Background: The inactivation of tumor-suppressor p53 plays an important role in second generation anti-androgens (SGAs) drug resistance and neuroendocrine differentiation in castration-resistant prostate cancer (CRPC). The reactivation of p53 by blocking the MDM2–p53 interaction represents an attractive therapeutic remedy in cancers with wild-type or functional p53. Whether MDM2-p53 inhibitor could overcome SGAs drug resistance in CRPC is still needed further research. Here, we investigated the anti-tumor efficacy and mechanisms of a novel MDM2-p53 inhibitor XR-2 in CRPC.Methods: To investigate the functions and mechanisms of XR-2 in prostate cancer, in vitro and in vivo biofunctional assays were performed. Western blot and qRT-PCR assay were performed to detect the protein and mRNA expression levels of indicated genes. CCK8, colony formation, flow cytometry and senescence assays were performed for cell function identifications. RNA-sequencing and bioinformatics analysis were mainly used to identify the influence of XR-2 on prostate cancer cells transcriptome. Subcutaneous 22Rv1 derived xenografts mice model was used to investigate the in vivo anti-tumor activity of XR-2. In addition, the broad-spectrum anti-tumor activities in vivo of XR-2 were evaluated by different xenografts mice models.Results: XR-2 could directly bind to MDM2, potently reactivate the p53 pathway and thus induce cell cycle arrest and apoptosis in wild-type p53 CRPC cell lines. XR-2 also suppresses the AR pathway as p53 regulates AR transcription inhibition and MDM2 participates in AR degradation. As a result, XR-2 efficiently inhibited CRPC cell viability, showed a synergistic effect with enzalutamide and overcame enzalutamide resistance both in vitro and in vivo. Moreover, results illustrated that XR-2 possesses broad-spectrum anti-tumor activities in vivo with favourable safety.Conclusion: MDM2-p53 inhibitor (XR-2) possesses potently prostate cancer progresses inhibition activity both in vitro and in vivo. XR-2 shows a synergistic effect with enzalutamide and overcomes enzalutamide resistance. Globally, prostate cancer (PCa) is one of the most common malignancies and the fifth leading cause of cancer-related death in men. . DespiteTP53 gene; p53 regulates some fundamental cellular processes under cellular stress, DNA damage or oncogene activation.  efficacy in vivo. . While, in vivo. . In addiistance. . TherefoThe LNCaP , 22Rv1 , DU145 , C4-2 , MCF7  and SJSA1  cell lines were purchased from American Type Culture Collection . The HepG2 (SCSP-510) and NCI-H460 (TCHu205) cell lines were purchased from the National Collection of Authenticated Cell Cultures of China. LNCaP, C4-2, 22Rv1, SJSA1 and NCI-H460 cell lines were cultured with Roswell Park Memorial Institute (RPMI)-1,640 supplemented with 10% foetal bovine serum . DU145 and HepG2 cell lines were cultured with DMEM supplemented with 10% FBS. MCF7 was cultured with Eagle’s minimum essential medium supplemented with 10% FBS. LNCaP-R cells were derived from LNCaP cells, as LNCaP cells were cultured with RPMI-1640 supplemented with 10% FBS and 5 μM enzalutamide for 2 months, and then these 5 μM enzalutamide-treated LNCaP cells were cultured with RPMI-1640 supplemented with 10% FBS and 10 μM enzalutamide for another 6 months.in vitro experiments, and XR-2 was dissolved in Cremophor EL , 95% ethanol  and saline solution (1.5:1.5:7) for in vivo experiments. Enzalutamide and RG7388 (idasanutlin) were purchased from Selleck .XR-1, XR-2 and XR-3  were dissolved in dimethyl sulfoxide  for 5 cells per well in a 6-well plate and incubated for 48 h. Then, DMSO (Sigma-Aldrich) or test compounds were added to each well at the designated concentrations. After another 24 h of incubation, cells were collected and lysed with radio-immunoprecipitation assay lysis buffer supplemented with protease inhibitor and phosphatase inhibitor on ice for 30 min. Then, the protein lysis buffer was treated with 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes for Western blot analysis. The following antibodies were used for the detection of proteins: rabbit anti-MDM2 , rabbit anti-p53 , mouse anti-PARP1 , rabbit anti-cleaved PARP1 , mouse anti-AR  and rabbit anti-PSA . Mouse anti-actin  was used as a loading control. Proteins were visualised using anti-mouse, anti-rabbit or anti-rabbit HRP-conjugated secondary antibodies  and ECL-Plus . The resulting bands were analysed and quantified using ImageJ® 1.49 g software . Each experiment was repeated at least twice.Logarithmic growth-phase cells were seeded at a density of ∼4 × 104 cells per well in a 96-well plate. LNCaP and 22Rv1 cells were cultured in a complete RPMI-1640 growth medium. DU145 cells were cultured in DMEM growth medium. After 24 h incubation, 1 µL of DMSO (Sigma-Aldrich) or 1 µl of the indicated concentrations of XR-2 were added to each well. After 72 h of incubation, 20 µl of MTT  solution ) was added per well and incubated for another 4 h. The MTT formazan formed by metabolically viable cells was dissolved in 100 µl of isopropanol. The absorbance was measured at 570 nm wavelength on a plate reader . Experiments were performed in triplicates. The value of the DMSO group was 100%.PCa cells were seeded at a density of 1 ×104 cells per well in 6-well plates (n = 3) and treated with the required drugs/compounds or vehicle for 12–14 days. The culture media were changed every 3 days. After removal of the culture media, cells were washed by PBS twice and colonies were fixed by methanol for 10 min. Thereafter, colonies were stained using 1% (w/v) crystal violet  for 10 min. Each well was washed with distilled deionised water until the background was clean; after another 30 min of airing, colony pictures were generated. To quantify staining, the stained wells were washed with 1 ml of 10% acetic acid, and absorbance at 590 nm wavelength was detected on a plate reader .Logarithmic growth phase cells were seeded at a density of ∼1 ×105 cells per well in a 6-well plate and incubated for 48 h. Then, cells were treated with vehicles (DMSO) or test compounds at designated concentrations. After another 24 h of incubation, total RNA was extracted by TRIzol  according to the manufacturer’s instructions. Complementary cDNA synthesis was performed using a cDNA reverse transcription kit  and total mRNA templates. Relative mRNA levels of AR, PΜMA, p53 and p21 were quantified by qRT-PCR using a SYBR Green Premix Pro Taq HS qPCR Kit (AGbio) on the qPCR instrument . The mRNA expression levels were normalised to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All reactions were performed in triplicates. The gene-specific primers are listed in All kinds of cells were seeded at a density of ∼4 × 10TP53 were designed and synthesized by Sangon Biotech . siRNA stocks (20 µM) and Lipofectamine™ RNAiMAX Transfection Reagent  were diluted in opti-MEM, the mix was incubated for 20 min and then added to cells according to the instruction. After another 24 h, cells were treated with indicated compounds. The siRNA primers are listed in The Small interfering RNAs (siRNAs) of 5 cells per well in a 6-well plate and incubated for 48 h. Then, DMSO (Sigma-Aldrich) or test compounds were added to each well at designated concentrations. For the cell cycle analysis, after compounds were treated for 24 h, cells were harvested and fixed with ice-cold 70% (v/v) ethanol for 24 h. Then, cells were centrifuged at 1,000 rpm for 5 min. Cell pellets were then washed once with phosphate-buffered saline (PBS) and stained with propidium iodide. A total of 30,000 events were acquired by flow cytometer , and proportions of cells in each phase of the cell cycle were calculated using FlowJo software (BD Biosciences). For apoptosis analysis, after compound treatment for 24 h, cells were harvested and washed once with cold PBS. Then, cells were incubated for 15 min at room temperature with Annexin V-FITC-PI in a binding buffer. Cells were then analysed on a flow cytometer  using FlowJo software. Results were expressed as percentages of Annexin V+ cells. Experiments were performed in duplicate.Logarithmic growth phase cells were seeded at a density of ∼4 ×104 cells per well in 24-well plates. After incubation for 24 h, cells in each well were co-transfected with 100 ng of PSA-luc and 3 ng of Renilla plasmids using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s protocol. After 24 h from the transfection, the medium was changed to phenol red-free RPMI-1640 supplemented with 10% charcoal-stripped FBS, containing 5 nM of DHT (1 µl) and 1 µl of test compounds at designated concentrations. After 24 h, the cells were lysed in 100 µl of passive lysis buffer per well, and 20 µl of the cell lysates were used for the detection of the luciferase activity using a dual-luciferase assay system  on a plate reader . All experiments were run in triplicates.As previously described, plasmid PSA-luc was a reporter gene plasmid in which the firefly luciferase expression is dependent on the PSA promoter; plasmid Renilla was a Ranilla luciferase reporter gene plasmid. These two plasmids were kindly provided by Dr Cen . LNCaP cells were seeded at a density of 6–7 × 106 cells using Invitrogen TRIzol® RNA Isolation Reagent. Sequencing libraries were generated using VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina® , following the manufacturer’s recommendations. Index codes were added to attribute sequences to each sample. Then, the library was examined successively. The clustering of the index-coded samples was performed on a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS  according to the manufacturer’s instructions. After cluster generation, the libraries were sequenced on an Illumina platform and 150 bp paired-end reads were generated. The passed raw reads were aligned and assembled by STAR . Differentially expressed genes were analysed by HTseq-count (https://pypi.org/project/HTSeq) and edgeR (https://bioconductor.org/packages/edgeR). The KEGG analysis was performed by EnrichR (https://cran.rproject.org/). Heatmaps were generated by Hiplot website (https://hiplot.com.cn/). Original data is available in The Genome Sequence Archive for Human .LNCaP cells were treated by compounds at designated concentrations for 24 h. Total RNA was extracted from approximately 1 × 105 cells per well in 6-well plates and incubated for 24 h. Then, DMSO or test compounds were added to each well at designated concentrations. After another 72 h of incubation, cell senescence was evaluated by visualising β-galactosidase activity using senescence β-galactosidase staining kit  according to the manufacturer’s instructions. Three fields of each well were photographed for each of the three independent replicates for each treatment condition.Cells were seeded at a density of ∼1 × 106 cells with Matrigel at a ratio of 1:1) were injected subcutaneously in the flank of the male SCID mice. SJSA-1 cells (2 × 106 cells with Matrigel at a ratio of 1:1), NCI-H460 cells (2 × 106 cells with Matrigel at a ratio of 1:1), or HepG2 cells (2 × 106 cells with Matrigel at a ratio of 1:1) were injected subcutaneously in the flank of female BALB/c nude mice. When the average tumor volume reached 100–150 mm3, the mice were randomised and divided into the indicated groups (n = 8/group). XR-2 was administered once per day intraperitoneally. RG7388 and enzalutamide were administered once per day by oral gavage. Combination therapy studies were performed in a blinded manner. The tumor volume and mice bodyweight were monitored every other day, and the tumor volume was calculated according to the formula W2 × L/2 (mm3), wherein W was the short diameter and L was the long diameter. Data are expressed as mean ± SD. Whole blood was collected 24 h later after the last administration from the orbit. Blood cell analysis was performed using a haematology analyser .All animal experimental procedures were conducted in the animal facility of KeyGEN BioTECH . All experimental procedures involving the care and use of mice were approved by the KeyGEN BioTECH Institutional Animal Care and Use Committee. The mice were housed five per cage in an environmentally controlled SPF room  on a 12-h light/dark cycle. The mice were fed commercial rodent chow  and received filter-purified water ad libitum. All studies utilised 4–6-week-old mice purchased from the Shanghai Laboratory Animal Center . 