase 4. Additionally, in animal experiments, the curdione-treated group showed severe necrosis of tumor cells, as displayed by HE staining. Furthermore, compared with the control group, levels of m6A modifying factors  were increased in the tissues after drug intervention. METTL14 knockdown was followed by an increase in CRC cell activity and glutathione levels. However, the levels of reactive oxygen species, malondialdehyde, and iron ions decreased. The expression levels of SLC7A11, SLC3A2, HOXA13, and GPX4 were all increased after METTL14 knockdown.A dose-dependent decrease in activity was observed in curdione-treated cells. Curdione increased the accumulation of reactive oxygen species in CRC cells and tumor tissues, greatly enhanced the levels of malondialdehyde, lipid hydroperoxide and FeThe results suggest that curdione induces ferroptosis in CRC by virtue of m6A methylation.The online version contains supplementary material available at 10.1186/s13020-023-00820-x. Curcumae Rhizoma is the dried rhizome of Curcuma phaeocaulis Val., Curcuma kwangsiensis S.G. Lee et. C. F. Liang or Curcuma wenyujin Y.H. Chen et C. Ling. Curdione, as the most abundant sesquiterpene in Curcumae Rhizoma, has activities including anti-platelet aggregation [With consideration of the data acquired, colorectal cancer (CRC) has the third and second highest incidence and fatality rates , respectregation , contribregation . NeverthIn 2012, ferroptosis was first reported by Dixon et al. . As a nom6A (N6-methyladenosine), a methylated form of the adenosine N6 site, was first discovered in the 1970s . The preBased on our previous experiments and literature studies, curdione increases the expression of METTL14 (a methylation transferase), enhances the methylation of XC-system subunit SLC7A11 mRNA and HOXA13 mRNA, lowers the stability of reading protein YTHDF2, and reduces HOXA13, causing a decline in another subunit of the XC-system SLC3A2 expression, thus activating ferroptosis.The CT26 murine-derived CRC cell line and SW480 human-derived colorectal cell line were both purchased from Cell Resource Centre, the Chinese Academy of Sciences. BALB/c nude male mice (7 weeks old) were obtained from Spelford (Beijing) Biotechnology Co., Ltd, with license number SCXK (Beijing) 2019–0010. Curdione was purchased from Macklin , with a purity exceeding 99%. Oxaliplatin was acquired from Sigma . Fetal bovine serum, dulbecco's modified eagle medium, trypsin digest and penicillin‒streptomycin combination were obtained from Gibco (America). METTL4  antibody was purchased from Absin. Antibodies against METTL3 , YTHDF2  and GPX4  were purchased from Abcam. Antibodies against METTL14 , SLC7A11 , SLC3A2 , GAPDH , and IgG (H + L)  were purchased from CST. The antibody against HOXA13  was acquired from Abways. SAB5600251  was provided by Merck. A reactive oxygen species assay kit (S0033M) was provided by Beyotime. An Iron Assay Kit (MAK025) was obtained from Sigma. A malondialdehyde assay kit (BC0025), lipid hydroperoxide assay kit (BC5245), and reduced glutathione assay kit (BC1175) were purchased from Solarbio.2) in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin‒streptomycin in volume fractions. When the number of cells reached 70–80%, curdione, prepared from DMSO at different concentrations , was added to intervene CRC cells for 48 h.Murine-derived CT26 cells and human-derived SW480 cells were routinely cultured , thus ensuring 90–95% confluence at the time of transfection. With biosystems, the recombinant vector plasmid METTL14 shRNA was designed and constructed. To lower the expression of METTL14, the coding sequence of METTL14 was cloned into the vector pLV3ltr-ZsGreen-Puro-U6 by using BamH1 and EcoR1 as enzyme cleavage sites . ConsideIn the logarithmic development stage, cell suspensions were prepared by counting and processing CT26 cells. Successively, 96-well plates were obtained by counting and punching cell suspensions for culture. Various doses of curdione were used to treat the cells, thereby evaluating cell activity with MTT, DMSO reagent, and a microplate reader. .After being exposed to low, medium, and high concentrations of curdione for 48 h, CT26 cells were stained with dichlorofluorescein diacetate  to assess the total quantity of intracellular ROS. Afterward, the cells were rinsed with phosphate-buffered saline (PBS) followed by treatment with 10 μM DCFH-DA for 30 min at 37 °C. Before flow cytometry, the cells were washed twice in PBS. The detailed steps were performed by referring to the manual offered by Beyotime Biotechnology .Total proteins were extracted from CRC cells or tissues, followed by separation with a 10% SDS‒PAGE system. Subsequently, the proteins were transferred to the membranes at a constant pressure of 100 V for 2 h  and blocked with 5% skimmed milk for 2 h at room temperature. After adding primary antibody, the membranes were incubated with primary antibodies overnight at 4 °C, washed three times with PBS, and incubated with secondary antibody corresponding to horseradish peroxidase (HRP). Afterward, the membranes were visualized with Gel imaging systems , and ImageJ software was employed to quantify the individual band intensity.2+ assay. The supernatant was centrifuged at 4 °C for 10 min, the absorbance value was measured on a microplate reader, and the amount of Fe2+ was calculated by applying the formula. In the same way as Fe2+, the contents of MDA, LPO and GSH were determined by complying with the kit instructions offered by the manufacturer of Solarbio.The recommended tissues and cells were homogenized  in an ice bath by adding extracts in accordance with the manufacturer's procedure for the Fe−△△CT relative quantification approach. The primer sequences applied in the qPCR experiments are shown in Table The TRIzol technique was adopted to extract total RNA from each group of CRC samples. Equipment  was also used to identify the RNA concentration in all test samples, followed by reverse transcription and cDNA synthesis. Two microliters of cDNA was used to perform qPCR experiments. After 40 cycles, GAPDH was used as an internal reference gene, and the relative expression of the target gene was measured with the 27/mL was injected into one side of the axilla of nude mice to generate a CT26 subcutaneous transplantation tumor model, followed by daily tumor development monitoring. Nude mice, successfully affected with CRC, were randomly divided into five groups, with six mice in each group and different doses of curdione for the administered groups. The five groups included one control group, one low-dose group , one medium-dose group , one high-dose group , and one positive group  administered via tail vein injection. The tumor volume of the xenograft mice was measured every three days. After 22 days, the mice were sacrificed under anesthesia.The animal experiments were approved by the Ethics Committee of Nanjing University of Chinese Medicine, with ethics approval number A220607 for the use of animals in this study. A 200 μL CT26 cell suspension at a concentration of 0.5 × 10The stripped CRC tumors were fixed with 4% paraformaldehyde solution, dehydrated , embedded , and sectioned (RM2235), followed by routine HE staining , microscopic observation , and image acquisition for analysis .To isolate total RNA, TRIzol reagent was used. RNA was concentrated and diluted, followed by heating for five minutes at 95 °C to denature it. An nylon membrane (Amersham Hybond-N +) was loaded with 2 μL RNA. After UV cross-linking, the membrane was blocked with 5% skim milk for an hour at room temperature. Afterward, the membrane was treated with m6A antibody  overnight at 4 °C and then with secondary antibody for an hour. By applying a chemiluminescence technique, the signal was discovered following PBST washing (Bio-Rad). As a loading control, membranes were colored in 0.3 mol/L sodium acetate (pH 5.2) with 0.02% methylene blue.To explore m6A gene differentiation, the Magna MeRIPTM m6A kit was adopted for MeRIP, with the following procedures. RNA was extracted from CRC tissue and fragmented; a fraction of the RNA sample was employed as the input group, and the remainder of the RNA sample was regarded as the IP group. IP buffer and antibodies were added to the IP group, followed by incubation for 4 h in a vertical mixer at 4 °C. Afterward, the IP group