The aim of this study was to determine the grain composition and (poly)phenolic profile of pollen from Abrantes , as well as its antioxidative and antidiabetic properties, and abilities to protect human erythrocytes against induced hemoglobin oxidation, lipid peroxidation, and hemolysis. The phytochemical profile of the Abrantes’ bee pollen revealed twenty phenolic compounds, identified by high-performance liquid chromatography with electrospray ionization mass spectrometry coupled with photodiode array detection. Among them, quercetin derivatives were the most abundant. Concerning the biological potential, the pollen extract showed notable capacity for 2,2-diphenyl-1-picrylhydrazyl, nitric oxide, and superoxide radicals, as well as for inhibition of α-glucosidase action, and protection of human erythrocytes against oxidative damage. Non-cytotoxic effects regarding the NHDF normal cell line, human adenocarcinoma Caco-2, and human liver HepG2 cells were observed. The results obtained contributed to further research on modes of action related to oxidative damage and metabolic health problems, to generate deeper knowledge of potential health-promoting effects to develop novel pharmaceutical drugs, nutraceuticals, and dietary supplements. Apis mellifera, with the purpose of feeding larvae in their early stages of development [Nowadays, there is an interest in more balanced, functional, and healthier food. In this context, natural plants and their derivatives have received a lot of attention owing to their health-promoting properties with little or no adverse effects . Bee polelopment ,5.Bee pollen is constituted of about 200 biologically active substances, collected from plants and transported to the hive in the form of pollen loads . O-rutinoside) [Regarding phenolics, pollen has higher concentrations of flavonols, namely quercetin derivatives, with values ranging from 24.0 (quercetin) to 956 µg/g (quercetin 3-inoside) . In factinoside) . In geneCastanea sativa, Cytisus, Echium, Quercus and Rubus plants) which confer to this regional honey unique characteristics, including a dark color and intense flavor, while not being too sweet, it is not surprising that honey production is increasing in this region [●), nitric oxide (●NO), and superoxide (O2●−) radicals and to inhibit the α-glucosidase enzyme. Additionally, its protective effects on different cellular models were also studied: (1) human erythrocytes, studying hemoglobin oxidation, lipid peroxidation, and hemolysis induced by peroxyl species (ROO●); (2) human colon carcinoma cells (Caco-2); and (3) liver carcinoma cells (HepG2). Erythrocytes were selected not only for being the most abundant cells in vertebrates, but also for their crucial role in immune reactions and respiratory gas exchange, which in turn, makes them highly susceptible targets to oxidation and inflammatory conditions. On the other hand, Caco-2 and HepG2 cancer cells were chosen as they present many morphological characteristics of intestinal epithelium and liver parenchymal cells, respectively. For comparative purposes, the effects on primary normal human dermal fibroblasts (NHDF) were studied.Therefore, given the current interest in pollen and the existence of few studies on this natural product, the aim of this work was to determine pollen grain composition and the phenolic profile of multifloral bee pollen from Abrantes . This city is located in the province of Ribatejo and belongs to Santarém district. Considering its favorable edaphoclimatic conditions and flora variety , N-(1-naphthyl)ethylenediamine dihydrochloride, sodium nitroprusside dihydrate (SNP), and sulfanilamide were acquired from Alfa Aesar . Acetonitrile and methanol were purchased from Fisher Chemical . The remaining ones were from Sigma-Aldrich . Water was deionized using a Milli-Q water purification system . Caco-2, HepG2 and NHDF cell lines were from American Type Culture Collection .The bee pollen sample (REF: M08AG006), composed of at least 500 pollen grains, was directly provided by a local company (Colmeicentro) in Alferrarede, Abrantes region, in 2017. The sample was stored in the dark under desiccating conditions to avoid alterations until use.g for 10 min in order to separate the pollen grains. Then, the obtained sediment was again dissolved in water and centrifuged for 5 min. Finally, two aliquots of the sediment were examined microscopically at 45×, using a bright-field microscope . Each aliquot was composed of a minimum of 800 pollen grains. The results were expressed as percentages.The botanical origin of the sample was determined according to the melissopalynological method described by Louveaux, Maurizio, & Vorwohl . Brieflyv/v), ultrasonicated for 1 h and centrifuged at 2900× g during 10 min at room temperature. Then, the supernatant was evaporated under reduced pressure to complete dryness at 40 °C. The resulting concentrate residue was stored at −20 °C, and protected from light until use. The obtained extraction yield from the starting dry material was 64.5 ± 0.16%. The extractions were performed in triplicate.The extract was prepared according to Moita et al. . BrieflyThe identification of phenolic compounds from bee pollen was performed according to Gonçalves et al. . They weρ-coumaric acid. Quercetin 7-glucoside-3-O-rutinoside, quercetin derivatives 1 and 2, quercetin hexoside and quercetin acethylrhamnoside were quantified as quercetin. Kaempferol di-hexoside, kaempferol 3-O-rutinoside-O-heoxide, kaempferol acethylhexoside, myrcetin rhamno-hexoside, and myrcetin derivative were quantified as kaempferol 3-O-rutinoside. On the other hand, isorhamnetin 3-O-rutinoside and isorhamnetin acetyl hexoside were quantified as quercetin. Analyses were carried out in triplicate.Concerning phenolics quantification, 20 µL extract was injected on an LC model Agilent 1260 system , according to Gonçalves et al. . Spectra●, ●NO and O2●−, was performed spectrophotometrically through in vitro microassays using 96-well plates. Microplate reader Bio-Rad Xmark spectrophotometer  was used to measure the absorbances. The results were expressed as 25% and 50% maximal inhibitory concentration  values (µg/mL). Each experiment was performed in triplicate, and seven different concentrations were tested.The capacity of bee pollen hydroethanolic extracts to scavenge free radicals, namely DPPH● was performed according to Gonçalves et al. [The capability of bee pollen hydroethanolic extract to quench DPPHs et al. . Each we●NO were based on the work of Silva & Teixeira [3PO4) was added to each well and incubated for 10 min in a dark (blanks received 100 µL H3PO4). After this time, the absorbance was read at 560 nm. Ascorbic acid was used as positive control.The effects of bee pollen extracts in capturing Teixeira . Briefly2●− generated by the NADH/phenazine methosulfate system was done according to a previous method [β-nicotinamide adenine dinucleotide (NADH), 150 µL nitrotetrazolium blue chloride (NBT), and 50 µL phenazine methosulfate (PMS). The control was composed of phosphate buffer instead of diluted extract. After the addition of PMS, each plate was immediately collocated in the spectrophotometer and absorbance readings were collected every 10 seg over 2 min at 562 nm. Ascorbic acid was used as a positive control.The possibility of pollen extracts being able to reduce Os method . Each weα-glucosidase was determined based on a previous method [α-D-glucopyranoside (PNP-G). Control was only composed of phosphate buffer and PNP-G. The reaction was initiated by the addition of 25 µL α-glucosidase in each well, followed by a period of incubation of 10 min of incubation at 37 °C. Finally, and after that time, the absorbance of 4-nitrophenol released from PNP-G was read at 405 nm. Acarbose was used as positive control. Three experiments were performed in triplicate.The inhibition of s method . Seven d3EDTA vacuum tubes, in accordance with the protocol approved by the Health Ethical Commission of Cova da Beira Hospital Centre (no. 25/2018) without the need for written informed consent of the patients. Therefore, erythrocytes from blood collected in the early morning already processed by the hospital and without any need for more studies. Then, they were isolated according to Gonçalves et al. [g for 5 min at 4 °C. This procedure was repeated three more times, and the resulting supernatant from each centrifugation was always discarded. The number of cells (cells/mL) and viability  were obtained by the Trypan blue exclusion method. The suspensions of isolated erythrocytes were kept on ice until use.Venous human blood was collected from randomized patients of Cova da Beira Hospital Centre  by antecubital venipuncture into Kno. 25/208 without● species in human erythrocytes, six different concentrations were prepared and dissolved in PBS. Five experiments were performed in duplicate in 