22Rv1 cells  for 2 h at room temperature, followed by incubation with HPR-conjugated secondary antibody for 1 h at room temperature. The immunoreaction products were visualized with 3, 3-diaminobenzidine (DAB)/H2O2 solution.Antibodies to Ki67  and cleaved caspase 3  were used for immunostaining on formalin-fixed paraffin-embedded xenograft tumor tissues. Generally, the rehydrated slides were microwave-heated for 20 min in citrate buffer  for antigen retrieval. Then incubated in 1% Hhttps://www.rcsb.org). Chain A of the MDM2 crystal structure remained and was modified by the “protonate 3D” module of Discovery Studio 3.5. The 2D chemical structure of XR-2 drawn by the Chemdraw 18.1 software was generated in 3D structure using the “prepare ligands” module by Discovery Studio 3.5. As described previously,  was downloaded from the PDB database ethyl--5'-((4-carbamoyl-2-methoxyphenyl)carbamoyl)-6-chloro-4'-(3-chloro-2-fluorophenyl)-2′-neopentyl-2-oxospiro-1′-carboxylate (XR-1):Compound 2 was synthesized as previous explained. (plained. . Compoun2-(2-(2-methoxyethoxy)ethoxy)ethyl--5'-((4-carbamoyl-2-methoxyphenyl)carbamoyl)-6-chloro-4'-(3-chloro-2-fluorophenyl)-2′-neopentyl-2-oxospiro-1′-carboxylate (XR-2): Compound 2  was dissolved in DMF (100 ml), then anhydrous KCO3 (4.0 mmol) was added with drops of 2- (2- (2-methoxyethoxy) ethane-1-ol , and stirred overnight at room temperature. After the reaction, water (20 ml) was added and extracted by ethyl acetate. The organic phase was separated and washed with brine, 1M hydrochloric acid and water successively, and then concentrated through the column to obtain the target product (Yield: 80%), which was a white solid. 1H NMR : 10.6 , 8.45 , 7.8 , 7.55 , 7.48 , 7.35 , 7.20–7.40 , 7.04 , 6.1–6.6 , 5.4–5.8 , 4.69 , 4.40–4.55 , 3.04 , 3.83 , 3.69–3.77 , 3.62–3.69 , 3.50–3.58 , 3.36 , 3.18–3.30 , 1.24–1.36 , 0.95 ; 13C NMR : 174.41, 171.63, 168.82, 157.69, 155.21, 149.52, 148.46, 140.10, 134.91, 130.57, 130.01, 128.73, 127.35, 125.51, 124.81 (Jc-F = 4.3 Hz), 123.62, 123.36, 121.40 , 119.95, 118.22, 116.15, 109.98, 72.01, 70.83, 70.81, 70.68, 68.61, 68.06, 66.60, 66.57, 65.51, 59.12, 55.76, 50.96, 42.79, 30.45, 29.89; HRMS (ESI-TOF): m/z calculated for C39H45Cl2FN4NaO9+ [M + Na]+: 825.2445, found: 825.2456.2,5,8,11,14-pentaoxahexadecan-16-yl--5'-((4-carbamoyl-2-methoxyphenyl)carbamoyl)-6-chloro-4'-(3-chloro-2-fluorophenyl)-2′-neopentyl-2-oxospiro-1′-carboxylate (XR-3): Compound 2  was dissolved in DMF (50 ml), followed by the addition of anhydrous KCO3 (2.0 mmol) and the addition of 1-chloroethyl  carbonate , stirred overnight at room temperature. After the reaction, water (20 ml) was added and extracted by ethyl acetate. The organic phase was separated, washed with brine, 1M hydrochloric acid and water successively, and then concentrated through the column to obtain the target product (Yield: 79%), which was a white solid. 1H NMR : 10.56 , 8.41 , 7.79 , 7.54 , 7.49 , 7.20–7.40 , 7.00–7.15 , 6.30–6.60 , 5.19 , 4.71 , 4.40–4.60 , 4.25–2.34 , 3.93 , 3.83 , 3.78 , 3.60–3.75 , 3.50–3.58 , 3.35–3.40 , 1.20–1.37 , 0.86–1.08 ; HRMS (ESI-TOF): m/z calculated for C43H53Cl2FN4NaO11 [M + Na]+: 913.2970, found: 913.2975.p < 0.05.Statistical analysis was performed using GraphPad Prism . Comparisons between groups were performed with a two-tailed Student’s t-test, except where specified, and differences were considered significant at cis-cis isomer of PSD XR-2  based on previous reported spiroindolinone pyrrolidinecarboxamide compound 2 . Shu et. Then, wPSD XR-2  led to tmpound 2 . Therefop21, PUMA and MDM2 but not p53 mRNA expression levels in both LNCaP and 22Rv1 cells  using Discovery Studio software. We found that XR-2 could suitably locate into the p53 binding site of MDM2 and form hydrogen bond interactions with Leu54 and His96 . The homv1 cells . Not sur45 cells . Notably45 cells . Taken tp21 and PUMA could regulate cell cycle arrest, apoptosis and senescence in various cancer cells. Subsequently, we investigated the CRPC inhibition activity of XR-2; as shown in 50 of 0.073 and 0.74 μM, respectively. Conversely, p53-mutated DU145 cells were quite less sensitive to XR-2. Furthermore, to estimate the effects of XR-2 on CRPC cell clonogenic activity, we exposed LNCaP, 22Rv1 and DU145 cells to different concentrations of XR-2 for about 2 weeks. As a result, XR-2 strongly decreased the number of LNCaP and 22Rv1 cell colonies in a dose-dependent manner compared with the untreated group, while specific treatment concentrations did not influence the number of DU145 cell colonies  cells through long-term enzalutamide treatment . Comparein vitro. Then, we investigated the tumor inhibition efficacy of XR-2, enzalutamide and XR-2 plus enzalutamide in vivo by using 22Rv1 xenografts in male SCID mice. In this study, 5 ×106 22Rv1 cells were injected into the left flank of each male mouse, and the tumor volume was allowed to increase to approximately 100 mm3. Then, the tumor-bearing mice received intraperitoneal injection of the vehicle control, 30 mg/kg of XR-2, 30 mg/kg of enzalutamide and 30 mg/kg of XR-2 plus 30 mg/kg of enzalutamide once a day for 4 weeks. The results indicated that enzalutamide could only inhibit 22Rv1 tumor growth weakly, mainly due to ARV7 overexpression, while both XR-2 monotherapy and combination therapy suppressed 22Rv1 tumor progression and decreased tumor weight significantly  possesses potently prostate cancer progresses inhibition activity both MDM2, modification murine double minute-2; qRT-PCR, quantitative reverse-transcriptase polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. DHT, dihydrotestosterone; CRPC, castration-resistant prostate cancer Contributions.


Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40 °C to 70 °C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on β-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of  In this context, lignocellulosic waste is a promising renewable resource owing to its abundance and affordability, and it can be used as a raw material to recover high-value-added products from degradation of its complex structure . . A. acidA. mali FL18 growth is pH 4, the purified AmCel9 showed its pH optimum at pH 6.0 and displayed more than 50% activity within the pH range from 5.0 to 7.0  [It is of note that the temperature activity profile A revealed Cel9D) .A. acidocaldarius and CenC of B. licheniformis despite having their temperature optimum at 70 °C, lost more than 40% of their activity after 2 h of incubation at 60 °C. Moreover, BlCel9 of B. licheniformis also showed long-lasting thermostability at 50 °C, retaining more than 80% of its activity after 24 h; however, at 60 °C, its enzymatic activity dropped significantly to about 40% after only 3 h, whereas AmCel9 was still over 60% active after 10 h [Regarding the thermo-resistance, the enzyme retained more than 60% and 40% of activity after being incubated at 50 °C and 60 °C for 16 h, respectively, therefore showing good stability B. The coter 10 h ,51. A. acidocaldarius or the endo-β-1,3(4)-glucanase of Alicyclobacillus sp. A4 [C. thermocellum [T. fusca with endo and exo-activities; or bifunctional Cel9X of C. cellulolyticum with cellulase/xylanase activities; or the cellulase/mannanase/xylanase activities of P. curdlanolyticus B-6 that hydrolyze different polysaccharides [GH9 enzymes generally exhibit heterogeneous hydrolytic capabilities on cellulose; most of them are mainly endoglucanases such as the endo β-1,4-glucanase Cel9A of s sp. A4 ,48, but mocellum ,53. Thischarides ,54,55.KM and Vmax values  comparable with those determined for other GH9s [cat) calculated for lichenan was about two-fold higher than that on CMC. Therefore, the kcat/KM of AmCel9 on this soluble substrate is about three times lower than that on lichenan; the lower enzyme activity could be due to the presence of methyl groups on CMC side chains. Overall, since most other GH9 members have not been characterized to date, the determination of the kinetic parameters of AmCel9 gives new insight into the biochemical features of GH9 family [The AmCel9 kinetic parameters indicate a very efficient enzyme on both substrates ; in facther GH9s . The tur9 family .A. acidocaldarius  [C. thermocellum (30 U/mg on CMC) [Alicyclobacillus sp. A4 (33.2 U/mg on lichenan) [Unfortunately, an all-embracing comparison of activity values within characterized GH9 members is not feasible because of their heterogeneity as each protein has different specific activity and substrate specificity on several polysaccharides . Howeverctively) . In addi on CMC) , GH9b an on CMC) , Alg9 ofichenan)  and PcMuichenan) . Interesichenan) ,43, and P. curdlanolyticus and BlCel9 of B. licheniformis, both harboring CBM modules, have specific activities towards Avicel of 28.57 U/g and 40 U/g, respectively [A. acidocaldarius, Agl9A of Alicyclobacillus sp. A4 or BP_Cel9 [® filter paper) and microcrystalline cellulose (Avicel PH101), showed a good enzyme capability to release glucose with values of specific activity of 68.7 ± 0.02 and 62.98 ± 0.05 U/g, respectively, comparable to those reported for PcMulGH9 or BlCel9 [Family GH9 also embraces catalysts that are able to degrade insoluble cellulose; to date, the presence of CBM modules is known to be correlated to the enzyme processivity on microcrystalline cellulose ,40; for ectively ,50. On t BP_Cel9 ,44,48. Vr BlCel9 ,50. It cr BlCel9 .The unexpected activity towards insoluble cellulose along with the broad substrate specificity and high catalytic efficiency make AmCel9 a promising multifunctional catalyst, that might more efficiently break down lignocellulosic material without requiring non-eco-friendly pre-treatments.A. mali FL18 was previously annotated by the Rapid Annotation Subsystem Technology (RAST) and deposited in NCBI (JADPKZ000000000) and IMG-IM (https://img.jgi.doe.gov/cgi-bin/mer/main.cgi?section=TaxonDetail&page=taxonDetail&taxon_oid=2931470914 accessed on 10 November 2022). CAZyme searches were performed using dbCAN metaserver (https://bcb.unl.edu/dbCAN2/ accessed on 10 November 2022) with integrated: (i) HMMER database; (ii) DIAMOND for fast blast hits in the CAZy database and (iii) HMMER for dbCAN-sub [The genome of bCAN-sub . ® filter paper (75 mm × 100 mm) was cut into circle paper units of 0.5 mm × 0.5 mm. Kanamycin and chloramphenicol were purchased from PanReac AppliChem .Carboxymethyl cellulose sodium salt (CMC), Avicel (PH-101), glucose, cellobiose, salicin, locust bean gum mannan and laminarin were purchased from Sigma-Aldrich . Konjac glucomannan (KGM), birchwood xylan, beechwood xylan, lichenan, guar galactomannan, carob galactomannan, larch arabinogalactan and wheat arabinoxylan were purchased from Megazyme . WhatmanA. mali FL18, with the Accession No. MBF8377998.1, was compared to sequences of GH9s already characterized available in the GeneBank database by utilizing BLASTp server (https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 14 September 2022).The aminoacidic sequence of the putative GH9 of A. acidocaldarius; Alg9 from Alicyclobacillus sp. A4; CED1 from B. fibrisolvens; PcMulGH9 from P. curdlanolyticus; E4 from T. fusca and of Cel9T from C. thermocellum [https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb accessed on 14 September 2022); this server predicts protein structures from their sequences using a simplified version of AlphaFold v2.0 [To identify homologous proteins and conserved domains in AmCel9, a multiple alignment and a phylogenetic tree were performed with CLC Main Workbench 22.0.2 software  by using the amino acid sequences of: Cel9A from mocellum ,46,48,54old v2.0 .AmCel9, which contains the gene encoding AmCel9 with a six-His tag at its N-terminus, was purchased from GenScript Biotech  and used to transform E. coli BL21-CodonPlus(DE3) cells . These cells were cultured in LB medium supplemented with kanamycin (50 μg/mL) and chloramphenicol (33 μg/mL). Protein expression was induced for 16 h by adding 1 mM of IPTG  when the cultures reached 0.7 OD600/mL. Cells were harvested by centrifugation at 6750× g and resuspended in 20 mL of buffer A  supplemented with lysozyme 0.2 mg/mL  and with protease inhibitor cocktail 1X . The resuspended cells were lysed with one cycle of freeze-thaw with the following procedure: (i) incubation for 90 min on an orbital shaker at 37 °C; (ii) freezing with ethanol and dry ice for 15 min and (iii) re-incubation for 15 min at 37 °C. After a centrifugation step at 35,000× g for 45 min at 4 °C, the purification of AmCel9 was set up through a two-step procedure. A first thermal precipitation at 60 °C for 10 min was followed by centrifugation at 35,000× g for 45 min at 4 °C then followed by affinity chromatography on a His-Trap™ column  connected to an AKTA Explorer system. The elution