was pretreated with magnetic beads and then incubated with produced Protein A/G magnetic beads in a vertical mixer at 4 °C for 1 h. After adsorption of the beads, the supernatant was removed. Subsequently, the wash buffer was added to the EP tube and gently blown into the tube. The tube was then washed for 5 min at 4 °C in a vertical mixer. The beads were adsorbed on a magnetic stand, followed by supernatant removal. After completing the previous process twice, 200 μL elution buffer was added to the input material, and then RNA extraction was performed by employing phenol‒chloroform. Finally, qPCR was adopted to detect identical amounts of reverse transcribed RNA.Apoptosis in SW480 cells was detected by employing the TUNEL kit. Following dewaxing at room temperature, soaking in gradient ethanol, rinsing with PBS, and incubation, adherent cells were cultured. Afterward, apoptosis was induced, anti-fluorescence quenching and sealing were performed, and the cells were blocked for microscopy and analysis.GraphPad Prism 8.0  was applied to analyze and interpret the collected data, with the mean ± SD representing the results. One-way analysis of variance (ANOVA) was adopted to analyze the difference between the treatment group and the control group. All experiments were repeated at least three times. P < 0.05 was deemed statistically significant.P < 0.01) , medium-dose (P < 0.01), and high-dose (P < 0.001) groups exhibited considerably increased fluorescence intensity of ROS. This indicates that curdione can tremendously promote intracellular ROS production.As ferroptosis was accompanied by an increase in the number of ROS and an expansion of lipid peroxides, the DCFH-DA fluorescent probe was employed to determine the level of intracellular ROS. Compared to the control group , 25 μM (P < 0.01), and 50 μM (P < 0.01) dramatically lowered the concentration of GSH while increasing the levels of MDA and divalent iron ions in a dose-dependent manner. Curdione enhanced ferroptosis in cells by promoting lipid peroxidation, increasing the accumulation of the lipid peroxidation product MDA, and decreasing the intracellular GSH concentration, which are all indications of ferroptosis. Furthermore, with an increase in the concentration of curdione, the LPO level was promoted in CT26 cells .With the reagent kit, GSH, MDA, LPO, and divalent iron ions in the cells were measured, with results presented in Fig. M P < 0.0, and 50 M P < 0.0, and 50 P < 0.01). The relative expression levels of SLC7A11, SLC3A2, HOXA13, and GPX4 were reduced in a dose-dependent manner (P < 0.01). Thus, 50 μM curdione was selected for further mechanistic study.As depicted in Fig. P < 0.01) , the cells displayed more obvious wrinkling and nuclear chromatin border set, accompanied by necrosis and fibrous hyperplasia, as well as reduced cellular arrangement density. Considering that the therapeutic effect of the high dose of curdione was close to that of oxaliplatin with no side effects, the oxaliplatin group was not subsequently added  Fig. a–c. Accoded Fig. d–h. Curdded Fig. d–h.Fig. P < 0.05, P < 0.01), medium-dose (P < 0.01), and high-dose (P < 0.01) groups compared to the control group  than in the medium-dose curdione group (P < 0.05).Spot blotting was performed on CRC tissues and SW480 cells, thereby determining the overall m6A levels of RNA in the four groups, with the results exhibited in Figs. P < 0.01), as displayed by the bar graph, with CRC mice administered a high dose of 50 mg/kg intervention having nearly five times more METTL14 expression compared to the control group. Curdione treatment increased the expression of YTHDF2, a reading protein related to m6A, particularly (P < 0.01) in a dose-dependent manner. As SLC7A11 and SLC3A2 are important components of the antioxidant system cysteine glutamate transporter receptor (system XC-), the reduction in HOXA13 decreased the expression of another subunit of the XC- system, SLC3A2, thereby activating ferroptosis. According to the graph, the protein expression levels of SLC7A11, SLC3A2, and HOXA13 were measured in comparison to the control. The expression of all three proteins showed a remarkable decrease with increasing dose when compared to the control (P < 0.01). Additionally, the expression of GPX4 was measured and was obviously reduced by curdione intervention (P < 0.01).According to Fig. P < 0.05, P < 0.01). SLC7A11, SLC3A2, and HOXA13 mRNA levels exhibited considerable increases . Compared to the control group, GPX4 mRNA expression levels were considerably lower (P < 0.01). As PTGS2 is also involved in ferroptosis pathology [P < 0.05, P < 0.01).To determine whether ferroptosis played a role in the therapeutic impact of curdione in CRC mice, qPCR was adopted to assess the mRNA levels of METTL3, METTL4, METTL14, SLC3A2, SLC7A11, HOXA13, YTHDF2, GPX4, and PTGS2 in the tissues of four groups of CRC mice, as shown in Fig. athology , 29, PTGP < 0.01) and notably higher in the shRNA-METTL14 + high-dose group than in the high-dose group. (P < 0.01), as shown in Fig. P < 0.01). The same high-dose curdione therapy was administered, and knockdown of METTL14 led to a notable decrease in ROS content (P < 0.01). As displayed in Fig. P < 0.01). With ELISA kits, the cellular Fe2+ and MDA contents of each group were measured, as well as the GSH activity. Compared to the control group, the addition of curdione at a concentration of 50 μM increased Fe2+ and MDA contents while decreasing GSH activity (P < 0.01). This conformed to earlier findings. Nonetheless, after knocking down METTL14, the levels of Fe2+ and MDA in the cells remained considerably decreased as predicted, whereas GSH activity was notably elevated in the shRNA-METTL14 + high-dose group compared to the high-dose group (P < 0.01). From another perspective, by knocking down the methylation transferase METTL14, Fe2+ and MDA levels were reduced, and GSH activity was increased, as displayed in Fig. P < 0.01), as shown in Fig. The shRNA-METTL14 was transfected into CT26 cells, with the transfection effect shown in Fig. P < 0.01), as shown in Additional file 2+, MDA, LPO and GSH were measured. Our results indicate that curdione promoted the development of ferroptosis (P < 0.01). However, when combined with Z-VAD-FMK, there was no statistically significant difference. Nonetheless, a significant difference was observed between the Z-VAD-FMK group and the curdione + Z-VAD-FMK group (P < 0.01). Notably, the combined treatment group exhibited higher levels of ferroptosis, suggesting that apoptosis does not affect curdione-induced ferroptosis.As reported, curdione can induce the onset of apoptosis , 30. AnoCurcumae Rhizoma, a Chinese medicine, has anticancer effects against a variety of cancers. The remarkable effects of its various active ingredients in inhibiting CRC development have been confirmed by many studies [CRC is a highly fatal malignancy responsible for causing approximately nine million deaths annually, as reported by the Lancet . In rece studies –35. None studies . There i2+ and ROS, decreasing GSH levels in CRC cells or inactivating GPX4. However, inhibition of ferroptosis may result in tumor progression and treatment resistance in CRC [As a kind of necrosis induced by extramitochondrial lipid peroxidation, ferroptosis is primarily driven by iron-dependent ROS growth . Ferropte in CRC . A numbee in CRC , 40. Hene in CRC . Neverthe in CRC . Low expe in CRC . AccordiThis study discovered a novel component (curdione) for CRC treatment in vitro and in vivo, elucidating that curdione induces ferroptosis by upregulating the expression of METTL14 and YTHDF2 via N6-methyladenosine. These results provide a reference or a new idea for the study of ferroptosis in CRC or other cancers.However, it is important to acknowledge the limitations of this study. It has been proposed that ROS drive apoptosis and ferroptosis . FerroptIn conclusion, our study provides experimental evidence that curdione regulates ferroptosis in colorectal cancer by m6A methylation, revealing that curdione might be a promising treatment for colorectal cancer.Additional file 1: Fig. S1. Curdione induces ferroptosis in colorectal cancer. Fig. S2. Curdione-induced apoptosis does not affect the induction of ferroptosis. Fig. S3. Curdione promoted ferroptosis in colorectal cancer through the upregulation of METTL14.