48-well microplates.For evaluating the protective effects of pollen hydroethanolic extract against ROO6 cells/mL, final density). Controls and blanks were performed by replacing the sample with 100 µL PBS. The mixture was then incubated in a water bath for 30 min at 37 °C, under slow agitation. After the incubation, 200 µL AAPH  prepared in PBS were added to the media (except in the blank), followed by another incubation using the same conditions described above for 4 h. After that time, the entire volume of the reaction mixture was transferred to conic Eppendorf tubes and centrifuged at 1500× g for 5 min at 4 °C. Finally, 300 µL supernatant was placed in a 96-well plate and the absorbance was measured at 630 nm. Quercetin was used as a positive control.The capacity of pollen extracts to prevent methemoglobin oxidation induced by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) was based on a previous work . Six dil6 cells/mL, final density) and incubated at 37 °C for 30 min, under slow agitation. After this period, 200 µL tert-BHP  was added to the media, and each well was further incubated under the same conditions for 30 min. Finally, the entire content was transferred to conic Eppendorfs, mixed with 250 µL trichloroacetic acid 28% (w/v) to incite protein precipitation, and centrifuged at 16,000× g for 10 min at 18 °C. Then, the supernatant was collected in conical test tubes (with screw caps) and 125 µL thiobarbituric acid 1% (w/v) was added to generate TBARS. The resulting mixture was heated for 15 min at 100 °C in a water bath. Finally, test tubes were cooled in ice and the absorbance was taken at 532 nm. Quercetin was used as a positive control.The ability of the extract to avoid lipid peroxidation was indirectly monitored through the formation of thiobarbituric-acid-reactive substances (TBARS) . Six dif6 cells/mL, final density) and 100 µL of each concentration. Controls and blanks were composed of PBS. In a first step, the reaction mixture was incubated at 37 °C in a water bath for 30 min, under slow agitation. After this period, 200 µL AAPH  was added to the mixture (except in the blank), followed by another period of incubation for 3 h, using the aforementioned conditions. Lastly, the entire volume was then transferred to a conic Eppendorf and centrifuged at 1500× g, for 5 min at 4 °C. After that, 300 µL supernatant was transferred to 96-well plates and the absorbance was read at 540 nm. Quercetin was used as a positive control.The capacity of pollen extracts to avoid erythrocyte lysis induced by AAPH was evaluated by monitoring the release of hemoglobin to the media after membrane disruption, according to a previous work . Briefly2 culture flasks and incubated at 37 °C in a humidified atmosphere of 5% CO2. Caco-2 and HepG2 cells were cultured as a monolayer in DMEM containing 10% FBS, 1% penicillin/streptomycin, while NHDF cells were cultured in RPMI 1640 medium supplemented with FBS (10%), l-glutamine (2 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (10 mM), sodium pyruvate (1 mM), and penicillin/streptomycin (1%). The medium was changed every 2 days. After a few passages, cells were washed twice with PBS and detached by gentle trypsinization. Then, viable cells were counted and suitably diluted in the adequate complete culture medium  [v/v) DMSO ) and plates were again incubated for a further 24 h. At the end of this period, the metabolic activity of cells was evaluated concerning their ability to reduce yellow tetrazolium 3--2,5-diphenyltetrazolium bromide (MTT) to a blue formazan product and to ensure membrane integrity. Cell culture conditions and procedures were consistent through all assays. All studies were conducted when cells were in the logarithmic growth phase.To understand the possible cytotoxicity effects, Caco-2, HepG2, and NHDF cell lines were cultured in 75 cmF cells) . After cAfter 24 h of cell treatment with different concentrations of pollen extracts, the medium was rejected, and each well was washed with PBS. Then, cultures were incubated with 200 µL MTT solution (dissolved in appropriate serum-free medium) for 4 h at 37 °C. After this period, the medium containing MTT was removed, and the purple crystals of formazan were dissolved in DMSO. The absorbance of the different solutions was read at 570 nm. Cell proliferation values were expressed as percentages from the relative absorbance measured in the treated wells versus control wells . A totalIn order to evaluate the release of the stable cytosolic enzyme lactate dehydrogenase (LDH) into the medium, after 24 h of cells incubation with the extracts, 50 µL culture medium was placed to a 96-well plate. Then, 200 µL NADH (252.84 mM) and 25 µL pyruvate 14.99 mM) were added to each well, according to Gonçalves et al. [4.99 mM wp < 0.05. To determine the contribution of the total phenols, on their antioxidant activity, Pearson’s correlation coefficients were calculated. All analyses were carried out using Graph Pad Prism Version 6.01 .Statistical comparison was determined using one-way ANOVA and the means were classified by Tukey’s test at a 95% level of significance. Differences were considered significant for Echium plantagineum, Cistus ladanifer and Quercus species were the predominant ones, with relative abundance percentages of 26, 23, and 19%, respectively. Other pollen types detected include Rubus spp., Eriobotrya spp., Raphanus raphanistrum, Crepis capillaris, Anthyllis spp., Trifolium spp., Cytisus spp., Lavandula stoechas, Calendula arvensis, Salix spp. And Lotus criticus. The obtained results are in line with the wide plant diversity observed in the Abrantes region. In fact, this area has a mixed forest of Echium, Castanea sativa and Quercus spp., Leguminosae scrubs (Cytisus spp.), and Rubus plants, which often colonize abandoned agricultural fields [Echium plantagineum pollen grains [Lavandula and Echium types  [Eucalyptus spp. [C. sativa is the most abundant grain type in Bragança samples [Rubus, C. sativa, and Cytisus species are the predominant pollen grains in Spanish honeys [Arecaceae, Asteraceae spp., and Brassicaceae grains [As expected, grains from different plant species were identified, which allows the pollen sample to be classified as heterofloral  Figure . Particul fields . These pl fields ,9. Partin grains , while hctively) . On the  samples . RegardiIn this study, twenty phenolic compounds were tentatively identified by the interpretation of their fragmentation patterns obtained from mass spectra and by comparison with literature. Particularly, three hydroxycinnamic acids and seventeen flavonols were detected, as listed in m/z 179, 342 and 135, which indicates the presence of a caffeic acid and the consequent decarboxylation of this moiety [Three hydroxycinnamic acids were found , exhibiting a molecular ion at ompounds ,16. Otheompounds ,14,16.In accordance with the literature, it is possible to state that hydroxycinnamic acids, even in vestigial amounts, increase the antioxidant potential of bee-pollen products, essentially due to their CH = CH-COOH group .Seventeen different flavonol glycosides were analyzed, including seven quercetin glycosides , five kaempferols , two myricetins (peaks 6 and 10), and three glycosides of isorhamnetin .m/z 463 or 609 and fragment ion at m/z 300/301, which corresponds to quercetin aglycon losses [Seven isomers of quercetin were identified. The majority displayed molecular ions at n losses ,14,15,16n losses ,13,14,16m/z 447 and 285, respectively, which are characteristics of this compound, as previously reported [Additionally, five kaempferol derivatives were found. Most of them showed molecular ions and fragment ions at reported ,14,15,16reported .m/z 316 and 271, which suggest a myricetin aglycon losses [Castanea sativa [Two myricetin compounds were also identified. Both showed fragment ions at n losses . Myricetn losses ,14 and aa sativa .m/z 623 (519 for isorhamnetin acetyl hexoside) and a fragment pattern at m/z 315, which is coherent with isorhamnetin aglycon losses [Finally, three forms of isorhamnetin were detected, exhibiting a molecular peak at n losses ,13,14. TThe abundance of flavonols in pollen, namely quercetin derivatives, is in accordance with previous reports, being considered the main reason for the biological potential exhibited by this matrix, including abilities to capture free radicals and reactive species, chelate metals, inhibit cytochrome P450 action, and increase antioxidant defenses ,29.