was performed with buffer A supplemented with a linear gradient of imidazole (0–500 mM), and all the peak fractions were pooled and then dialyzed against a storage buffer  using an Ultracel® 10 kDa ultrafiltration disc  connected to an Amicon® stirred cell. Bradford assay was used to estimate protein concentration using bovine serum albumin as standard. The purity degree of AmCel9 were appraised by SDS-PAGE analyses (12%) after staining the gel with Coomassie brilliant blue R-250 .The plasmid pET28b(+)/The purified AmCel9 was further analyzed by using a size-exclusion chromatography system connected to Mini DAWN Treos light-scattering system  equipped with a QELS module (quasi-elastic light scattering module) and with mass value and hydrodynamic radius (Rh) measurements. One milliliter of protein (0.6 mg/mL) was loaded on a Superdex S200 column  equilibrated in buffer 50 mM Tris-HCl pH 8, 200 mM NaCl, 1mM DTT, with a flowrate of 0.5 mL/min. Data were analyzed using Astra 5.3.4.14 software  .w/v) CMC in assay buffer . A total of 1 µg of enzyme was added to the substrate mix, pre-incubated for 10 min at 50 °C and the reaction stopped after 1 min at 50 °C in ice for 5 min. Then, 160 µL of 3,5-dinitrosalicylic acid (DNS) was added to the reaction mix, which was transferred in a 96-well microplate and placed in a thermomixer  at 100 °C for 20 min [540nm in a 96-well microplate reader  and by interpolating absorbance data with a standard curve that was prepared according to Kim et al. [AmCel9 standard assay was set up at 50 °C and using CMC as substrate; the assay mixture  contained 1.6% , with increments of 0.5 pH units at 50 °C for one minute, using the DNS assay described above. Citrate buffer (pH 3.0–5.5), Na-P buffer (pH 6.0–7.5) and glycine-NaOH buffer (pH 8.0–10) were used. Temperature optimum was measured by assaying enzyme activity at pH 6.0 from 40 °C to 90 °C with an increase of 10 °C; each experiment was performed in triplicate and results were reported as relative activity (%). To test the pH stability of recombinant AmCel9, it was preincubated in various buffers from pH 4.0 to 8.0 at 4 °C for 4 and 16 h; residual activity of the enzyme was measured under standard assay conditions. Thermo-resistance was determined by measuring residual activity up to 16 h , pre-incubating the purified enzyme without the substrate at temperatures of 50 °C, 55 °C, 60 °C, 65 °C and 70 °C. All the experiments were performed in triplicate and the 100% was the enzyme activity in the absence of pre-incubation.The substrate specificity of AmCel9 was tested by using the standard assay  on several polymers: cellobiose (1.5%), lichenan (1.4%), laminarin (1.5%), locust bean gum mannan (0.5%), KGM (0.7%), birchwood xylan (0.9%), beechwood xylan (0.9%), guar galactomannan (0.7%), carob galactomannan (0.7%), larch arabinogalactan (0.7%) and wheat arabinoxylan, prepared in assay buffer  or in water, according to the specific data-sheet of each substrate. The released reducing monosaccharides were quantified using the DNS method described above, except for cellobiose, for which the amount of free glucose was estimated by using the D-Glucose Assay Kit  according to the manufacturer’s protocol. The assay on CMC (1.6%) was used as the control; one unit (U) was defined as the amount of enzyme necessary to release 1 μmol of reducing sugars per minute at 50 °C and pH 6; each experiment was performed in triplicate.M was calculated by non-linear regression analysis using GraphPad 7.0 (Prism software) and each experiment was performed in triplicate.The kinetic parameters were determined using CMC and lichenan as substrates: 0.4 μg of AmCel9 was tested with CMC  and 0.2 μg with lichenan . The Michaelis–Menten constant K® filter paper) with 35 μg of purified enzyme in the assay buffer  at 55 °C for 1 h on an orbital shaker at 200 rpm. To measure the amount of glucose released, 500 μL of DNS solution were added to each sample and the mixture boiled for 10 min. After a cooling step at 4 °C for 30 min, the A540nm was measured and data were interpolated with a glucose standard curve to measure the concentration of the reducing sugar in solution [The hydrolytic activity of the enzyme was also investigated on insoluble celluloses; the assays were performed in a total volume of 500 μL, by incubating 2.5 mg of Avicel (PH-101) or 1 circle paper unit of 0.5 mm × 0.5 mm (Whatmansolution ,61. For A. mali FL18 as a reservoir of CAZymes was investigated. Genome-wide analysis and functional annotation were performed to identify the key contributors in the lignocellulose decomposition and to provide insight into the genes correlated with lignocellulolytic activity. In particular, the analysis of CAZome revealed the presence of 32 putative GHs, indicating that A. mali FL18 possesses a palette of interesting activities for the biodegradation of the holocellulosic component of the cell wall. In silico analyses suggest that at least four of these GHs might be involved in cellulose degradation; among them, a novel thermophilic GH9, AmCel9, was biochemically characterized. AmCel9 exhibits a peculiar activity in a broad range of temperatures, as well as thermo-resistance and stability in a wide range of pH values. Moreover, AmCel9 hydrolyses β-glycosidic linkages of synthetic and natural celluloses with relevant specific activities, and, to the best of our knowledge, is the first GH9 member without a CBM module that is also able to hydrolyze insoluble celluloses. Overall, the data presented in this work prove that AmCel9 is a catalyst deserving further exploitation; in fact, it can be employed either as multifunctional enzyme to ameliorate the degradation of lignocellulosic biomass for the conversion of recalcitrant residues into value-added products or to specifically hydrolyze crystalline cellulose.In this study, the potential of the thermoacidophilic bacterium


R2 ≥ 0.999), sensitivity (limit of quantification in the range of 0.50–5.00 μg kg−1), recovery (89.35 ± 2.67% to 110.93 ± 1.56%), and precision , and it was then successfully applied to investigate a total of 60 feed samples. Among them, 50 samples were found to be contaminated with ZEN (an incidence of 83.3%) at levels ranging from 0.63 to 615.24 μg kg−1, whereas 22 samples were contaminated with ZEN-14G (an incidence of 36.7%) in the range of 0.89–15.31 μg kg−1. The developed method proved to be a specific and reliable tool for intensive monitoring of ZEN and ZEN-14G in complex feed matrices.A reliable ultra-high-performance liquid chromatography–tandem mass spectrometry method (UHPLC-MS/MS) was developed for the simultaneous determination of two mycotoxins, that is, zearalenone (ZEN) and zearalenone-14-glucoside (ZEN-14G) in formula feed, concentrated feed, and premixed feed products. An improved sample pretreatment was achieved with the hydrophilic–lipophilic balance (HLB) cartridges efficiently removing the impurities and enriching the target analytes in different feeds. The critical parameters affecting the performance of the solid-phase extraction (SPE) procedure were carefully optimized, and 20% acetonitrile in water as the loading solution, 50% methanol in water as the washing solvent, and 5 ml of methanol as the elution solvent yielded the optimal purification efficiencies. The established method was thoroughly validated in terms of linearity ( Fusarium species and has attracted more and more attention due to safety concerns and economic impacts , one of the most prevalent mycotoxins, is produced by  impacts . As a li impacts . ZEN can impacts . Based oacts , enzyme-linked immunosorbent assay (ELISA), liquid chromatography coupled with mass spectrometry (LC-MS) or a fluorescence detector (FLD), gas chromatography (GC) –MS, and high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS), have been established for detecting and quantifying a variety of mycotoxins in cereals and cereal products . TLC is To face these analytical challenges, the aim of our study was to develop a reliable and sensitive ultra-HPLC-MS/MS (UHPLC-MS/MS) method for the simultaneous determination of ZEN and ZEN-14G in various feeds. The established analytical method based on an efficient hydrophilic–lipophilic balance–solid-phase extraction (HLB-SPE) clean-up approach was extensively validated by determining the linearity, sensitivity, recovery, and precision and then applied for monitoring the two analytes in different feed samples to reveal the real contamination situation in China.18 and HLB-SPE cartridges were acquired from NanoChrom Co. . PriboFast®MFC 260 cartridges were purchased from Pribolab . Methanol and acetonitrile were purchased from Merck . Water used throughout the whole analysis was Milli-Q quality water . All reagents and chemicals were of analytical or HPLC grade.The ZEN and ZEN-14G standards were obtained from Sigma-Aldrich  and HPC standards GmbH , respectively . C18 andA total of 60 feed samples were randomly collected from different formal enterprises in Shanghai, Jiangxi province, and Guangdong province in China, consisting of three types: formula feed products (28), concentrated feed products (14), and premixed feed products (18). It was verified that neither ZEN nor ZEN-14G was detected in samples used as blank matrices. All samples were finely ground using a high-speed grinder , passed through a 0.45 mm sieve, and stored at 4°C until analysis.g for 5 min, 1 ml of the supernatant was collected and dried with nitrogen gas  at 40°C. The residues were redissolved in 5 ml of acetonitrile/water (20:80 v/v) and passed through the HLB-SPE cartridges, which were pre-equilibrated with 2 ml of methanol and acetonitrile/water  at a flow rate of 1–2 drops/s successively. Then, the cartridges were washed with 5 ml of methanol/water . All target analytes were eluted with 5 ml of methanol, dried with nitrogen gas at 40°C, and redissolved in 1 ml of acetonitrile/water containing 5 mmol L−1 ammonium acetate . Finally, the eluent was passed through a 0.22 μm nylon filter , ready for UHPLC-MS/MS analysis.Each sample (2.0 g) was accurately weighed into a 15 ml centrifuge tube and was macerated with 10 ml of acetonitrile/water/formic acid  for 5 min, followed by ultrasonic extraction for 30 min at 40°C . After centrifugation at 13,000 via a Waters ACQUITY HPLC system  at 40 °C using a Poroshell 120 EC-C18 column  . A linear gradient elution was carried out at a flow rate of 0.3 ml min−1 using (A) methanol and (B) 5 mmol L−1 ammonium acetate aqueous solution as the mobile phase, and the gradient program was designed as follows: 0.0–1.0 min 10% A, 1.0–3.0 min 10–25% A, 3.0–6.0 min 25% A, 6.0–7.0 min 25–90% A, 7.0–7.5 min 90% A, 7.5–7.6 min 90–10% A, and 7.6–9.0 min 10% A. The injection volume was 3 μL.UHPLC analysis was performed to separate ZEN and ZEN-14G +) for ZEN-14G and the negative mode (ESI−) for ZEN. The parameters were set as follows: ion spray voltage, 5.5 kV (ESI+) and 4.5 kV (ESI−); block source temperature, 500°C; nebulizer gas, 50 psi; turbo gas pressures, 50 psi; curtain gas pressure, 35 psi; and collision gas value, medium. Multiple reaction monitoring (MRM) was performed for the quantification of ZEN and ZEN-14G  equipped with an electrospray ionization (ESI) interface operating in the positive mode (ESI ZEN-14G . Data ac−1), and the standard solutions. A series of standard solutions for ZEN and ZEN-14G with concentrations of 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, and 200 ng ml−1 were prepared in neat solvents  and blank matrices. Linear calibration curves (1/x weighted) were obtained for ZEN and ZEN-14G by plotting MS responses (peak areas) versus the concentrations of the analytes. The sensitivity of the method was evaluated by the determination of LOD and LOQ, defined as the concentrations of the analytes generating signal-to-noise (S/N) ratios of 3 and 10 in each matrix, respectively. Blank samples spiked with 1, 5, 20, 50, and 100 μg kg−1 for ZEN and 5, 10, 20, 50, and 100 μg kg−1 for ZEN-14G were utilized to investigate the recovery, intra-day, and inter-day precisions (n = 5). The recovery of the overall process efficiency (PE) was calculated by comparing the concentration levels measured by the matrix-matched calibration curves with the spiked levels according to the following formula , limit of quantification (LOQ), recovery, and precision for each matrix . The selectivity of the method was investigated by comparing the MRM chromatograms of the uncontaminated feed samples, contaminated feed samples, blank matrices spiked with ZEN and ZEN-14G (50 ng ml formula . RecoverThe matrix effect (ME) was evaluated by determining the signal suppression/enhancement (SSE) values, calculated by comparing the slope of the matrix-matched calibration curves with that constructed in the neat solvent according to the following equation :ME=SlopData analyses were performed using the software SPSS Statistics 19 . The results were described by means ± standard deviations  methanol–water containing 0.1% formic acid and 5 mmol L−1 ammonium acetate. The results of multiple injections indicated that the responses of ZEN and ZEN-14G were greatly improved and the highest sensitivity was obtained when methanol–water containing 5 mmol L−1 ammonium acetate was selected. Under such a situation, a satisfactory separation efficiency and a nice peak shape were also achieved (−1 ammonium acetate) were further investigated  methanol–water, 2) methanol–water containing 0.1% formic acid, 3) methanol–water containing 5 mmol Lachieved . Subsequstigated . We founRE) according to the following formula using the spiked formula feed samples (50 μg kg−1)  . First, μg kg−1) . Then, fμg kg−1) . The neuμg kg−1) . Alternaμg kg−1) . Then, tμg kg−1) . FinallyA denotes the peak area of the analyte recorded for the sample spiked with the target compound(s) before extraction, and B denotes the peak area of the analyte recorded for the sample spiked with the target compound(s) after extraction.