Lonicera caerulea fruits are a rich source of vitamins, organic acids, and phenolic compounds, which are characterised by their health-promoting properties. The content of bioactive compounds in this fruit may vary depending on the cultivar and the harvest date. The fruits of the L. caerulea var. kamtschatica cultivars ‘Duet’ and ‘Aurora’ and the L. caerulea var. emphyllocalyx cultivars ‘Lori’, ‘Colin’ and ‘Willa’ were used in this study. L. emphyllocalyx fruit, especially the cultivar ‘Willa’, was characterised as having a higher acidity by an average of 29.96% compared to L. kamtschatica. The average ascorbic acid content of the L. kamtschatica fruit was 53.5 mg·100 g−1 f.w., while L. emphyllocalyx fruit had an average content that was 14.14% lower. The antioxidant activity  varied according to the cultivar and the species of fruit analysed. The total polyphenol content differed significantly depending on the cultivar analysed; fruits of the L. emphyllocalyx cultivar ‘Willa’ were characterised by the lowest content of total polyphenols—416.94 mg GAE·100 g−1 f.w.—while the highest content of total polyphenols—747.85 GAE·100 g−1 f.w.—was found in the fruits of the L. emphyllocalyx cultivar ‘Lori’. Lonicera caerulea fruits contained 26 different phenolic compounds in their compositions, of which the highest content was characterised by cyanidin 3-O-glucoside (average: 347.37 mg·100 g−1). On the basis of this study, it appears that both L. kamtschatica fruits and L. emphyllocalyx fruits, especially of the cultivars ‘Lori’ and ‘Willa’, can be used in food processing. Lonicera caerulea L.) belongs to the genus Lonicera (Caprifoliaceae), which contains more than 200 species, native to the cold lands of the Far East and central Asia. Most of them are ornamental plants; only about 17 are edible fruit-producing species [Lonicera caerulea has numerous varieties, several of which are widely cultivated, originating from Russia  and the island of Hokkaido in Japan . They are long-living (25–30 years) shrubs that can reach 0.8–3.0 m height. They need an outside pollinator to bear fruits one year after planting. After three years, approx. 500 g of fruit can be obtained from one plant. Berries are dark blue or dark purple in colour, with a size of 1.5–3.0 cm, and a mostly cylindrical shape with a wax coating. They are plants that are very resistant to frost, able to withstand temperatures up to −46 °C [Lonicera caerulea var. kamtschatica is a variety of honeysuckle, commonly known as Kamchatka berry, which is one of the most popular fruits in Poland, the Czech Republic, Canada, and Russia. Several varieties native to the species have been selected, which differ, among other things, in terms of flowering time, prolificacy, and content of bioactive substances [Lonicera caerulea var. emphyllocalyx (Maxim.) Nakai, commonly referred to as Japanese haskap. This is native to the island of Hokkaido in northern Japan and is also cultivated in China, Korea, or Russia [The blue honeysuckle  do not significantly affect the content of active compounds; fruit from different sites will be characterised by similar contents of bioactive compounds [Lonicera caerulea berries are characterised by a high content of phenolic compounds, ranging from 428.14 to 622.52 mg GAE·100 g−1 f.w. [L. caerulea is an important source of phenolic compounds such as anthocyanins, flavonoids, proanthocyanidins and phenolic acids [O-glucoside. This is a widely used compound in the plant world, accounting for about 79–92% of the the anthocyanin content of L. caerulea fruit [−1 [L. caerulea has many uses in food processing. Pulp made from L. caerulea berries has a high juice yield and tiny seeds, making it excellent source of fruit juice [L. caerulea; however, due to the high content of sugars, the amount of added sugar should be controlled during the production stage [The ompounds . Lonicerg−1 f.w. ,8. L. caruit [−1 . The berit juice . Due to it juice ,14. Cannon stage . The proon stage .L. caerulea var. kamtschatica ( ‘Duet’ and ‘Aurora’ cultivars) and L. caerulea var. emphyllocalyx  and the potential use of L. emphyllocalyx fruit in food processing.The purpose of this study was to compare the chemical compositions of the fruits of L. caerulea fruit are rich in organic acids  [L. kamtschatica and L. emphyllocalyx are rich in organic acids that give a taste resembling blueberries, with a distinct hint of sourness.These results are comparable to those obtained by other authors. In a study by Gerbrand et al. , the fruu et al. , the fruu et al.  and Oszmu et al. , we foun12–5.03) , red cur12–5.03) , sea buc12–5.03) , highbus12–5.03) , raspber12–5.03) . In geneL. kamtschatica fruit was at the level of 1.22–1.31 g·100 g−1, while the L. emphyllocalyx fruit, especially the ‘Willa’ cultivar, had a higher acidity by 29.96% on average compared to the L. kamtschatica fruit  [−1) [−1) [−1) and raspberry (0.63 g·100 g−1) [The difference in acidity between varieties may have been caused by different climatic conditions. The acidity of the ca fruit . The res100 g−1) , saskato100 g−1) .L. kamtschatica fruit was 53.5 mg·100 g−1 f.w., while the L. emphyllocalyx fruit had an average ascorbic acid content of 45.93 mg·100 g−1 f.w. , raspberries (average: 30.6 mg·100 g−1 f.w.), blueberries (average: 60.1 mg·100 g−1 f.w.) [−1 f.w.), blackberry (33.85 mg·100 g−1 f.w.) [−1–44.1 mg·100 g−1 f.w.) [−1 f.w.) [L. caerulea berries increase the concentration of ascorbic acid by 100%. Heating plant material before crushing may influence the stability of ascorbic acid, preserving its contents. This can be used in the food industry in the production of L. caerulea products [Ascorbic acid is a powerful antioxidant that is required for the activation of many enzymes. This compound is essential for the functioning of the human body, influencing the immune and circulatory systems, accelerating wound healing, slowing skin ageing, and regulating collagen production . The aveg−1 f.w. . The cona et al. , the con−1 f.w.) ,33, plum−1 f.w.) , sea buc−1 f.w.) . Accordi−1 f.w.) , spreadsproducts .Lonicera caerulea fruit extract containing anthocyanins is a highly effective antioxidant agent, effectively reducing reactive oxygen species (ROS), which are produced by immune cells as a result of inflammation. Persistent inflammation can lead to cell damage and chronic disease. The fruit extract further reduces lipid peroxidation, which affects cellular damage under oxidative stress conditions, which can contribute to the reduction in diseases related to oxidative stress [L. kamtschatica and L. emphyllocalyx fruit, determined by the DPPH method, was at the level of 68.68–89.62% inhibition, of which the L. kamtschatica cultivar ‘Duet’ had the highest free radical scavenging activity . These results are comparable to those obtained by Kula et al. [Lonicera caerluea were, respectively, 85% and 78.70%. The antioxidant activity of L. caerulea fruits is significantly higher compared to other species, e.g., sea buckthorn (74%), bilberry (37–91%), and garden rhubarb (48–98%) [L. kamtschatica range from 30.52 to 37.67 μM Fe2+·g−1 f.w. and L. emphyllocalyx range from 30.52 to 37.67 μM Fe2+·g−1 f.w. Results comparable to this study were obtained by Rupasinghe et al. [2+·g−1. According to studies, L. caerulea had more antioxidative properties (FRAP) compared to other species grown in Poland, e.g., strawberries (8.00 µM TE·g−1 f.w), blackberries (15.03 µM TE·g−1 f.w.), highbush blueberry (16.24 µM TE·g−1 f.w.), elderberry (29.56 mM Fe2+·g−1 f.w.), blackthorn (14.74 mM Fe2+·g−1 f.w.) and wild strawberry (10.99 Fe2+·g−1 f.w.) [Lonicera caerulea fruits are characterised by a higher antioxidant capacity. The antioxidant activity of the fruits of L. kamtschatica and L. emphyllocalyx, determined by the ABTS method, ranged from 1.91 to 2.21 mM TE·100 g−1 f.w. As reported by Rop et al. [Lonicera caerulea was 0.30 µM·g−1. The values obtained in this study are different to other species cultivated in Poland, e.g., highbush blueberry (16.87 mM TE·g−1 f.w.), elderberry (15.88 mM TE·g−1 f.w.), blackberry (9.55 mM TE·g−1 f.w.), strawberries (5.61 TE·g−1 f.w.) [−1 d.m.) [−1 f.w.) and raspberry (0.08 mM TE·100 g−1 f.w.) [L. kamtschatica and L. emphyllocalyx makes them very valuable in terms of bioactivity. Research on the antioxidant activity of extracts with L. caerulea revealed that the fruits of this plant are characterised by stronger antioxidant properties from other berries commonly regarded as effective antioxidants. Considering the relationship between modern-day diseases and long-term oxygen stress, strong antioxidant properties may indicate the potential importance of this fruit, not only in prophylaxis, but also in the treatment of many diseases [L. caerulea products more valuable for food industry.e stress . The anta et al. , in whic(48–98%) . The iro−1 f.w.) . Lonicerbilberry –91%, and−1 f.w.) , red cur−1 f.w.) . The higL. caerulea fruit extract exhibit anti-inflammatory effects. They reduce cellular damage under conditions of oxidative stress in in vitro cultures of rat microsomes and reduce ROS production in cultures of pro-inflammatory gingival fibroblasts [L. emphyllocalyx cultivar ‘Willa’ were characterised by the lowest total polyphenol content—416.94 mg GAE·100 g−1 f.w.—while the highest total polyphenol content of total polyphenols—747.85 GAE·100 g−1 f.w.