●. This revealed an ability to reduce the radical in a dose-dependent manner .We decided to perform a general screening of the antioxidative effects of the pollen extract against DPPHt manner A, exhibi●NO and O2●−. Although these radicals are products of normal cellular metabolism, their combination promotes the generation of more radicals, including peroxynitrite, which in turn oxidizes low-density lipoprotein and causes irreversible cell damage [●NO and O2●− species in a dose-dependent manner  .Additionally, we also tested its capacity against l damage . The extctively) B,C. Even●NO and O2●− species. Regarding DPPH● potential, pollen collected on Parque Nacional da Peneda of Gerês  revealed more effectiveness in scavenging DPPH● (IC50 = 5.87 µg/mL) [50 values of 428.6 and 6.09 µg/mL, respectively) [50 = 860 µg/mL, respectively) [50 values ranging from 7.5 and 109.0 mg/mL, depending on the country) [As far as we know, this is the first report concerning the pollen potential to scavenge 7 µg/mL) , as wellctively) . On the ctively) . Besidescountry) . The obt● experiment, caffeoyl hexose , quercetin 7-glucoside-3-O-rutinoside , quercetin derivative 1 , quercetin acetyl rhamnoside , and also with total phenolic compounds and total flavonoids . On the other hand, regarding ●NO scavenging potential, high correlations were found between this and caffeoyl hexose , quercetin 7-glucoside-3-O-rutinoside , quercetin derivative 1 , quercetin acetyl rhamnoside , and total phenolic compounds . These results are not surprising, since it was already reported that the CH = CH-COOH group presented by caffeoyl hexose, together with the catechol group on the B ring, the double bound and 4-oxo-group on the C ring, and the several hydroxyl groups found in quercetin derivatives make these compounds powerful natural antioxidants [● and ●NO antioxidant assays . Even so, negative correlations were found between the both experiments; isorhamnetin acetyl hexoside and quercetin hexoside . Concerning O2●− scavenging potential, mild correlations were found between this assay, isorhamnetin acetyl hexoside , quercetin 3-O-rutinoside , and quercetin hexoside , while negative correlations were verified between this experiment, total phenolics , flavonols , quercetin 7-glucoside-3-O-rutinoside , and quercetin derivative 1 , and also with DPPH●  and ●NO  assays. The obtained correlations are in accordance with others works [In fact, the antioxidant activities exhibited by pollen are mainly due to its richness in phenolics, which have well-known abilities due to their ease of transferring hydrogen atoms or electrons to free radicals and reactive species, neutralizing them . This heoxidants . Furtherrs works ,32.Nevertheless, it is also important to consider the presence of other natural antioxidant compounds, such as vitamins, organic acids, and carotenoids not determined in this work, whose presence also increase pollen’s biological potential .α-glucosidase activity, an important enzyme involved in carbohydrate digestion. In this study, this activity was dependent on the concentration, revealing an IC25 score of 1192.71 ± 8.14 µg/mL (25 = 113.81 ± 1.00 µg/mL).The pollen hydroethanolic extract also exhibits the capacity to inhibit 14 µg/mL D. Howeve50 values of 4510 and 600 µg/mL regarding α-amylase and α-glucosidase inhibitory activities, respectively. Comparatively to other bee products, propolis hydroethanolic extracts  already showed ability to reduce baker’s yeast α-glucosidase action, and rat intestinal sucrase and maltase enzymes  [The antidiabetic effects of pollen have already been described by Daudu et al. , who con2 µg/mL) . Besides2 µg/mL) . Further2 µg/mL) .β-cells from oxidative damage, incentive insulin production [α-glucosidase activity was already described, revealing inhibitory effects of 91% at 200 µM [O-rutinoside and α-glucosidase inhibitory action .Indeed, phenolics have already been proven to protect pancreatic oduction , and intoduction . The menoduction ,38. In p50 score of 311.50 ± 1.37 µg/mL (50 = 2.61 ± 0.15 µg/mL). Furthermore, it also prevented lipid peroxidation and hemolysis in a concentration-dependent manner, exhibiting 25% inhibitory concentration values of 277.03 ± 2.52 and 103.48 ± 2.23 µg/mL, respectively , and between lipid peroxidation, caffeoyl hexose , quercetin rutinoside , and quercetin acetyl rhamnoside . Furthermore, notable correlations were also described regarding the O2●− scavenging potential of pollen, anti-hemolytic effects , and between hemoglobin oxidation, lipid peroxidation , and hemolysis . Mild correlations were verified among hemoglobin oxidation and O2●− assay . Even so, negative correlations were obtained between hemoglobin oxidation and quercetin derivative 1 , and between hemolysis, total phenolics , total flavonols , quercetin 7-glucoside-3-O-rutinoside , quercetin derivative 1 , DPPH● , and ●NO experiments . Concerning lipid peroxidation, negative correlations were obtained with isorhamnetin acetyl hexoside  and quercetin hexoside . The obtained correlations reinforce previous results which report that the antioxidant ability of phenolics is strongly influenced by the catechol residue, and number and position of hydroxyl groups, and less by the glycosylation pattern [Indeed, phenolic compounds, mainly due to their hydroxyl groups and liposolubility, can penetrate the cytoplasm of erythrocytes, and hence scavenge free radicals before they can attack erythrocyte membranes, enhancing their integrity and preventing oxidative injury ,13,39. Bnd 37 µM , and avond 37 µM . Moreovend 37 µM . These f pattern . In factα-glucosidase activity [As already mentioned before, it was already reported that pollen has antidiabetic properties and is able to prevent lipid peroxidation ,34,35. Tactivity .tert-Butyl hydroperoxide. Furthermore, Oyarzún et al. [Considering other studies, Sousa et al.  verifiedn et al.  reportedGiven the current interest regarding natural products, the present study provides new insights about Abrantes multifloral bee pollen. This regional pollen was portrayed here for the first time and showed notable antioxidant capacity, inhibition of α-glucosidase enzyme action, and protection of human erythrocytes against hemoglobin oxidation, lipid peroxidation, and hemolysis. These health-promoting activities are strongly correlated with phenolic content, highlighting the presence of quercetin derivatives. Furthermore, and although non-anticancer effects were observed, the results obtained herein, namely regarding its antidiabetic action, prove that pollen may be used safely as an antidiabetic agent and incorporated in new pharmaceutical drugs, nutraceuticals, and dietary supplements. The obtained data, together with the verified correlations, can be extrapolated to other pollens with similar compositions. Notwithstanding, further investigation, namely in vivo studies, should be conducted to assess the full biological potential of this bee derivative and to reveal a safe dosage.


The average crystallite sizes estimated by the Rietveld method of LMO/GO nanocomposites were in the range of 18–27 nm. The electrochemical performance was studied using CR2013 coin-type cell batteries prepared from pristine LMO material and LMO modified with 5%wt. GO. Synthesized materials were tested as positive electrodes for Li-ion batteries in the voltage range between 3.0 and 4.3 V at room temperature. The specific discharge capacity after 100 cycles for LMO and LMO/5%wt. GO were 84 and 83 mAh g−1, respectively. The LMO material modified with 5%wt. of graphene oxide flakes retained more than 91% of its initial specific capacity, as compared with the 86% of pristine LMO material.In this work, a facile, wet chemical synthesis was utilized to achieve a series of lithium manganese oxide (LiMn The discovery of new innovative materials as well as adding new functionalities to well-known materials using various “bottom up” and “top down” techniques is highly important from the practical point of view for various applications, including the energy sector ,4,5,6,7.2O4, LMO) is recognized as an attractive candidate for a positive electrode material in lithium ion batteries and supercapacitors [2) or lithium nickel (LiNiO2) oxides [+), especially at elevated temperature regions of 50–60 °C, which limits the use of LMO in commercial LiBs [3+ -> Mn4+ + Mn2+ followed by Mn2+ dissolution into the commonly used standard liquid electrolyte [2O4 synthesis, fine, homogeneous, and non-agglomerated powders with small particles and outstanding electrochemical characteristics are typically obtained. A one-step hydrothermal approach without thermal treatment to synthesize LMO/graphene nanosheets (GNS) has been proposed by B. Lin et al. [−1 of initial discharge capacity at a 0.5 C rate, while after 200 charging and discharging cycles, 75.6% of initial capacity remained [−1 at 1 C, 50 C, and 100 C rates, respectively [2O4 with graphene by A. Li and co-authors [−1 of initial discharge capacity, while the after 100 charging and discharging cycles capacity retention rate was 96.2% [2O4 with graphene led to the enhanced electrochemical performances due to the establishment of fast Li+ channels and improved structural stability of the LMO material.Cubic lithium manganese oxide  oxides ,21,22. Hial LiBs ,24,25. Tctrolyte ,24,25. Tctrolyte ,39,40,41ctrolyte ,39,40,41n et al. . Their an et al. . K.