®MFC 260, C18-SPE, and HLB-SPE) were compared using the spiked formula feed samples (50 μg kg−1). As shown in 18-SPE cartridges were used, only ZEN-14G achieved a satisfactory recovery. With respect to HLB-SPE cartridges, satisfactory recoveries of 97.84 ± 7.36% for ZEN and 102.11 ± 7.14% for ZEN-14G were reached. This may be attributed to the fact that HLB cartridges contain apolar and polar regions  were compared . With thTo reduce the presence of possible interference caused by organic compounds in feed matrices, such as lipids, proteins, and carbohydrates, different methanol–water solutions as the washing solvents, 1) 20/80 (v/v), 2) 30/70 (v/v), 3) 40/60 (v/v), 4) 50/50 (v/v), 5) 60/40 (v/v), and 6) 70/30 (v/v), were tested . As showA total of five different elution solvents, that is, (A) methanol–water , (B) methanol–water , (C) methanol–water , (D) methanol–water , and (E) methanol, were compared . The eluThe elution volumes from 1 to 10 ml were also studied. As shown in To characterize and clarify the established clean-up method, the visual appearance characteristics and MEs of ZEN and ZEN-14G in different feed matrices, purified or unpurified with the HLB-SPE cartridges, were compared. As shown in R2) higher than 0.999 were obtained for ZEN and ZEN-14G in the neat solvent and in different feed matrices  reported in a global mycotoxin survey from 19,171 samples of 100 countries over 10 years  (−1)  (−1) (−1 were detected (an incidence of 36.7%).Feed sources are extraordinarily diverse, and their compositions vary with not only the target species but also the age. A total of 60 feed samples for pigs (50) and poultry (10) at different growth stages, namely, 28 formula feeds, 14 concentrated feeds, and 18 premixed feeds, were collected and analyzed by the established UHPLC-MS/MS method . Among t10 years . Howeverμg kg−1) , Poland −1) (−1) , South Kμg kg−1) , and Chi−1) (−1) . Differe−1 with a positive rate of 90.0%, but only two samples were contaminated with ZEN-14G at the levels of 1.8 μg kg−1 and 2.5 μg kg−1. Only one formula feed sample contained ZEN (615.2 μg kg−1) with a concentration higher than the limit established by China (500 μg kg−1)  (−1) (−1) (−1)  (−1)  (There were few differences in the incidence of ZEN among the formula feeds (82.1%), concentrate feeds (78.6%), and premixed feeds (88.9%). Poultry feeds were contaminated with ZEN in the range of 4.0–615.2 μg kgμg kg−1) . The conμg kg−1)  but lowe−1) (−1) , Kenya (−1) (−1) , and Sou−1) (−1) . Feeds fd by −1)  but lowe−1) (−1) . In a re(−1) (−1 . In the y limits . Compareits (−1) .−1 and 0.89–15.31 μg kg−1, respectively. Although there are no recommendations or regulations on ZEN-14G in feeds, the occurrence of ZEN-14G would certainly increase the adverse effects on animal and human health (It is worth noting that ZEN co-occurred with ZEN-14G in 21 feed samples (an incidence of 35.0%) with concentrations of 0.71–119.66 μg kgn health .A sensitive and reliable UHPLC-MS/MS method based on the commercially available HLB-SPE cartridges was established for the simultaneous determination of ZEN and its masked form, ZEN-14G, in formula feed, concentrated feed, and premixed feed products. After the optimization of the SPE procedure and UHPLC-MS/MS conditions, the established method was demonstrated to be rapid, sensitive, effective, accurate, and efficient to monitor ZEN and ZEN-14G in different feed samples, which also shows its feasibility for broader applications in the field of analytical chemistry. Considering the highly toxic characteristics of ZEN alongside its high co-occurrence with the masked form, continuous monitoring in various feeds using the developed UHPLC-MS/MS method is recommended.


Combretum micranthum (CM), a plant of the family Combretaceae in sub-Saharan Africa, has been utilized in folklore medicine to cure diverse human ailments. In order to develop a neuroprotective phytomedicine, the current research was undertaken to explore the antioxidant, anti-inflammatory, anticholinesterase and neuroprotective potential of CM extract.Brain damage is a severe and common pathology that leads to life-threatening diseases. Despite development in the research, the medical evidence of the effectiveness of potential neuroprotective medicines is insufficient. As a result, there is an immense and urgent demand for promising medication. For millennia, herbal remedies were a fundamental aspect of medical treatments. 2-AA) by assessing the lipoperoxidation biomarker malondialdehyde (MDA).Colorimetric methods were used to determine CM antioxidant activity, in-vitro protein denaturation and membrane destabilization assays were used to evaluate its anti-inflammatory capacity, anticholinesterase activity was carried out using Ellman’s method, and neuroprotective potential was assessed on brain homogenate stressed with ferric chloride and ascorbic acid (FeCl50 = 27.15 ± 0.06 µg/mL) and Total Antioxidant Capacity (IC50 = 31.13 ± 0.02 µg/mL), CM extract demonstrated strong antioxidant activity. Anti-inflammatory effect were improved in heat-induced Egg albumin and BSA denaturation (IC 50 = 46.35 ± 1.53 and 23.94 ± 1.10 µg/mL) as well as heat and hypotonia induced membrane destabilization (IC 50 = 20.96 ± 0.11 and 16.75 ± 0.94 µg/mL).In Ferric Reducing Antioxidant Power (IC50 = 59.85 ± 0.91 µg/mL). In an ex-vivo neuroprotective model, CM extract showed substantial inhibition (p < 0.001) of oxidative damage caused by FeCl2-AA in brain tissue.CM extract showed strong anticholinesterase activity (IC C. micranthum may be a good candidate for its probable neuroprotective potential. Its neuroprotective benefits might be attributed to its antioxidant, anti-inflammatory and anticholinesterase effects. Every 3tivities . Our pretivities . Phytochtivities  while sttivities , which ativities . Furthertivities . Recentltivities . There i22.1Malondialdehyde (MDA), ascorbic acid, methyl-2-phenylindole, nitric oxide, Ferric chloride, 2,6-di-tert-butyl 4-methylphenol, methanol, 5,5-dithiobis-2-nitrobenzoic acid, Bradford reagent, dimethyl sulphoxide (DMSO), acethylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI), acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and bovine serum albumin (BSA) were procured from Sigma-Aldrich. All other chemicals and reagents were of analytical grade.2.2C. micranthum leaves were harvested in the Alibi I zone of Tchamba's northwestern region. A voucher specimen (N° TOGO151088) was kept in the herbarium of the Institute. The extraction was done as before, with a yield of 12.15% w/w .2.42.4.12.4.1.1The method of 0 = absorbance value of control, Kt = absorbance value of CM extract or standards.Where, K50 values of the CM extract, aspirin, and diclofenac sodium were generated in GraphPad Prism-9  using Fit spline analysis.The IC2.4.1.250 values were calculated as mentioned in BSA denaturation inhibition assay procedure.This assay has been done as described previously . The rea2.4.2The anti-inflammatory activity was assessed using RBC membrane stability. The equal volume of rat blood and 0.9% w/v normal saline was taken in heparinized tubes. The blood was centrifugated for 10 min at 3000 rpm. Then the suspended cells were washed carefully with saline three times. Finally, 10% v/v cell suspension was prepared using saline  and used2.4.2.150 values were calculated as mentioned in above procedure.This experiment was executed using the method as described in the literature . The inv2.4.2.250 values were calculated.The assay mixture contained 1 mL of RBC suspension and 1 mL CM extract or standard drug solutions (25–400 µg/mL). The tube samples were kepet at 70 ˚C in the bath for 30 min. After cooling and centrifuging for 5 min at 3000 rpm, the absorbance was measured at 560 nm using UV spectrophotometer . In addi2.52.5.150 values of the CM extract, ascorbic acid and quercetin were calculated using GraphPad Prism-9 software using Fit spline analysis.The power of the extract to reduce ferric ions was analyzed using the reported method . Acetate2.5.250 values.TAC assay was carried out according the reported method . The ass2.62.6.12 and 100 μL of different amount of CM extract or quercetin. The samples were kept at 37 °C for one hour. After the incubation, the malondialdehyde (MDA) concentration in the samples was estimated as previously described using 1,1,3,3-tetra-methoxypropane to make a standard curve  were reported in 50 values indicated their anti-inflammatory power in heat-induced protein denaturation assay protocols. CM extract exhibited almost nearer IC50 values than that of standard tested drugs.The IC3.1.250 values as expressed in CM extract exhibited good RBC membrane stabilizing properties as evident by lower IC3.23.2.13+-TPTZ) to Fe2+-TPTZ complex by CM extract and the standard compounds was tabulated in 50 values of the CM extract (27.15 ± 0.06 µg/mL) was found to be better than quercetin (37.50 ± 0.28 µg/mL) and nearly similar to ascorbic acid (23.62 ± 0.02 µg/mL).The reduction of the ferric tripyridyltriazine complex (Fe3.2.250 (31.13 ± 0.02 µg/mL) when compared to quercetin (30.11 ± 0.14 µg/mL) and ascorbic acid (29.73 ± 0.05 µg/mL).TAC of the CM extract determined was tabulated in 3.33.3.12-ascorbic acid increased lipoperoxidation leading to MDA generation significantly (p 0.001). However, CM extract and quercetin significantly (p < 0.001) inhibited the lipid peroxidation in tested homogenate induced by FeCl2-AA  is reduced to molybdenum (V), generating a green phosphomolybdate (V) complex. . This acxidation . Ascorbixidation . Free raxidation . Becausexidation . As a rexidation . Many anxidation . The phyxidation . The poltroscopy . Our eartroscopy . The seatroscopy . Our fintroscopy . Findingtroscopy .in silico experiments demonstrated that polyphenolic bioactive chemicals found in the CM extract inhibited Nuclear factor kappa B (NF-κB), soluble epoxide hydrolase (sEH) and activated peroxisome proliferator-activated Receptor alpha (PPARα) and gamma (PPARγ) expression (The cholinergic system dysfunction might play a critical role in ND and could be the principal origin of neurocognitive problems, as well as the cognitive deficits that accompany them . The mospression . These fpression . These spression . In addipression . All the5, in order to validate the neuroprotective ability of CM extract, in vivo chronic cerebral hypoperfusion model of Alzheimer’s disease will be undertaken in our future studies.The current study demonstrated clearly that CM extract has promising antioxidant, anti-inflammatory and anticholinesterase activities. These pharmacological properties are potential targets in neuroprotection. The ex-vivo neuroprotective effects of CM extract obtained in this study could confirm its therapeutic potential in neurodegenerative disease. FurthermoreMabozou Kpemissi: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Resources, Writing - original draft.Yendube T. Kantati: Conceptualization, Methodology, Data curation.Zurina Hassan: Funding acquisition, Data curation, Formal analysis, Supervision.Kwashie Eklu-Gadegbeku: Data curation, Validation, Formal analysis, Supervision.Veeresh Prabhakar VEERAPUR: Data curation, Supervision, Validation.