—was also found in fruits of L. emphyllocalyx but of the cultivar ‘Lori’ , MS and MS/MS with available publications [The analysis of phenolic compounds using the UPLC-PDA-MS/MS method allowed for the determination of the differences between the contents of individual groups of polyphenolic compounds contained in the fruit of the analysed cultivars of llocalyx . Berriesications ,43,44,45of L. kamtschatica ‘Duet’, at 456.3 mg·100 g−1 (L. kamtschatica (509.29–597.29 mg GAE·100 g−1) and L. emphyllocalyx (416.94–747.85 mg GAE·100 g−1), anthocyanins represented, on average, 94% of all polyphenols, and the main representative was cyanidin 3-O-glucoside—C3G . Among the varieties analysed, the L. kamtschatica fruits of the ‘Duet’ variety contained the highest concentration of C3G in their composition—382.18 mg·100 g−1 , blueberry (3.0 mg·100 g−1), and the cranberry (0.7 mg·100 g−1) [L. caerulea had higher concentrations of C3G  in comparison to fresh fruit. The fruits of this species play important roles in a wide range of physiological processes, e.g., protective effect against UV radiation for skin, protection against pathogenic strains, etc..The highest anthocyanin content in the analysed fruits was found in the berries ·100 g−1 . Among t·100 g−1 . The resb et al. , and the100 g−1) . In the 100 g−1) , spreads−1 in the L. emphyllocalyx cv. cultivar ‘Colin’ to 24.4 mg·100 g−1 in the case of the L. kamtschatica cv. cultivar ‘Duet’  and significantly higher compared to the other cultivars  to 2750 mg·100 g−1  in the year 2022. Fruits of the L. caerulea var. emphyllocalyx cultivars ‘Lori’, ‘Willa’ and ‘Colin’ were obtained from ‘Korfanty’  in 2022. Both species were grown in containers filled with a peat substrate containing sand and perlite in a ratio of 20:1:1, with the addition of fertilizer Osmocote Exact 3–4 m  at concentration of 2.0 kg for 1 m3 of substrate.Fruits of the The average monthly temperatures in the period from March to June in Tyczyn were, respectively, 3.3, 7.0, 14.8, and 19.8 °C, and in Grabownica Starzeńska were, respectively, 2.2, 6.0, 13.7, and 18.5 °C. The average monthly rainfall values in the period from March to June in Tyczyn were, respectively, 20 mm, 60 mm, 50 mm, and 20 mm, and in Grabownica Starzeńska the average monthly rainfall in the period from March to June was 50 mm.The fruits of the analysed cultivars were harvested by hand at the stage of their harvest maturity (first decade of June), 1000 g each. Immediately after harvest, the fruits were subjected to chemical analysis. L. kamtschatica and L. emphyllocalyx fruit were determined through the potentiometric titration of the sample for analysis with a standard 0.1 M NaOH solution at pH = 8.1 using a TitroLine 5000  according to the method given in PN-EN 12147:2000 [The total acidity (as citric acid) and the pH of the 147:2000 . The res−1 f.w.) was determined using the Folin–Ciocalteu method according to the methodology described by Bakowska-Barczak et al. [L. kamtschatica and L. emphyllocalyx fruit was determined according to the methodology reported by Gorzelany et al. [Vitamin C (ascorbic acid) was determined according to PN-A-04019:1998 . Total py et al. . 2+·g−1 f.w. The antioxidant activity of the fruit was determined using DPPH methods according to the methodology given by Jurčaga et al. [−1 f.w. All analyses were performed in triplicate. The ability of the fruit to reduce iron ions (FRAP method) was determined according to the methodology given by Rupasinghe et al. , and thea et al. , and thev/v). All determinations were performed in triplicate. The sugar content was measured using the HPLC method with refractive index detection. The chromatographic equipment SYKAM , consisting of sample injector S5250, pump system S1125, column oven S4120 and RI detector S3590, was used. Separation was carried out using a Cosmosil Sugar-D column 250 × 4.6 mm . The separation was achieved with a mobile phase of 70% of acetonitrile in water in isocratic mode. The flow rate was 0.5 mL/min at column temperature set at 30 °C. The volume of injected sample was 20 µL and 15 min was needed to complete the analysis. Samples before injection were centrifuged at 5000 rpm for 10 min using Centrifuge 5430  and diluted with mobile phase 1:4 , a statistical analysis of the results obtained was performed that included the analysis of variance (ANOVA) and NIR significance test at a significance level of α = 0.05. L. caerulea cultivars. The fruits of the L. kamtschatica cultivar ‘Aurora’ contained the highest amount of ascorbic acid in their composition, approximately 22% more compared to ‘Lori’, which had the highest content of this compound among L. emphyllocalyx cultivars. The highest total polyphenol content was found in the L. emphyllocalyx cultivar ‘Lori’, while the predominant polyphenolic compound was cyanidin 3-O-glucoside. On the basis of the results obtained, it can be concluded that the L. emphyllocalyx cultivars ‘Lori’ and ‘Willa’ can find applications in various food industries. Despite the diversity of chemical composition, the fruits have high antioxidant properties compared to the well-known varieties ‘Duet’ and ‘Aurora’. The variety ‘Lori’ has the highest content of phenolic compounds among the tested varieties. ‘Willa’ has the highest concentration of C3G comparable to the widely known varieties of L. kamtschatica and the highest sugar content among L. emphyllocalyx. The use of Lonicera as an ingredient of functional foods, natural colourant or source of natural antioxidant seems to be promising. Compared to other types of berries grown in Poland, e.g., raspberries and red currant, L. kamtschatica and L. emphyllocalyx can be a good source of bioactive substances and sugar, making them good in food industry and processing, including juices, wines, spreads and dried fruits.Based on this study, differences in fruit composition were found in both individual species and the


Spirulina platensis is a microalga with a high protein content essential for human and animal nutrition. The present study aimed to utilize the beet filter cake extract (BFCE) to produce Spirulina platensis commercially. However, the cultivation of S. platensis on BFCE to produce economically single-cell protein has not been reported previously.Beet filter cake (BFC) is a by-product of sugar beet processing, which is difficult to dispose of and involves severe environmental concerns. S platensis showed an increase in dry cell weight and protein content from 25 to 75% BFCE  with an insignificant variation compared to Zarrouk’s medium (p ≤ 0.05), indicating that S. platensis can be economically produced when cultivated on 75% BFCE The predicated parameters from response surface methodology were NaNO3 (2.5 g/L), NaHCO3 (0.67 g/L), BFCE (33%) and pH = 8, which resulted in biomass yield and protein content  closer to that achieved using the standard Zarrouk’s medium (0.6 g/L and 55.11%). Moreover, the total essential amino acid content was slightly higher in the optimized medium (38.73%) than SZM (36.98%).The batch experiment revealed the maximum dry weight at Zarrouk’s medium (0.4 g/L) followed by 0.34 g/L in the treatment of 75% BFCE. The highest protein content was 50% in Zarrouk’s medium, followed by 46.5% in 25% BFCE. However, adding a higher concentration of 100% BFCE led to a protein content of 31.1%. In the adaption experiment, Therefore, BFCE supplemented medium could be used as a novel low-cost alternative growth medium for producing a single-cell protein with acceptable quantity and quality compared to the standard Zarrouk’s medium.The online version contains supplementary material available at 10.1186/s12934-023-02146-7. Sugar cane and sugar beet account for most of the sugar produced in Egypt and worldwide. Sugar beet was grown on 207,527 hectares in 2019 and is Egypt's largest source of sugar production. The sugar industry starts with sucrose extraction from sugar beet using hot water resulting in raw juice, which is then purified, filtered, and concentrated by cyclic rinsing and evaporation. During the clarification of beet juice, milk of lime and sulfur dioxide are added to neutralize the juice's acidity and precipitate the non-sugar components. The residue is settled in a clarifier, and the settled sludge is filtered using a rotary vacuum filter [Solid waste is produced as a by-product of the clarification of juice before its concentration and crystallization, called beet filter cake (BFC), also known as press mud, which is relatively rich in organic matter and some minerals causing a disposal problem . The sugSpirulina platensis is an economically crucial filamentous cyanobacterium naturally grown in alkaline water and warm regions and commercially produced for human and animal consumption [Spirulina deserves special attention as a microbial cell factory for edible protein production (70% of dry biomass), essential amino acids, fats, and carbohydrates [S. platensis commercial cultivation on the standard Zarrouk’s medium as a selective medium that minimizes contamination requires excessive nutrients, especially NaHCO3, for both carbon source and pH control. The production cost of the growth medium is relatively high because NaHCO3 concentration is 16.8 kg/m3, which is a limiting factor for large-scale cultivation and economic feasibility [sumption , 8. The hydrates . Moreovehydrates , 11 S. psibility .S. platensis [S. platensis as a microbial cell factory for single-cell protein has not been reported in the literature. Therefore, this study aims to formulate a new cost-effective medium using BFC supplementation which can minimize environmental pollution and production costs as possible while maintaining high biomass and protein content by optimization of the different growth variables incorporated in the medium for maximum biomass and protein yield in both photobioreactors and outdoor open ponds.Single-cell protein (SCP) is a dried biomass of high protein content that can be used as an alternative protein in animal feed or human nutrient supplements. Different microorganisms like yeast, bacteria and cyanobacteria can produce SCP, such as latensis . The comlatensis . UtiliziS. platensis alga products in food or feed applications. Since this waste is from one of the clean food industries' wastes, the result of the analysis of the various elements in the extract using inductively coupled plasma mass spectrometry (ICP-MS) was as expected and at a minimum level; moreover, mercury, cadmium, and copper were not detected. On the other hand, the Ca++ and Na+ were at 565 ppm and 391 ppm, respectively, which are acceptable values. Also, ammonia, nitrate, and phosphate were 2.05 ppm, 4.84 ppm and 50.1 ppm, respectively  and BFCE was measured every day to assess the impact of BFCE on photosynthesis performance. Richmond et al. [nd day of the experiment due to the stress induced by high concentrations of BFCE at the beginning of the experiment. Later, the alga adapted to this change in the medium nutrient content for 75% and 100% treatments. At the end of the experiment on the 7th day, the lowest activity was in the SZM, and this may be due to low nutrient availability caused by S. platensis utilization or due to a self-shading effect caused at the end of log growth phase, which effect on photosynthetic potential [In this experiment, the photosynthetic activity of d et al.  reportedd et al. , 17. In otential , 19, whiNumerous studies have shown that Fv/Fm values obviously change by stress conditions during growth, such as high light intensity, nutrient deficiency, oxygen, radiation, and excess heavy or toxic elements –23.S. platensis growth rate increases in all concentrations until the fifth day, but the growth decreases in the 75% BFCE on the sixth day and then increases again. Similarly, the growth declined in the 100% BFCE , followed by 75% BFCE (0.34 ± 0.03 g/L), while the minimum dry biomass was at 50% BFCE (0.25 ± 0.007 g/L). The variation in dry cell weight was significantly decreased (p ≤ 0.05) in different concentrations of BFCE except for 75%, which gave a non-significant decrease compared to the SZM.As shown in Fig. a concentration is directly related to biomass concentration and protein content. The maximum chlorophyll-a concentration (19.284 ± 3.6 mg/g) was obtained in 50% BFCE, followed by the SZM (12.0 ± 0.6 mg/g), while the concentration of chlorophyll-b in SZM and 50% BFCE was almost similar as shown in Fig. The chlorophyll-S. platensis is proteins (50–70%), carbohydrates (15–25%), lipids (6–8%) and minerals (7–13%). These significant components' percentages alter with the cultivation conditions of the alga [The standard nutritional profile of the alga . In agreproteins 0–70%, caS. platensis biomass cultivated in raw piggery effluents medium was 45.31%, while supplementation with ferrous sulfate caused an increase in the protein content up to 55.15%. Similarly, direct cultivation of S. platensis at different concentrations of BFCE had a significant negative impact on the protein content at p ≤ 0.05 by increasing the BFCE concentrations. Because the BFCE is limited in nutrient content compared to the standard growth medium, and as a result, these conditions cause a nutrient deficiency, especially nitrogen concentration, which affects the protein content. In agreement, Uslu et al. [3 limited protein synthesis, which resulted in a protein productivity decline.In a relevant study , the prou et al.  concludeS. platensis culture, which was cultivated in this experiment, showed a generally increasing trend, as shown in Fig. Chlorella vulgaris, Phaeodactylum tricornutum, and Micractinium pusillum in other mixotrophic growth conditions [The carbohydrate content in nditions –29.Fig. S. platensis grows autotrophically using only carbon dioxide and sodium bicarbonates as the sole carbon source for carbohydrate accumulation. However, in SZM supplemented with BFCE with different ratios, S. platensis grew mixotrophically, simultaneously accumulating more carbohydrates from other carbon sources in BFCE.The carbohydrate content was enhanced by increasing the BFCE concentrations to 75% (15.07 ± 0.58%), which significantly differs from SZM (7.28 ± 0.5%). In comparison, the pure BFCE 100% led to decreased carbohydrate content. In SZM, The lipid content represented in Fig. S. platensis lipid contents showed relatively low proportions; therefore, higher BFCE concentrations showed no remarkably significant impact on lipid accumulation in the lyophilized biomass. These results were lower than that obtained by Chang Y, et al. [S. platensis on synthetic human urine for biomass production and nutrient removal. Also, Li et al. [S. platensis performance cultivated mixotrophically.In general, , et al.   as shown in Additional file S. platensis showing that combining nutrients from SZM and BFCE is better for S. platensis biomass production on a large scale than BFCE alone, which is limited in its nutritional components.The behaviour of end Fig. , indicatS. platensis increased with the gradual rising of BFCE up to 75% (1.122 ± 0.063 g/L) with a non-significant difference. Excessive BFCE concentrations, up to 100%, had a remarkable negative impact on the cell dry weight of S. platensis (0.49 ± 0.034 g/L), revealing that S. platensis dry weight increasing trend is similar to the optical density, up to 75% BFCE.As shown in Fig. S. platensis biomass depends on medium composition, particularly the kind and amount of nitrogen [a increased in S. platensis cultivated in SZM during the entire experiment period. However, it raised to (5.6 ± 0.3 mg/g) when the BFCE concentration was increased to 50%, but it dropped again when the concentration was raised to 100% (3.5 ± 0.4 mg/g). Moreover, the chlorophyll-b increased when using 25% and 50% BFCE over the SZM, then decreased again in 100% BFCE concentration  compared to SZM Fig. . MoreoveS. platensis may be attributed to the utilization of organic carbon in the BFCE as a carbon source in addition to photosynthesis. Likewise, a similar study reported an improving in carbohydrate content in S. platensis growing on tofu wastewater enriched with various organic carbon [The enhanced carbohydrates content in c carbon .The lipid content increased from 4.92 ± 0.07% to 6.55 ± 0.13% in treatments 25% and 75% of BFCE, which was almost similar to that of SZM (3.85 ± 0.38% to 7.71 ± 0.5%). In contrast, 100% BFCE resulted in only 2.66 ± 0.4%. The lipid content of the biomass was not significantly affected by the different concentrations of BFCE Fig. . The dif3 on the lipid accumulation in S. platensis, the maximum lipid production was 4.7% at 15 °C in a nitrogen-free medium. In contrast, the best growth and lipid production was at 35 °C and 1.25 g/L of sodium nitrate [In another study investigating the effect of nitrogen concentration as NaNO nitrate .In general, a slight difference between all SZM during the four weeks of the experiments is owing to the variation in the inoculum dry weight (g/L) which coincide with Pelizer et al. , they st3, NaHCO3, BFCE and PH were predicted to affect the S. platensis growth and protein yield. Moreover, NaNO3 and NaHCO3 are the main costly elements in the SZM and highly considered in the economic feasibility of large-scale production of S. platensis while BFCE is a massive sugar industry waste with non-significant application and cheap prices. On the other hand, it is a source of carbonates and still contains impurities from the sugar industry [S. platensis. A CCD matrix was performed to detect the appropriate NaNO3, NaHCO3, BFCE and PH controlling the maximum biomass and protein yield. Biomass growth is expressed as dry weight (g/L) and protein yield (%). The optical density and photosynthetic activity (Fv/Fm) of S. platensis cultivated using CCD matrix are shown in Additional file 3 was reduced to 25% of the initial content (4.2 g/L), the protein content and dry biomass were nearly the same as SZM. This contradicts the conclusion that appeared with Raoof et al. [3 reduced to 1.075 g/L led to a decrease in protein content compared to the SZM and the response surface quadratic model for biomass yield and protein concentration are tabulated in Table Factors such as NaNOindustry  which cof et al.  reportedp-value < 0.05 was considered significant, as shown in Table Analysis of the main effect, quadratic effect and interaction between factors with Second-order polynomial models that predict the biomass dry weight and protein yield as a function of the experimental variable are shown in Eqs.  and 2.For biomass dry weight:For % protein:p-value of 0.0074 and 0.04, respectively. The different independent factors with a p-value less than 0.05 indicate that they are significant model terms. Regarding biomass yield, the BFCE (C) and pH (D) significantly affected the biomass yield with p-values of 0.004 and 0.030, respectively. The interaction of BFCE with pH (CD) also significantly affected the biomass yield with p-values of 0.030.From the ANOVA analysis Table , The mod3 (A) and NaHCO3 (B), the later optimum concentration was 4.2–16.8 g/L. While in NaNO3 (A) and waste (C), the optimum waste concentration was 55–75%. At the same time, NaNO3 range was 0.6–1.47 g/L in both interactions. However, in its interaction with waste or pH, its optimum range was 0.6–1.93 g/L, and the optimum ranges for waste and pH were 55–75% and 9.2–11, respectively. Finally, in the interaction between NaHCO3 (B) and waste, the optimum range for waste was 65–75%. While in NaHCO3 (B) and pH (D), the optimum pH was 9.8–11. Moreover, a NaHCO3 concentration of 0–16.8 g/L was the optimum concentration in the interaction with waste and pH.The interaction between the tested variables on the dry biomass was