Camellia petelotii (Merr.) Sealy and Camellia impressinervis Chang & Liang belong to the golden subgroup of Camellia (Theaceae). This subgroup contains the yellow-flowering species of the genus, which have high medicinal and ornamental value and a narrow geographical distribution. These species differ in their tolerance to high light intensity. This study aimed to explore the differences in their light-stress responses and light damage repair processes, and the effect of these networks on secondary metabolite synthesis. Two-year-old plants of both species grown at 300 µmol·m-2·s-1 photosynthetically active radiation (PAR) were shifted to 700 µmol·m-2·s-1 PAR for 5 days shifting back to 300 µmol·m-2·s-1 PAR for recovery for 5 days. Leaf samples were collected at the start of the experiment and 2 days after each shift. Data analysis included measuring photosynthetic indicators, differential transcriptome expression, and quantifying plant hormones, pigments, and flavonoids. Camellia impressinervis showed a weak ability to recover from photodamage that occurred at 700 µmol·m-2·s-1 compared with C. petelotii. Photodamage led to decreased photosynthesis, as shown by repressed transcript abundance for photosystem II genes psbA, B, C, O, and Q, photosystem I genes psaB, D, E, H, and N, electron transfer genes petE and F, and ATP synthesis genes ATPF1A and ATPF1B. High-light stress caused more severe damage to C. impressinervis, which showed a stronger response to reactive oxygen species than C. petelotii. In addition, high-light stress promoted the growth and development of high zeatin signalling and increased transcript abundance of adenylate dimethylallyl transferase (IPT) and histidine-containing phosphotransferase (AHP). The identification of transcriptional differences in the regulatory networks that respond to high-light stress and activate recovery of light damage in these two rare species adds to the resources available to conserve them and improve their value through molecular breeding.  Camellia oleifera which belongs to the same family as the golden subgroup of Camellia has been studied for photosynthesis because of its wide cultivation area and the development of related industries. The photosynthetic characteristics of Camellia oleifera have been measured, and it was found that the main functional leaf for photosynthesis in Camellia oleifera is the 2-year-old leaf, which has the highest chlorophyll content and photosynthetic rate  , it was found that the net photosynthetic rate of C. petelotii was significantly higher than that of C. impressinervis. C. impressinervis (204 mm-2) was 1.7-fold higher than that of C. pete-lotii (120 mm-2).Based on the results of Chai et al. wild  flowers . It prefronments . HoweverC. impressinervis than those of C. petelotii , total phenolics (TP), proanthocyanidin (PA), hydrolyzable tannin, and condensed tannin were significantly higher in C. impressinervis than the five other species. Remarkably, the flavone concentration was about 10-fold higher in C. impressinervis than in C. petelotii. Flavonoids such as flavonols, dihydro flavonoids, chalcones, and anthocyanins are the plant secondary metabolites that vary widely in concentration among plants. They have complex and diverse structures, mainly based on a C6-C3-C6 structure  Sealy and Camellia impressinervis Chang & Liang. The seedlings were to grow 8-10 leaves during the experiment.The test seedlings were 2-year-old C. petelotii leaves under gradient light intensity were carefully tested, and three light intensities of 700, 300, and 100 µmol·m-2·s-1 were set in the plant culture room for 7 days, it was found that under strong light stress (700 µmol·m-2·s-1), the thylakoid morphology is abnormal in the leaves, the antioxidant system is damaged, 300 µmol·m-2·s-1 is a relatively appropriate growth light condition and that strong light stress (700 µmol·m-2·s-1) causes damage to the photosystem of C. petelotii. , the stress phase at a light intensity of 700 (µmol·m-2·s-1), and the recovery phase at a light intensity of 300 (µmol·m-2·s-1). The chamber temperature was set to 25°C, with a humidity of 70%. The plants were treated for 5 days. Measurements were taken and leaves were harvested after 5 h of light exposure on day 2 of each treatment. Some leaves were kept fresh while others were snap-frozen in liquid nitrogen, depending on the intended use.Therefore three stress-repair trial phases were set up in the CONVIRON CG72 walk-in plant growth chamber, the initial phase at a light intensity of 300 . Pigments were extracted from fresh leaves in the dark at 24°C by grinding 0.3 g sample in 95% ethanol with quartz sand and calcium carbonate powder in a mortar and pestle until the tissue became white. After allowing it to stand for 5 min, the homogenate was filtered and diluted to 25 mL with 95% ethanol. The absorbance was determined at 665 nm, 649 nm, and 470 nm. The concentrations of Chl a, Chl b, and carotenoids were calculated according to the method of Leaf photosynthetic rate was measured by gas exchange , while Fvia exonuclease/polymerase activities. After adenylation of 3’ ends, an adaptor with a hairpin loop structure  was ligated onto the cDNA to prepare for hybridization. To select cDNA fragments of 250 bp to 300 bp in length, the library fragments were purified by magnetic bead separation . The size-selected, adaptor-ligated cDNA was subjected to uracil-specific excision  at 37°C for 15 min followed by 5 min at 95°C. Amplification by PCR was performed using a high-fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer . The PCR products were purified by magnetic bead separation  and library quality was assessed .RNA extraction, sequencing, and bioinformatic analyses were done commercially from snap-frozen leaves . Messenger RNA was purified from total RNA using oligo-dT attached to magnetic beads. Fragmentation was carried out using divalent cations at elevated temperatures in a commercial buffer , Ipswich, MA, USA). First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (NEB). Second-strand cDNA synthesis was performed using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends Clustering of index-coded samples was performed  according to the manufacturer’s instructions. After cluster generation, libraries were sequenced  to generate 150 b paired-end reads.Differential expression analysis was performed (DESeq2  in R pachttps://blast.ncbi.nlm.nih.gov/Blast.cgi), Eukaryotic Orthologous Groups (kog) (http://www.ncbi.nlm.nih.gov/COG/), Protein Families (Pfam) (http://pfam.xfam.org/), Gene Ontology (GO) (http://geneontology.org/) and KEGG pathway enrichment analysis, was done. The GO enrichment analysis was implemented through the cluster Profiler package in R. The GO terms that were differentially abundant with a corrected P-value ≤ 0.05 were considered to be significantly enriched in a set of DEGs. The KEGG database resource was used to assign high-level functions and utilities to the DEGs (http://www.genome.jp/kegg/).Case-specific analysis of differentially expressed genes (DEGs), including NCBI non-redundant (nr) (According to A) sample pre-treatmentThe samples to be tested were first killed at 105°C for 15 min, then dried at 60°C, crushed, and set aside.B) sample extractionWe took the sample to be tested and ground it to a fine powder. We weighed about 0.3 g of ground sample and performed reflux extraction with 60% ethanol twice (20 mL each time), and filtered it. The filtrates were combined and evaporated to dryness. The dry filtrate was dissolved with 60% ethanol and fixed the volume to 25 mL.C) standard curve drawing2 solution, and kept for 6 min. Then, 0.5 mL of 10% Al (NO3)3 solution was added, and kept for 6 min, and added 4 mL of 4% Al(NO3)3 and NaOH solution, with 60% ethanol to fill the volume to the marked line, and kept for 15 minutes. Finally, we determined the absorbance at 510 nm and drew the standard working curve.We precisely measured 0 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL, and 1.6 mL of rutin reference solution in a 10 mL volumetric flask, and added 60% ethanol to make the final volume of 2.0 Ml. We added 0.5 mL 5% NaNOD) sample determination2 solution, the absorbance was measured, and the rutin content was checked in the sample solution from the standard working curve, followed by calculation of total flavonoids in the sample.Accurately measured 0.5-2.0 mL of sample solution was added to a 10 mL volumetric flask. According to the method of drawing a standard working curve, after adding 0.5 mL of 5% NaNO3 standard solutions for the solvent, and the standard curve in practice linear outliers was excluded from the equation. The data acquisition system mainly included high-performance liquid chromatography   and tandem mass spectrometry  . Mass spectral data were processed using the software Analyst.The samples were ground in liquid nitrogen to dry powder. The 1.5 g of powder was put in a glass test tube and added isopropanol-water-hydrochloric acid mixed extract into it, followed by shaking at low temperature for 30 min. Then, dichloromethane was added, followed by shaking at low temperature for 30 min and centrifugation at 13000 r/min for 5 min at low temperature. The lower organic phase was removed, protected from light, and dried with nitrogen. The organic phase was reconstituted with methanol (0.1% formic acid); centrifuged at 4°C for 10 min (13000 r/min), and the supernatant was taken to 0.22 µm filter membrane. For HPLC-MS/MS detection, the extraction process was performed on an ice box at 4°C throughout the operation. Methanol (0.1% formic acid) was used to prepare different gradients of IAA, ABA, Zeatin, and GAWe took 0.5 g of leaves in different treatment groups, according to weight (g): volume (mL) ratio=1:5-10, and added 0.2 mol/L PBS buffer (PH 7.2-7.4). The homogenate was prepared under ice bath conditions, centrifuged at 2500 r/min for 10 min, and used the supernatant and diluted with PBS buffer. CAT enzyme activity was determined according to the method of Tub1.Total RNA was extracted using Trizol reagent  and used for reverse transcription . Complementary DNA was diluted 10-fold for real-time RT-PCR. The PCR cycling was done under the following conditions: 95°C for 15 s, 56°C for 30 s, 72°C for 30 s, and 40 cycles . Primers used in this study are shown in Data were processed and plotted with Excel 2010 software, and statistical and ANOVA analyses were performed with SPSS 19.0 software.C. petelotii and C. impressinervis to changes in light intensity were examined in the leaves of 2-year-old plants. To determine the time course of the light response, plants were moved to a growth chamber at 300 µmol·m-2·s-1 light for 5 days, then transferred to 700 µmol·m-2·s-1 for 5 days, followed by recovery at 300 µmol·m-2·s-1 for 5 days. SPAD values were the highest on the second day of each light treatment. At each point, SPAD values were higher for C. petelotii than for C. impressinervis (C. petelotii leaves decreased during each of the three treatments of 300 - 700 - 300 R (µmol·m-2·s-1), while carotenoid (Cr) concentration only decreased during the 300 R µmol·m-2·s-1 treatment  than after the treatment at 300 µmol·m-2·s-1  or at 700 µmol·m-2·s-1   were identified with a log2 relative expression level ≥ 1 and ≤ −1  in pairwise comparisons of samples A with d, B with e, and C with f. There were more unique DEGs between the two species during the recovery period at 300 µmol·mC. petelotii and C. impressinervis were identified by comparing sample A individually with samples B and C, and sample d individually with samples e and f, respectively  and during the recovery process  than C. impressinervis  , but the CAT activity was significantly higher in leaves of C. petelotii than those of C. impressinervis (P < 0.05). The magnitude of these changes was accompanied by changes in the expression of a large number of genes related to flavonoid synthesis during a light stress response followed by damage repair and was consistent with the GO and KEGG enrichment analysis of the DEGs, showing significant enrichment in the oxidation-reduction process and flavonoid bio-synthesis during the light treatments , abscisic acid (ABA), methyl salicylic acid (MeSA), methyl jasmonic acid (MeJA), and gibberellic acid (GA) were determined in leaves . Camellia petelotii was relatively unstressed by the changes in light regimens, with an Fv/Fm of about 0.8 in all treatments. This species seemed to be least stressed at 700 µmol·m-2·s-1, since it had a slightly higher Fv/Fm at this PAR intensity than at 300 µmol·m-2·s-1. However, C. impressinervis showed more stress responses at both 300 and 700 µmol·m-2·s-1, with an Fv/Fm value lower than 0.8, which was further dropped during the recovery phase. The differences in stress levels at low light intensity would likely cause differences in the long-term growth and development of these species.i.e., PsbA, B, C, O, Q), Photosynthetic electron transport , Photosystem I  and F-type ATPase  showed differential expression in C. impressinervis between 300 µmol·m-2·s-1 and recovery from 700 µmol·m-2·s-1 , than the initial 300 µmol·m-2·s-1 treatment. The proteins encoded by these genes include PsbA (thylakoid located D1) and PsbB (chlorophyll A binding protein CP47), PsbC (CP43 protein), which are located in the PSII reaction center  decreased the photosynthetic rate of C. impressinervis, probably by affecting the formation and stability of PSII and PSI complexes and altering electron transfer. The differences in the effects of light transitions on the photosynthetic apparatus are likely to be translated to long-term growth effects on plants, where C. petelotii would be favored in higher light environments than C. impressinervis.From the results, it seems likely that there are no significant differences in photosynthetic indices between the two species in response to light stress, while the photodamage repair capacity of Camellia spp. This finding suggests that an expansion in gene numbers explains why Camellia spp. is rich in flavonoids , the first enzyme in the flavonoid biosynthetic pathway. In contrast, over 31 PAL unigenes were identified in the avonoids . Total l-2·s-1 actually caused a reduction in the concentration of leaf flavonoids in C. petelotii, while a significant elevation was observed in C. impressinervis. During the recovery phase, the flavonoid concentration in C. petelotii increased within 2 days, and the flavonoid concentration in C. impressinervis decreased, but the concentration exceeded the level present before the higher light treatment in both species. A comparison of the transcript levels encoding the rate-limiting enzymes in the flavonoid biosynthesis pathway showed that the majority of genes were down-regulated under light stress, consistent with the decrease in flavonoid concentration during light stress repair. However, C. impressinervis showed similar levels of PAL transcripts during high-light stress and recovery phases. It is noteworthy that PAL activity is upstream of the rate-limiting enzymes, such as C4H, in the flavonoid synthesis pathway .We thank professor Wuming Qin (Guangxi University) and all members  for their helpful assistance during the research, and professor Patrick M. Finnegan  for helpful discussions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Morus alba L. Studies have shown that it has antitussive, antiasthmatic, tyrosinase and antioxidation activities. However, little is known about the metabolism of it in vitro and in vivo. In our study, an integrated strategy on the basis of UHPLC-Q-Exactive Plus Orbitrap MS and network pharmacology was established to comprehensively research the metabolic characteristic of mulberroside A for the first time. Plasma, urine, feces and liver tissues of rats in the blank group and drug group were collected after intragastric administration of mulberroside A at a dose of 150 mg/kg, and rat liver microsomes were cultured for in vitro metabolism experiment. The biological samples were processed by different methods and analyzed in positive and negative ion modes using UHPLC-Q-Exactive Plus Orbitrap MS. A total of 72 metabolites were finally identified based on the accurate molecular mass, retention time, MS/MS spectra and related literatures combined with the Compound Discoverer 3.1. The metabolic pathways were mainly hydrolysis, glucuronidation, hydrogenation, sulfation, hydroxylation, methylation and their composite reactions. In addition, a network pharmacology method was used to predict the mechanism of action of mulberroside A and its metabolites. In the end, 7 metabolites with high gastrointestinal absorption and drug-likeness and 167 targets were screened by Swiss ADME and Swiss Target Prediction. 1702 items of GO analysis and 158 related signaling pathways of KEGG were enriched using Metascape. This study established a novel integrated strategy based on UHPLC-Q-Exactive Plus Orbitrap MS and network pharmacology, which could systematically analyze the metabolism behavior of mulberroside A in vivo and in vitro of rats and provide basis for the further research of mulberroside A.Mulberroside A is a polyhydroxylated stilbene active component of  Morus alba L.  coupled with mass spectrometry (MS), especially high-resolution mass spectrometry (HRMS), has been widely used in drug metabolism analysis . UHPLC h18-Low solid-phase extraction cartridges  were purchased from Grace Davison Discovery Science .The reference standard, mulberroside A, was purchased from Chengdu Must Biotechnology Co., Ltd. , with purity greater than 98% determined using HPLC-UV analysis. Rat liver microsomes were purchased from Wuxi Xinrun Biotechnology Co., Ltd. . HPLC grade methanol, formic acid, and acetonitrile were provided by Thermo Fisher Scientific . Water was obtained from Watsons . Grace Pure SPE CSix male SD rats (200 ± 10 g) were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. . The animals were housed in a controlled environment with the temperature held at 24 ± 2 °C, relative humidity held at 60±5%, and kept on a 12 h light/12 h dark regime. After 7 days of acclimation, the rats were randomly divided into drug group (n=3) and control group (n=3). Before the experiment, all rats were fasted for 12 h with free access to water. Mulberroside A was dissolved in normal saline. The rats in the drug group were given with a dose of 150 mg/kg/d orally for 3 days. A same dose of normal saline was administered to the rats in the control group.0.5 ml of blood samples were taken from the suborbital venous plexus of rats at 0.5, 1, 1.5, 2, 4, 6, 12, and 24 h after the last administration. The blood samples collected at different times were mixed and placed in a centrifuge tube coated with sodium heparin. After that, they were centrifuged for 10 min at 3,500 rpm to separate plasma sample. Then, it was frozen in a refrigerator at -80°C. The centrifugation was performed on Centrifuge 5430R .24 h after the last administration, liver tissue was obtained and quenched in liquid nitrogen and then stored at -80°C. 1 g of liver tissue was added to 10 ml of normal saline to homogenize at 4°C and then centrifuged at 3,500 rpm for 10 min to obtain the supernatant.Two groups of rats were housed in the metabolic cage. The urine and feces were collected within 24 h after the last administration. The urine samples were frozen in the refrigerator at -80°C after centrifuged at 12,000 rpm for 15 min, and the feces samples were freeze-dried and ground into powder.2 and liver microsomes were dissolved in PBS (pH=7.4), formulated into a solution with a MgCl2 concentration of 3 mM and a protein concentration of 1 mg/ml. The solution was used to dissolve mulberroside A with a final drug concentration of 0.1 mg/ml. Drug group and blank group were set up. 900 µl of the above mixture was added to each well of the 6-well plate in the drug group while the blank group was given an equal volume of drug-negative solution. After preheating at 37°C for 5 min, 100 µl NADPH  was added to start the reaction and continue to incubate at 37°C. At 5, 10, 15, 30, 45, 60, 120, 240 min after the start of the reaction, 100 µl of the system solution was mixed with 200 µl of cold acetonitrile to terminate the reaction. Then, the supernatant was obtained after centrifuged at 20,000 rpm for 10 min.MgCl2 at 4°C. The residue was then redissolved in 300 µl methanol and centrifuged for 15 min at 20,000 rpm (4°C).The SPE method for precipitation and concentration of protein and solid residues was applied to pretreat all the biological samples. Before loading and elution, 2 g feces was added to 10 ml pure water, sonicated for 0.5 h, and centrifuged to obtain the supernatant. Then, plasma, urine, feces, and liver tissues samples (1 ml) were added to SPE cartridges pretreated with methanol (3 ml) and deionized water (3 ml), respectively. After that, the SPE cartridges were successively washed with deionized water (3 ml) and methanol (3 ml). The methanol eluent was collected and dried under N2 at room temperature. The residue was then redissolved in 300 µl methanol and centrifuged for 15 min at 20,000 rpm (4°C).In addition, plasma samples were treated with methanol (3 ml) and acetonitrile (3 ml), respectively. Then, they were centrifuged at 3,500 rpm. The obtained supernatant was dried under NAll the biological samples from the same group were merged into a collective sample for the further instrumental analysis.18 column . The flow rate was 0.3 ml/min and the injection volume was 5 µl. The column temperature was maintained at 35°C. The mobile phase consists of acetonitrile(A) and 0.1% formic acid-water (B). The gradient elution condition was set as: 0–5.0 min, 95% B; 5.0–10.0 min, 95%–70% B; 10.0–15.0 min, 70%–50% B; 15.0–32.0 min, 50%–10% B; 32.0–32.1 min, 10%–95% B; 32.1–35.0 min, 95% B.The separation was performed on Vanquish UHPLC  with a ACQUITY UPLC BEH Cm/z 80–m/z 1,200 at a resolution of 70,000 with AGC target at 3e6. The dd-MS2 data were obtained at a resolution of 17,500 with AGC target at 1e6. Mass spectrometric detection was performed in positive and negative ion modes. The ion source parameters were set as follows: sheath gas and auxiliary gas of nitrogen (purity ≥99.99%) with the flow rate of 45 arb and 10 arb; capillary temperature of 320 °C; spray voltage of 3,800/3,500 V (+/-); probe heater temperature of 320°C. The stepped normalized collision energy (NCE) was set at 15, 30 and 45. S-Lens RF Level was 50.00.The MS analysis was performed on Q-Exactive Plus Orbitrap MS  with a heated electrospray ionization (HESI) source. A high-resolution mass spectrum was acquired at full scan in a mass range of http://www.swissadme.ch/) and the targets were predicted using Swiss Target Prediction (http://www.swisstargetprediction.ch/). All genes were submitted to Metascape (http://metascape.org) for GO and KEGG analysis and Cytoscape 3.8.2 was used for the construction of metabolite-target network and metabolite-target-pathway network. The visual analysis of GO and KEGG enrichment results was performed using Bioinformatics online platform (http://www.bioinformatics.com.cn/).Thermo Scientific Xcalibur 3.1 workstation and Compound