created by response surface plots Fig. . The dee3 (B) showed a significant effect with p-value of 0.025. The interaction of NaNO3 and NaHCO3 (AB) and NaNO3 and BFCE (AC) and the quadratic terms of NaHCO3 (B2), also significantly affected the protein yield with p-value of 0.047, 0.04 and 0.0203, respectively.Regarding the protein, as shown in Table 3 2.5 g/L and NaHCO3 from 2.1 to 6.3 g/L. The interaction between NaNO3 and waste showed that the optimum NaNO3 and waste range was 2.12–2.5 g/L and 35–75%, producing 50–60% protein. Moreover, an enhanced protein response was observed at the interaction between NaNO3 and pH, with optimum ranges of 2.4–2.5 g/L and 8–8.9, respectively.The interactive effect of variables on protein content was revealed by response surface plots Fig. . It can This result is in agreement with Colla et al. , they st3 and waste, the optimum NaHCO3 and waste ranges for maximum protein production were 4–12.6 g/L and 60–75%, respectively. Additionally, in the interactions between NaHCO3 and pH, the optimum NaHCO3 and pH ranges were 1.05–8.4 g/L and 8–9.5, respectively. Ultimately, the protein response was enhanced by the interaction between waste and pH, with optimum ranges of 67–75% and pH 8–8.9.Regarding the interaction between NaHCO3 (2.5 g/L), NaHCO3 (0.678 g/L), BFCE (33.08%) and pH = 8.During the optimization process, the statistical tool produced 31 trials with several combinations of all independent variables. The predicated combination of independent variables showing the highest desirability value was selected as the predicted optimization value. The best biomass yield (0.8 g/L) and protein (54.15%) were with the following level of independent variables: NaNOThe model was verified experimentally. The actual results were then compared with the predicted value of the models in Eqs.  and 2),,2), and S. platensis depends mainly on its amino acid content, especially essential amino acids [S. platensis cultivated in SZM and optimized medium are shown in Table S. platensis protein exhibited; was mainly leucine (8.36%) followed by valine (5.71%), whilst the lowest percentage was noted for histidine (1.62%) in the optimized medium. Likewise, leucine (8.16%) and valine (5.4%) were the major essential amino acids in SZM. Fortunately, the total essential amino acid was higher in the optimized medium (38.73%) than in SZM (36.98%). The results for non-essential amino acids in S. platensis protein indicated that glutamic acid was the major amino acid up to 14.65% and 14.26% in optimized and SZM, respectively. In contrast, cysteine was the lowest content (0.31%). It is evident from the results that TEAA in S. platensis protein from the optimized medium using BFCE was a promising source of leucine, valine, isoleucine, lysine, glutamic acid, aspartic acid, alanine and arginine compared to the SZM. These levels and profile of amino acids in Spirulina are similar to that reported by Bashir et al. [The protein quality of no acids . The resr et al. Table 5ThS. platensis can adapt to high concentrations of BFCE up to 75% substitution of SZM. Furthermore, the biomass and protein content similar to that of SZM. In contrast, direct cultivation on higher concentrations of BFCE led to low protein content and biomass compared to the SZM. The CCD predicated the optimum variables of NaNO3 (2.5 g/L), NaHCO3 (0.6 g/L), BFCE (33%), and pH = 8 to maximize the biomass yield and protein content. The resulting biomass yield was 0.56 g/L, and protein content was up to 52.5%. Also, protein quality was determined by its total essential amino acids content, which was 38.73% in the optimized medium and 36.98% in SZM. Economically, the newly optimized medium significantly saves approximately 96% NaHCO3 compared to SZM without affecting the protein content and biomass production, and these results indicating that BFCE could be used as a promising feedstock for the cost-effective large-scale cultivation of Spirulina as a microbial cell factory for protein.Microalga ® bottle (w1) containing 1 L distilled water, autoclaved at 121 °C, 1.5 atm for 20 min. After autoclaving, the bottle was left to settle down, and then the clear beet filter cake extract (BFCE) was separated under aseptic conditions. Complete characterization of BFCE ingredients, including nitrate, ammonium, and phosphorus, was carried out. To confirm the toxic pollutants' absence, the concentration of the minerals was detected using inductively coupled plasma mass spectrometry (ICP-MS) before S. platensis inoculation.The BFC was collected from Dakahlia Sugar Factory, Dakahlia province, Egypt. A hundred grams of solid waste was transferred into a pre-weighed 2 L screw cap Schott Duran, while SZM only served as the control treatment. Two strategies were applied in this study as following.The BFCE-supplemented cultures were prepared by adding different volumes of beet filter cake extract (BFCE) to the standard Zarrouk’s medium (SZM) , while SS. platensis was cultivated in SZM supplemented with BFCE. The constituents of SZM were eliminated stepwise and substituted with increasing percentage volume of BFCE . The experiment was conducted in a replicate for each treatment. All treatments proceeded with a total volume of 400 mL in a 500 mL Erlenmeyer flask, then inoculated with 10% (v/v) exponentially growing culture (O.D = 0.8 at 680 nm). At the same time, the incubation temperature was 26 ± 2 °C, 50 µmol/s/m2 illuminations, 16:8 h light: dark cycle, and continuous bubbling with sterile air flow for seven days.S. platensis inoculum was inoculated in the SZM and the 25% BFCE. Then after one week, each flask in the experiment was equally divided into two fractions (200 mL each) and centrifuged at 6000 rpm for 10 min. The first fraction’s pellet was used for further analysis after lyophilization. In contrast, the other fraction’s pellet was used as an inoculum in the next run at an increment BFCE medium ratio equal to 25% BFCE higher than the current concentration. At the same time, the control flasks followed the same steps but with the same SZM for the next run. As a result, a total of four runs with BFCE concentrations of 25, 50, 75 and 100% for 28 days was the whole duration of the experiment, while the control for each run was still the same SZM constituents.In this design, the culture was subjected to a gradual increment in the BFCE concentration for each run, while the control was only SZM. In the first run, S. platensis grown in SZM and BFCE using a pulse amplitude modulation fluorometer (PAM) (AquaPen AP 110-C). The maximum photochemical efficiency of photosystem II (Fv/Fm) was measured [S. platensis was harvested by centrifugation at 6000 rpm for 10 min and washed with distilled water to remove any salt remains. The pellet of the S. platensis was dried in a lyophilizer for 48 h at − 50 °C and weighed [For the photosynthetic performance assessment, chlorophyll fluorescence was measured daily in dark-adapted cultures of measured , 54. Themeasured . For dry weighed .a, b and carotenoids) was extracted by a known volume of 80% acetone and then measured spectrophotometrically at wavelengths 664, 647, 630 and 452 nm, respectively. The pigment concentrations were calculated by Jeffrey St, et al. [The pigment fraction  as shown in Table Central composite design (CCD) is the most popular RSM design and was applied to determine the combined influence of two or more variables used in the experiment. Based on the results of the previous experiments, four independent variables, namely (nitrate concentration (g/L), sodium bicarbonate concentration (g/L), BFCE concentration (%), and pH value) and two dependent variables, namely: biomass yield (g/L) and protein content (%), were chosen for designing the experiment by CCD in MinitabS. platensis inoculum and incubated under optimum culturing conditions. The photosynthetic activity and optical density at OD = 680 nm were measured daily, and at the end of the experiment the biomass yield was determined by dry weight and protein content by Lowry’s method [CCD design generated 31 runs with seven replicates of central points, in addition to the control flask containing only SZM. Each flask was inoculated with 40 mL of s method .The predicted optimum results were calculated according to the following equation .3\documeThe data obtained were analyzed using analysis of variance (ANOVA) using Design Expert 8 software. Based on the analysis of results, validation experiment for CCD was carried out.S. platensis grown in SZM and optimized medium was determined at Regional Center for Food and Feed (RCFF), Egypt, by using AOAC Method No. 994.12E [The amino acids content of  994.12E  using hi© software. CCD consisting of variables and responses were analyzed statistically and subjected to analysis of variance (ANOVA) to evaluate “the fit of the model” and the R2 (coefficient determination) value was calculated by the regression equations.The mean, and standard error were calculated from the triplicate experimentations. The one-way ANOVA was applied to identify the significant differences between treatments using Duncan’s test at a significant level p ≤ 0.05. Growth medium optimization was performed through CCD method using MinitabAdditional file 1: Table S1. Growth parameters  of S. platensis cultivated under different concentrations of NaNO3, NaHCO3, BFCE and pH in Central Composite Design. Fig. S1. Growth of S. platensis cultivated in the SZM and different BFCE treatments. Fig. S2. Growth of S. platensis cultivated in the SZM and BFCE treatments for incremental waste acclimatization for 28 days. Fig. S3. Optimization of growth parameters using Central Composite Design (CCD) for eight days of incubation.


Polymyxin B (PB) is a dose-dependent drug used to treat multidrug-resistantgram-negative bacteria, for which a suitable method is needed to determine clinical samples. A simple, economical, and efficient high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) method was developed and validated for polymyxin B1 (PB1), polymyxin B1-Ile (PB1-I), polymyxin B2 (PB2), and polymyxin B3 (PB3) in human plasma. Chromatographic column was Waters BEH C18 column . Phase A was water with 0.2% formic acid (FA), and phase B was acetonitrile containing 0.2% FA. The elution method is gradient elutio. The total analysis time was 5 min. The pretreatment method involved protein precipitation using acetonitrile containing 0.2% trifluoroacetic acid and 0.1% FA as the precipitant. The recovery rate was 92–99%. The total quantity of PB1 and PB1-I was measured in the linear range of 100–8000 ng/mL. Simultaneously, the total amounts of PB2 and PB3 were measured in the linear range of 11.9–948.5 ng/mL. This validated method was successfully applied to the pharmacokinetics of PB in critically ill patients. Bacillus polymyxa μg/mL. The quality control (QC) working solutions were 140, 80, 20, and 4 μg/mL. The IS solution, polymyxin E2, was diluted in 1% FA water to 7.5 μg/mL. All solutions were stored at −80°C.Standard and IS stock solutions were prepared in Milli-Q water containing 1% FA (v/v) at 1 mg/mL  and subpacked in EP tubes. Working solutions were diluted from the stock solution. The concentrations of PB1 in the PB1-I's working solution were 160, 120, 100, 48, 24, 12, 6, and 2 μL) and 10 μL working solution were mixed to prepare calibration curves of 8000, 6000, 5000, 2400, 1200, 600, 300, and 100 ng/ml for PB1/PB1-I, and 948.5, 711.4, 592.8, 284.6, 142.3, 71.1, 35.6, and 11.9 ng/mL for PB2/PB3. Similar to the QC standards, for PB1/PB1-I, the low QC (QCL), medium QC 1 (QCM1), medium QC 2 (QCM2), and high QC (QCH) were 200, 1000, 4000, and 7000 ng/ml, respectively. For PB2/PB3, QC standards were 23.7, 118.6, 474.3, and 830.0 ng/ml. First, a 100 μL calibration sample or QC sample was removed; 10 μL IS solution, 200 μL ACN with 0.1% FA (v/v), and 0.2% TFA (v/v/v) were added successively. The samples were vortexed for 2 min to make precipate the protein fully, then centrifugated at 18800 g at 4 ℃ for 15 min. About 200 μL of the supernatant was sucked and transferred into 96-well plates for analysis.Blank plasma  and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH).μL solvent and 200 μL precipitant to 95 μL of different matrices. LLOQ samples were prepared with working solutions of LLOQ, IS solution, and matrices. The responses attributable to interfering components in the retention time should not be more than 20% of PB and not more than 5% of IS in the LLOQ sample of each matrix. The matrix effect was evaluated by analyzing at least three replicates at low and high QC concentrations. The QCL and QCH of plasma samples were prepared using their working solutions with the eight matrices in QC sample preparation. QCL and QCH of the solvent samples were prepared using 5 μL working solution, 95 μL solvent, 10 μL IS solution, and 200 μL precipitant. The ratio of analyte and IS in the matrices and solvent, respectively, is calculated as the matrix factor (MF) of the analyte and IS. The IS‐normalized MF was then calculated to evaluate the matrix effect. The coefficient of variation (CV) of the IS‐normalized MF of the eight matrices should not be greater than 15%.Six blank matrices obtained from different individual sources, one high-fat matrix obtained from volunteers with abnormally high triglyceride levels, and the hemolytic matrix prepared by adding hemolytic whole blood  to the blank matrix were selected to comprehensively evaluate the selectivity and matrix effect of this method. For selectivity, the lower limit of quantification (LLOQ) and blank-level samples were used for evaluation. Eight blank samples were prepared in the above matrices by adding 15 The extraction recovery was calculated by the peak area ratios of samples recovered from plasma, extracted blank plasma, and IS spiked with the same concentrations of PB. Samples at four concentrations were analyzed in triplicates.Eight calibration-level samples, a blank sample, and a zero sample were used to establish the curve. The preparation was the same as that described in Accuracy and precision were determined using five QCs: LLOQ, QCL, QCM1, QCM2, and QCH. Five replicates at each concentration level were paralleled for each run. The between-run accuracy and precision were evaluated in three runs over two days. The accuracy of the LLOQ should be within ±20% and that of other QCs should be within ±15% overall. The precision of the LLOQ should not exceed 20%; the other QCs should not exceed 15% overall.During the investigation of dilution integrity, the dilution QC concentration was 10000 ng/mL, which was diluted with blank plasma. The two dilution factors investigated were two and four, respectively. There were five replicates for each dilution factor. The mean accuracy of the dilution QC samples should be within ±15%; the precision should not exceed 15%.QC samples  were used to investigate the stability of the stock solutions, working solutions, and samples. Each QC was set with three parallels. The stability of plasma samples stored at room temperature (25°C) for 4 h, at 4°C in a refrigerator for 24 h, at −20°C for 37 days, and at −80°C for 97 days was investigated. The freeze-thaw stability of −20°C and −80°C freeze-thaw cycles at three times each was investigated. The stability of the stock and working solutions stored at −80°C for 97 days was investigated. The accuracy of the quality control sample should be within ±15% of the nominal concentration; the precision should be within 15%.Plasma samples were collected from critically ill patients who had received at least a third dose of polymyxin B in EDTA-K2 blood collection tubes. Blood samples were collected at seven time points: 10 min before drug administration, 5 min, 1, 2, 4, and 8 h after infusion, and 10 min before the next drug administration. Blood samples were centrifuged at 996 g at 4°C for 10 min, separated into EP tubes, and frozen at −80°C. This study was approved by the institutional review board of Guangdong Provincial People's Hospital. A total of 350 clinical plasma samples were collected. The PB content was measured using the method developed in this study. The results were used to study the population pharmacokinetics (PPK). Alternatively, this method could be used for clinical drug concentration monitoring. Plasma samples of the peak and valley concentrations of PB were collected and measured using HPLC-MS/MS after validation.2+ 602.7. The iron product is shown in 2+. The product scan of iron is shown in PB standards include PB1, PB1-I, PB2, and PB3. In this method, PB1 and PB1-I were not separated by chromatography, similar to PB2 and PB3. PB's structural formula is shown in For the measurement of clinical samples, PPE is simple, more convenient, and more economical than SPE. Therefore, PPE was adopted in this method. Comparing the extraction recovery and the peak area of ACN, ACNe containing 0.1% FA, ACN with 0.2% TFA and 0.1% FA, and ACN with 0.1–2% TFA, we found that the addition of TFA can increase the response of polymyxin, which was consistent with what has been reported . When thμm) [μm), and Waters BEH C18  were evaluated. The results showed that the carry-over of Waters BEH C18 was the smallest. Therefore, this column was selected for analysis.During the experiment, the Waters HSS T3 column  , AgilentThe entire time was 5 min, including an analysis time of 3.2 min and a posttime of 1.8 min. The retention time of PB1/PB1-I was 2.06 min; PB2/PB3 was 2.04 min. At 0–0.5 min, the aqueous phase was the main phase for keeping PB on the column and eluting substances with large polarity in the sample. In the first minute, they were almost salt. Therefore, no MS was conducted. Subsequently, the organic phase ratio was increased to elute analytes. A high organic phase (90% phase B) was used to elute impurities with low polarity. Then, returned to the initial proportion and postrun for 1.8 min to stabilize the pressure.X2 was the weight factor. The correlation coefficient R2 was greater than 0.99. For PB1/PB1-I, the linearity was y = 1.2477x − 0.0214; for PB2/PB3, the linearity was y = 2.0285x + 0.0050 . The accuracy of the three intrabatch analyses was within ±15%; the CV was within 10% . The intThe area of the analyte at the retention time in the blank sample was lower than 20% in the LLOQ. The IS area was lower than 5%, indicating high selectivity. As showed in The investigation of the two dilution factors, two and four, is presented in Stability was investigated for short-term, freeze-thaw, and long-term stability. The established HPLC-MS/MS method was used to measure more than 300 samples collected clinically. A steady-state metabolic curve of polymyxin B was obtained, as shown in In this study, a precise, accurate, and convenient method for the determination of PB was developed and validated with a linearity range of 100–8000 ng/mL for PB1/PB1-I and 11.9–948.5 ng/mL for PB2/PB3 within 5 min. To our knowledge, this is the first study to measure PB1, PB1-I, PB2, and PB3 together, which is convenient. The method was successfully applied to a PPK study of 350 samples. Therefore, it was also suitable for TDM.