Discoverer 3.1 software were used to process and analyze the collected mass spectrometry data. The chemical formulas attributed to the selected peaks were calculated using a formula predictor by setting the parameters as follows: C [5–40], H [5–60], O [2–30], S [0–2], N [0–3] and the ring double bond (RDB) equivalent value [3–20]. The HRMS data obtained from UHPLC-Q-Exactive Plus Orbitrap MS was imported into the Compound Discoverer, and the structure information of mulberroside A was added to Compound Discoverer at the same time. Then, Compound Discoverer gave the information of possible metabolites according to the structural characteristics of mulberroside A, HRMS information and the corresponding databases. In addition, the metabolites were selected by Swiss ADME , m/z 245 [M+H-2Glu]+, m/z 227 [M+H-2Glu-H2O]+, m/z 199 [M+H-2Glu-H2O-CO]+, m/z 135 [M+H-2Glu-C6H6O2]+. Representative ions were detected in negative ion mode as follows: m/z 405 [M-H-Glu]-, m/z 243 [M-H-2Glu]-, m/z 225 [M-H-2Glu-H2O]-, m/z 215 [M-H-2Glu-CO]-, m/z 199 [M-H-2Glu-CO2]-, m/z 181 [M-H-2Glu-CO2-H2O]-, m/z 175 [M-H-2Glu-C3O2]−, m/z 157 [M-H-2Glu-C3O2-H2O]−. The mass fragmentation behavior of mulberroside A in positive and negative ion modes are shown in We summarized the mass spectrometry fragmentation behaviors of mulberroside A according to related literatures reported and the MSbstances . As showA total of 76 metabolites were finally identified based on the accurate molecular mass, retention time, MS/MS spectra and related literature reports in the plasma, urine, feces, liver tissues and liver microsomes. The chromatographic and MS data of these detected metabolites were summarized in M0 was identified as the prototype of mulberroside A with the [M-H]- ions at m/z 567.1707  in negative ion modes. The fragment ions at m/z 243, m/z 567, m/z 405, m/z 225 were detected, which were consistent with the mass fragmentation behavior of the reference substance of mulberroside A. Therefore, M0 was characterized as the prototype of mulberroside A. M27 possessed the same theoretical [M-H]- and product ions with M0, but compared with M0, it was eluted later than M0. So, M27 was deduced as isomer of mulberroside A.M1 and M21 generated [M-H]- ions at m/z 285.0607  and m/z 311.0767  respectively. The neutral loss of 176 Da (m/z 285→m/z 109 and m/z 311→m/z 135) was generated in their ESI-MS/MS spectra, indicating that their structures contain glucuronic acid (Glu A). In addition, the product ion at m/z 175 ([Glu A-H]-) was detected, which also proved the existence of Glu A. According to pubchem database (https://pubchem.ncbi.nlm.nih.gov/) and molecular formulas, M1 was identified as β-D-Glucopyranosiduronic Acid-3-Hydroxyphenyl and M21 was characterized as isomer of 6-(4-Ethenyl-2-hydroxyphenoxy)-3,4,5-trihydroxyoxane-2-carboxylic acid. They were the glucuronidation metabolites of compounds formed by the rupture of C1-α bond or C1′-β bond in mulberroside A.M2 and M6 possessed the same theoretical [M-H]- ions at m/z 583.1652  with the retention time of 7.72 and 8.20 min, respectively. They were 16 Da more massive than M0, which suggested that they might be the hydroxylation products of mulberroside A. The fragment ion at m/z 259 ([M-H-2Glu]-) was 16 Da more massive than m/z 243, providing evidence for hydroxylation reaction. The product ions at m/z 421 ([M-H-Glu]-) was formed by losing Glu. Therefore, it was speculated that M2 and M6 were hydroxylation products of mulberroside A.M7 was eluted at 8.20 min with the [M-H]- at m/z 599.1602 . It was 32 Da (2O) more massive than M0, indicating that it might be the dihydroxylation product of mulberroside A. The fragment ions involved m/z 137, m/z 599, m/z 125, m/z 437, and m/z 275. The [M-H]- ion lost Glu to form m/z 437 ([M-H-Glu]-), and lost another Glu to form m/z 275 ([M-H-2Glu]-). We first assumed that two benzene rings were each bound to a hydroxyl group. Then it was split by Chem Draw, and we found that the α-β bond of the product ion at m/z 275 ([M-H-2Glu]-) broke to form C7H5O32-, which molecular weight was exactly m/z 137. From these, it was speculated that M7 was dihydroxylation product of mulberroside A and two hydroxyl groups were bonded to the benzene rings on both sides.M5 and M29, with the molecular formula of C27H33O16, showed the same theoretical [M-H]- ions at m/z 613.1763 and they were 46 Da (CH2O2) more massive than M0. The loss of 30 Da (m/z 435→m/z 405) suggested the existence of methoxy. In addition, the product ions at m/z 243, m/z 405, m/z 567 were representative ions of mulberroside A. As a consequence, it was deemed that M5 and M29 were dihydroxylation and methylation products of mulberroside A.M9 eluted at 8.24 min and generated the [M-H]- at m/z 601.1769 . It was 34 Da (H2O2) more massive than M0, which indicated that it was hydrogenation and hydroxylation product of mulberroside A. The fragment ions at m/z 137, m/z 243, m/z 601, m/z 567, m/z 439 ([M-H-Glu]-) were observed in its ESI-MS/MS spectrum. The α-β bond of M9 was broken and then lost Glu to form m/z 137 (C7H5O3−), while m/z 243 and m/z 567 were characteristic ions of mulberroside A. Therefore, M9 was identified as vicinal diols formed by dihydroxylation at α-β bond of mulberroside A.M54, with the retention time of 10.92 min, was 58 Da (C2H2O2) more massive than M0. The fragment ion at m/z 579 was fragmented by losing of CO and H2O from [M-H]- ion, while m/z 271 was identified as C12H15O7− formed by the fracture of the bonds C-1 and α. The neutral loss of 30 Da (m/z 330→m/z 300) indicated that methoxy was contained in the structure. Hence, M54 was finally characterized as hydroxylation and acetylation product of mulberroside A.M57 was 42 Da (C2H2O) more massive than M0 with the molecular formula of C28H33O15. It was speculated that M57 was acetylation product of mulberroside A. In the ESI-MS/MS spectrum of M57, the fragment ion at m/z 463 removed Glu to form m/z 301, then lose -CH3 to form the ion m/z 286. The product ion at m/z 297 was 30 Da more massive than m/z 267 ([M-H-2Glu]-), which proved the existence of methoxy.M60 owned the [M-H]- ion at m/z 595.2017  with the retention time of 11.39 min and was 28 Da (C2H4) more massive than M0. We speculated that M60 was dimethylation product of mulberroside A. In its ESI-MS/MS spectrum, the loss of 30 Da (m/z 449→m/z 419) was detected, which confirmed our prediction. In addition, the fragment ion at m/z 297 was identified as C14H18O7− formed by the rupture of α-β bond.M67 was 2 Da more massive than M0 and generated the [M-H]- at m/z 569.1871  with the retention time of 13.13 min, which indicated that it might be hydrogenation product of mulberroside A. The product ion at m/z 407 ([M-H-Glu]-) was 2 Da more massive than that of m/z 405. Thus, M67 was ultimately characterized as hydrogenation product of mulberroside A.M4 and M28, with the [M-H]- ions at m/z 665.1384 (0.239 ppm) and m/z 665.1390 (1.156 ppm) respectively, was 98 Da more massive than M0, suggesting that they might be the hydrogenation, hydroxylation and sulfation metabolite of mulberroside A. The fragment ions at m/z 96.96 ([SO4H]-) had the highest abundance, which was a base peak, indicating that S may be contained. In addition, m/z 243, m/z 567, m/z 405 were characteristic fragments of mulberroside A. Therefore, it was speculated that M4 and M28 were hydrogenation, hydroxylation and sulfation products of mulberroside A.Metabolites M52, M59 and M70 generated the [M-H]- ions at m/z 405.1180 (-0.145 ppm), m/z 405.1177 (-0.811 ppm), and m/z 243.0651 (-0.392 ppm), respectively. M52 and M59 were 162 Da less massive than M0 while M70 was 324 Da less massive than M0. Moreover, the product ion at m/z 225 was included in their ESI-MS/MS spectra and m/z 199 was detected in the ESI-MS/MS spectra of M70. It was speculated that they were hydrolysates of mulberroside A. M52 and M59 were formed by losing a molecule of Glu while M70 was formed by losing two molecules of Glu. In the experiment of Zhaxi et al., they isolated three metabolites of mulberroside A from rat intestinal contents and feces. The three metabolites were identified as oxyresveratrol-2-O-β-D-glucopyranoside, oxyresveratrol-3′-O-β-D-glucopyranoside and oxyresveratrol according to ESI-MS spectrum, 1H and 13C-NMR spectroscopy , which was 12 Da more massive than M0. The DPI at m/z 271 was deduced as C12H15O7− formed by the rupture of α-β bond.M3, M16 and M25, with the retention time of 7.98, 8.77 and 9.29 min, possessed the same theoretical [M-H]- ion at m/z 581.1501 . They were 176 Da more massive than M52 or M59, which suggested that they may be the glucuronidation products of M52 or M59. In the ESI-MS/MS spectrum, the product ions at m/z 243 ([M-H-Glu A-Glu]-), m/z 405 ([M-H-Glu A]-), m/z 225 ([M-H-Glu A-Glu-H2O]-) were detected. Therefore, M3, M16 and M25 were identified as glucuronidation metabolites of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside.M8, detected at 8.20 min, generated the [M-H]- ion at m/z 437.1079  and was 32 Da more massive than M52 or M59, indicating that it may be the dihydroxylation metabolite of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside. The fragment ions at m/z 275 ([M-H-Glu]-) and m/z 257 ([M-H-Glu-H2O]-) were detected in its ESI-MS/MS spectrum. In addition, the fragment of m/z 137 was also detected, which was presumed to be C7H6O3− formed by α-β bond breakage.M13 eluted at 8.57 min, was deduced as hydrogenation and glucuronidation product of M52 or M59. The successive loss of 162 and 176 Da (m/z 583→m/z 421→m/z 245) indicated that Glu and Glu A may be contained. Besides, the DPI at m/z 245 ([M-H-Glu-Glu A]-) was 2 Da more massive than m/z 243 (characteristic ion of M52 and M59). Eventually, M13 was identified as hydrogenation and glucuronidation metabolite of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside. In the ESI-MS/MS spectrum of M14, the successive loss of Glu A, Glu and Glu A (m/z 759→m/z 583→m/z 421→m/z 245) was detected, which indicated that two molecules of Glu A and a molecule of Glu may be contained. So, M14 was characterized as hydrogenation and bis-glucuronidation product of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside.M31, with the [M-H]- ion at m/z 485.0748 , was 80 Da more massive than M52 or M59, which implied that it might be the sulfation product. In its ESI-MS/MS spectrum, the product ions at m/z 405 ([M-H-SO3]-) and m/z 243 ([M-H-SO3-Glu]-) confirmed the metabolite identification. Hence, M31 was tentatively characterized as sulfation product of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside.M63 was 2 Da more massive than M52 or M59, indicating that they were hydrogenation metabolites of M52 or M59. The fragment ion at m/z 201 were detected in its ESI-MS/MS spectrums, which was formed by the successive loss of Glu and CO2. In addition, the product ion at m/z 227 ([M-H-Glu-H2O]-) was also detected.M41 possessed the [M-H]- ion at m/z 435.1271 , with the retention time of 10.00 min. The DPIs at m/z 417 [M-H-H2O]-, m/z 403 and m/z 193 were generated in its ESI-MS/MS spectrum. Therefore, it was speculated that M41 was hydroxylation and methylation metabolite of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside. The fragment of m/z 403 was presumed to be formed by the loss of H2O from m/z 421 (m/z 405+16) and m/z 193 may be C11H14O3− formed by benzene ring opening.M62, M64, M66, M68 and M71, with the same molecular formula of C20H23O10, had the same theoretical [M-H]- ion at m/z 423.1286. They were 18 Da more massive than M52 or M59, suggesting that they may be hydrogenation and hydroxylation