Patients with Down syndrome are at a higher risk of developing autoimmune disorders such as thyroiditis, diabetes, and celiac disease compared with the general population. Although some diseases are well known to be associated with Down syndrome, others such as idiopathic pulmonary hemosiderosis and ischemic stroke due to protein C deficiency remain rare.We report a case of a 2.5-year-old Tunisian girl with Down syndrome and hypothyroiditis admitted with dyspnea, anemia, and hemiplegia. Chest X-ray showed diffuse alveolar infiltrates. Laboratory tests showed severe anemia with hemoglobin of 4.2 g/dl without hemolysis. A diagnosis of idiopathic pulmonary hemosiderosis was confirmed by bronchoalveolar lavage showing numerous hemosiderin-laden macrophages, with a Golde score of 285 confirming the diagnosis of pulmonary hemosiderosis. Concerning hemiplegia, computed tomography showed multiple cerebral hypodensities suggestive of cerebral stroke. The etiology of these lesions was related to protein C deficiency.Idiopathic pulmonary hemosiderosis remains a severe disease, which is rarely associated with Down syndrome. The management of this disease in Down syndrome patients is difficult, especially when associated with an ischemic stroke secondary to protein C deficiency. Patients with Down syndrome (DS) are usually followed for several health problems, especially immunological ones such as celiac disease, thyroiditis, diabetes, and so on. Although some of these diseases are well known to be associated with DS, others rarely occur in patients with DS. Few cases of idiopathic pulmonary hemosiderosis (IPH) have been reported in patients with DS. It is a rare and severe disease that predominantly affects children younger than 10 years of age . It is cIschemic stroke is a rare event in childhood. However, its occurrence is considerably more common in patients affected by DS . Risk faThe association of IPH and DS has rarely been reported and the association between DS, stroke and IPH has, so far, never been reported.We report the first case, to our knowledge, of a child with DS who presented with IPH and ischemic stroke secondary to protein C deficiency in the same onset.3, mean corpuscular hemoglobin of 18.8 pg, white blood cells of 17,400/mm3, platelets of 169,000/mm3, and reticulocytosis of 223,400/mm3. The blood smear did not reveal schistocytes. Biochemical analysis showed no signs of hemolysis: total bilirubin of 10 μmol/l with a direct fraction of 2 μmol/l, haptoglobin of 2592.6 mg/l, and D-lactate dehydrogenase of 1295 UI/l. Blood urea nitrogen was 7.3 mmol/l, creatinine was 48 mmol/l, and serum ferritin was 71 ng/l. The level of transaminases was in the normal range . Microbiological analysis for bacteria, mycobacteria, and viruses, as well as visceral leishmaniasis serology and latex agglutination tests were negative. The direct antiglobulin test, electrophoresis of hemoglobin, and the erythrocyte fragility test showed no anomalies. Flexible bronchoscopy was performed. Bronchoalveolar lavage (BAL) showed 1,750,000 cells/ml . Numerous hemosiderin-laden macrophages were detected (Golde score: 285) confirming the diagnosis of hemosiderosis. Bacteriological culture of the bronchoalveolar lavage fluid isolated neither bacteria isolates nor mycobacterium tuberculosis. Echocardiography showed no lesions and abdominal sonography identified an isolated splenomegaly. However, brain natriuretic peptide (BNP) was 3656 pg/ml. Immunoglobulin E was of 16 UI/ml. Immunoserological markers of celiac disease and autoimmune hepatitis, as well as antinuclear and antiphospholipid antibodies, were all negative. Laboratory investigations of macrophage activation syndrome  were at normal ranges. Only the circulating anticoagulants were positive.A 2.5-year-old Tunisian girl with DS, was admitted to our pediatric department with dyspnea, fever, and motor weakness. She was born at term to nonconsanguineous parents. During the first days of life, she was diagnosed with persistent ductus arteriosus. At the age of 12 months, the congenital heart defect was closed with catheterization. When she was 11 months, she was diagnosed with hypothyroidism secondary to Hashimoto’s thyroiditis and was treated with levothyroxine. Her parents reported recurrent lung infections and the occurrence of recurrent bronchopneumopathies, but no history of hematuria or thrombotic events. On admission, physical examination revealed a normal stature and microcephalic child with height of 83 cm [−1.8 standard deviation score (SDS)], weight of 11.1 kg (−1 SDS), and a head circumference of 45.5 cm (−2.1 SDS). She was pale, febrile (temperature of 38.2 °C), and had severe dyspnea. She had tachypnea at 60 breaths/minute with oxygen saturation in air at 70%. Her heart rate was at 140 beats/minute, her blood pressure was at 110/63 mmHg, and she had no heart murmur. Pulmonary auscultation found crackles and wheeze. Abdominal palpation showed splenomegaly and neurological examination showed isolated left hemiplegia. Chest X-ray showed diffuse alveolar infiltrates and no cardiomegaly  identified cortical and subcortical hypodensities located in the right parietal lobe, and hypodensities of the periventricular deep white substance and semi-oval center Fig. . These lAfter 1 year of evolution, the patient developed cervical lymphadenopathies with persistence of splenomegaly, and recurrences of dyspnea onsets and anemia. Thoraco–abdominal CT showed multiple cervical and thoraco–abdominal lymphadenopathies and splenomegaly. CT also identified a lung nodule in the right upper lobe.Bone marrow examination showed no sign of malignancy and tuberculin skin test was negative. Histological analysis of one lymph node showed hyperplasia with evidence of epithelioid histiocytic granuloma and no areas of caseous necrosis. The etiological diagnosis of this lymphadenopathy was finally attributed to IPH. The patient was again put on corticosteroids (1 mg/kg/day) and inhaled corticosteroids (200 mcg/j) with no recurrence of his dyspnea and anemia during 1 year of follow-up.et al. [et al. [et al. [et al. [The association of IPH and DS has rarely been reported. The frequency of this association among children with IPH seems to be higher than that in the general population. Taytard et al.  reported [et al.  reported [et al. , 9. Neve [et al. , 7 and t [et al. . Several [et al. , 11. The [et al. . Based o [et al. . DS pati [et al. , 15. In  [et al. . Genetic