products of M52 or M59. The DPIs at m/z 405, m/z 201, m/z 263 and m/z 247 were detected, which provided evidence for the metabolite identification.M17, M24, M33, M36 and M46 were 256 Da more massive than M70 with the same theoretical [M-H]- ions at m/z 499.0541 . In the ESI-MS/MS spectra, the successive loss of Glu A and SO3  was observed. Therefore, they were eventually characterized as glucuronidation and sulfation products of oxyresveratrol.M34, M43 and M44 were tentatively identified as glucuronidation products of M70 according to the neutral loss of 176 Da (m/z 419→m/z 243). In addition, the fragment ion at m/z 175 ([Glu A-H]-) provided evidence for the existence of glucuronic acid group. M48 was 80 Da more massive than M70, with the [M-H]- ion at m/z 323.02209 , implying that it may be sulfation product of M70. The neutral loss of 80 Da (m/z 323→m/z 243) proved the existence of SO3 in its ESI-MS/MS spectrum.M50 was 2 Da more massive than M70 and it was preliminarily deduced as the hydrogenation metabolite of oxyresveratrol. It was produced by hydrogenation reduction reaction occur at the α-β bond according to the product ion at m/z 123, which was formed by the rupture of α-β bond. The fragment ions at m/z 203 ([[M-H-CO-CH2]−]-) and m/z 159 ([M-H-C3O2-H2O]−) were generated in its ESI-MS/MS spectra. Besides, the DPI at m/z 227 ([M-H-H2O]-) was also detected. M49 was 2 Da less massive than M70, indicating that it might be the dehydrogenation product of oxyresveratrol. The DPIs at m/z 197 ([M-H-CO2]-), m/z 179 ([M-H-CO2-H2O]-) and m/z 223 ([M-H-H2O]-) provided evidence for the metabolite identification. Finally, M50 was identified as hydrogenation metabolite of oxyresveratrol and M49 was characterized as dehydrogenation product of oxyresveratrol.M10, M51, M55 and M65, with the same theoretical [M-H]- ion at m/z 433.1129 , were 190 Da more massive than M70. The DPI at m/z 257 ([M-H-Glu A]-) and the continuous neutral loss of 30 Da (m/z 433→m/z 403→m/z 373) were detected, suggesting that Glu A and -OCH3 might be contained. Thus, they were eventually deduced as methylation and glucuronidation metabolites of oxyresveratrol.M11, M19 and M20, eluted at 8.43 min, 8.84 min and 8.94 min respectively, were deduced as hydrogenation and bis-glucuronidation products of M70. In the ESI-MS/MS spectra, the successive loss of 176 Da (m/z 597→m/z 421→m/z 245) indicated that two molecules of Glu A might be contained. Furthermore, the DPI at m/z 175 confirmed the existence of Glu A and the DPI at m/z 245 ([M-H-2Glu A]-) was 2 Da more massive than m/z 243 ([M-H]- ion of M70). Therefore, they were eventually identified as hydrogenation and glucuronidation products of oxyresveratrol.M12, eluted at 8.52 min, was 174 Da more massive than M70. The neutral loss of 176 Da (m/z 421→m/z 245 and m/z 417→m/z 241) was detected in both ESI-MS/MS spectra, which was suggested that Glu A might be contained in its structure. What’s more, the DPI at m/z 241 of M12 was 2 Da less massive than m/z 243 ([M-H]- ion of M70). Thus, M12 was characterized as dehydrogenation and glucuronidation metabolite of oxyresveratrol. M22 and M37, with the retention time of 9.19 and 9.82 min, were 178 Da more massive than M70. The neutral loss of 176 Da (m/z 421→m/z 245 and m/z 417→m/z 241) provided evidence for the existence of Glu A. In addition, the DPI at m/z 245 was 2 Da more massive than m/z 243. Finally, they were deduced as hydrogenation and glucuronidation product of oxyresveratrol.M15, M23 and M26 were identified as bis-glucuronidation products of M70 according to the successive loss of Glu A (m/z 595→m/z 419→m/z 243) with the retention time of 8.76 min, 9.25 min and 9.31 min, respectively. M18, M30, M32 and M35, with the molecular formula of C20H21O13S, generated the same theoretical [M-H]- ions at m/z 501.0697. The successive loss of 176 Da (m/z 421→m/z 245) and 80 Da  was detected in their ESI-MS/MS spectra, indicating that they might be the glucuronidation and sulfation products of M70. In addition, the DPI at m/z 245 was 2 Da more massive than the [M-H]- ion of M70 (m/z 243). So, they were deduced as the hydrogenation, glucuronidation and sulfation products of oxyresveratrol.M38, M47 and M58 were deduced as the dehydroxylation and glucuronidation metabolites of oxyresveratrol, with the molecular formula of C20H19O9. The DPIs at m/z 227 [M-H-Glu A]-  provided the evidence for the metabolite identification. The successive loss of 176 and 16 Da (m/z 435→m/z 259→m/z 243) was observed in the ESI-MS/MS spectrum of M39, indicating that Glu A and -OH may be contained. Therefore, M39 was identified as hydroxylation and glucuronidation product of oxyresveratrol. M40 was characterized as hydration and glucuronidation metabolite of oxyresveratrol based on the successive loss of Glu A and H2O (m/z 437→m/z 261→m/z 243).M42 and M45 were deemed to be the hydrogenation and sulfation products of oxyresveratrol while M56 was identified as dehydrogenation and sulfation metabolite of oxyresveratrol. The DPIs at m/z 245  and m/z 241  provided the evidence for the metabolite identification.M61 possessed the [M-H]- ion at m/z 271.0964 , which was 28 Da (C2H4) more massive than M70. It was presumed to be the dimethylation product of oxyresveratrol. The successive loss of 30 Da (m/z 197→m/z 167→m/z 137) provided evidence for the existence of methoxy.M69, with the [M-H]- ion at m/z 339.0176 , was eluted at 13.63 min. The neutral loss of 80 Da (m/z 339→m/z 259) was observed in the ESI-MS/MS spectrum, indicating that SO3 might be contained in its structure. Besides, the fragment ion at m/z 259 was 16 Da more massive than m/z 243, which suggested that it may be hydroxylation product of M70. Hence, it was speculated that M69 was hydroxylation and sulfation product of oxyresveratrol.in vivo and in vitro of rats were hydrolysis, hydrogenation, hydroxylation, methylation, acetylation, sulfation, glucuronidation and so on. In this paper, the product peak areas of each type of metabolic reaction were summarized, as shown in in vivo were mainly hydrolysis, glucuronidation and hydrogenation. The total peak area of hydrolysis metabolites accounted for the largest proportion. Therefore, it was preliminarily speculated that after entering the body, mulberroside A was hydrolyzed into aglycone firstly, and then a series of metabolic reactions occur. While in vitro, the most abundant compounds were hydrolysate and acetylation products of mulberroside A.The metabolic reactions of mulberroside A in vivo and in vitro of rats. (1) The metabolic reactions such as sulfation, hydroxylation and methylation were occurred on the basis of mulberroside A . In this pathway, glucuronidation reaction was undetected. (2) Mulberroside A was hydrolyzed to form oxyresveratrol-4-O-β-D-glucopyranoside (M52) or oxyresveratrol-3′-O-β-D-glucopyranoside (M59), on which metabolic reactions took place, such as M3, M8, M13, M14, M16, M25, M31 and so on. (3) Mulberroside A continued to be hydrolyzed and lose another Glu to produce oxyresveratrol (M70). Then, groups such as glucuronic acid, sulfate and methyl were bound to oxyresveratrol . Besides, β-D-Glucopyranosiduronic Acid 3-Hydroxyphenyl (M1) and isomer of 6-(4-Ethenyl-2-hydroxyphenoxy)-3,4,5-trihydroxyoxane-2-carboxylic acid (M21) were formed by the rupture of C1′-β bond. The proposed metabolic pathways of mulberroside A are shown in In this experiment, a total of 72 metabolites including mulberroside A were detected, 40 in blood, 37 in urine, 12 in feces, 9 in liver, and 10 in liver microsomes. The prototype component was only detected in rat plasma samples, which indicated that mulberroside A could be absorbed into the blood, and then a series of metabolic reactions occurred. There was no significant difference on the type of metabolites in plasma and urine, and the metabolites in feces were mainly glucuronidation, sulfation and methylation products of oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside. It was speculated that mulberroside A was metabolized in 3 pathways in vivo and in vitro metabolites of mulberroside A were mostly based on its hydrolyzed products oxyresveratrol-4-O-β-D-glucopyranoside or oxyresveratrol-3′-O-β-D-glucopyranoside and oxyresveratrol. The main metabolic reactions were hydrolysis, hydroxylation, methylation, sulfation, glucuronidation and their compound reactions. The metabolic result showed that the main forms of mulberroside A in vivo were the glucuronidation and sulfation products of its aglycone, while in vitro they were mainly the hydroxylation and hydrolysis products of its prototype. The in vitro pharmacokinetic study of mulberroside A also showed that the glucose group on mulberroside A was rapidly hydrolyzed to generate monoglycosides and its aglycone oxyresveratrol when incubated anaerobically with intestinal bacteria  and solid phase extraction method . In our 7 metabolites (N1–N7) with high gastrointestinal absorption and drug-likeness were selected by Swiss ADME. The information of the 7 metabolites is shown in p < 0.01). The top 10 clusters with their representative enriched terms (one per cluster) were selected and they are shown in The 167 targets were submitted to the Metascape for GO enrichment analysis, including molecular functions (MF), biological processes (BP) and cellular components (CC). A total of 1702 GO items were obtained finally  (p < 0.01). The result suggested that the pathways of mulberroside A metabolites were mainly involved Axon guidance, Alzheimer disease, Serotonergic synapse, NF-kappa B signaling pathway, Neuroactive ligand-receptor interaction, Epithelial cell signaling in Helicobacter pylori infection, TNF signaling pathway and so on , 167 targets and the top 20 pathways were imported to Cytoscape 3.8.2 for metabolite-target-pathway network. As a result, the metabolite-target-pathway network, with 194 nodes and 602 edges, was constructed, as shown in Studies have shown that mulberroside A has the effect of neuroprotection . From thHelicobacter pylori infection. RPS6KA5 was the key target of N2, which exerts anti-inflammatory effect by decreasing the expression of TNF-α and inhibiting the activation of NF-κB  were applied. We concluded that solid phase extraction was the most suitable method for plasma sample of mulberroside A. As a result, a total of 72 metabolites including mulberroside A were detected by UHPLC-Q-Exactive Plus Orbitrap MS. The metabolites and metabolic pathways of mulberroside A in vivo and in vitro of rats were also elaborated. The main metabolite reactions of mulberroside A were hydrolysis, glucuronidation and hydrogenation. Furthermore, the results of network pharmacology study showed that the component that exerts pharmacological effects in the body is not mulberroside A but its metabolites, which mainly include oxyresveratrol (aglycon of mulberroside A), dehydrogenation, hydrogenation, and demethylated products of oxyresveratrol.In the present study, an integrated strategy based on the UHPLC-Q-Exactive Plus Orbitrap MS and network pharmacology was firstly established to elucidate the metabolic mechanism of mulberroside A. In order to detect as many metabolites as possible, not only biological samples  were prepared in vivo and in vitro of rats more comprehensively, which would provide more theoretical basis for the study of mulberroside A. It would be conducive to better understand the metabolic action and promote research for the further potential therapeutic application of mulberroside A.The integrated strategy established in our study could elucidate the metabolic pathways and mechanism of mulberroside A