-Y. Jn et al.  used then et al. . Y. Chenn et al.  obtainedremained . Hybrid remained . The obtectively . The pre-authors . The graas 96.2% . All theas 96.2% ,44,45,462O4 with graphene oxide flakes. Contrary to other studies, our technique is relatively fast, non-toxic, and cost-effective. The synthesis was conducted without the use of advanced and expensive equipment, such as autoclave used for the hydrothermal or solvothermal synthesis. After the surface modification of LMO material with GO flakes, no destruction of the LMO structure was observed. All synthesized materials were characterized extensively by a number of methods, including X-ray powder diffraction (XRD), Raman spectroscopy, and scanning and transmission electron microscopy . Electrochemical tests were performed for pristine LMO and LMO modified with 5%wt. of graphene oxide.The main challenge in the battery technology is to achieve the highest capacity but it has yet to be reached as expected. In the present study, we have tried to modify the materials to address this issue. Herein, we present a facile, wet chemical approach based on a simple, wet chemical, low-temperature process of surface modification of LMO grains using graphene oxide flakes. A lithium-manganese oxide powder was first synthesized using a modified sol-gel method ,23,24,252O4 powder using citric and acetic acids as chelating agents. The synthesis description of LMO material was demonstrated in our earlier work [2O suspension GO was added. This part of the synthesis was performed for 4 h under constant magnetic stirring at room temperature to obtain a homogenously dispersed suspension. Then, the EtOH-H2O solution was slowly evaporated at 60 °C for 12 h. Afterward, the lithium manganese oxide with graphene oxide (1–5%wt. GO) samples were air-dried overnight at 150 °C. LMO/1–5% GO fine powders were reached after grinding all materials in agate mortar. The flowchart of facile, wet chemical synthesis is presented in A modified sol-gel method was applied to create nanocrystalline LiMnier work ,23,24,25ier work ,48,49,50XRD = 1.542 Å) radiation source was used to perform the structural analysis in order to identify the crystal structure, determine the crystallite sizes, and measure unit cell parameters of as-synthesized LMO/1–5%wt. GO samples. The XRD patterns in the range of 15° ≤ 2θ ≤ 60° were registered with a step size of 0.002° and acquisition time of 3 s per step. The average size of crystallites d was determined from the linewidths of XRD peaks using the Scherrer Formula (1) in its simplest form, assuming that XRD peak broadening was solely dependent on crystallite size [K is a shape factor between 0.9 and 1.1, λCuKα is the incident X-ray wavelength (here CuKα = 1.542 Å), β is the full width at half-maximum (FWHM) of the selected peak, and θ is the Bragg’s angle of the peak.The structural properties of LMO/1–5%wt. GO materials were investigated using powder X-ray diffraction (XRD) and Raman spectroscopy at room temperature. A SIEMENS D500 diffractometer  equipped with a Cu Kα  equipped with a 532 nm emission line of Nd:YAG laser. The surface morphology and particle size of synthesized LMO/1–5%wt. GO powders was examined by using a Carl Zeiss CrossBeam Auriga  scanning electron microscope (SEM). The powders were also analyzed using a FEI Tecnai F20  transmission electron microscope (TEM), operating at 200 kV accelerating voltage. TEM specimens were prepared by dispersing sample powders on commercial holey carbon-coated TEM copper grids.6-EC:DMC (1:2). Every cell was cycled using the constant current mode in a potential range between 3.0 V and 4.3 V at room temperature using VMP3 Bio-Logic Science Instruments .The electrochemical characterization was performed using CR2013 coin-type cells assembled in a dry argon-filled glove box. The cathodes were fabricated by mixing 10 mg of active electrode with 3 mg of teflonized acetylene black (TAB-2) as a conducting binder. The mixture was pressed onto stainless steel mesh. The average thickness of each cathode was 18–20 µm and Li foil was used as a counter electrode separated by a porous propylene film (ADVANTEC GB-100R). The cathodes were dried at 150 °C for 5 h under vacuum. The electrolyte used was 1 M LiPF0 = 8.24762 Å (V0 = 561.03 Å) for lithium manganese oxide (ICDD PDF-35-0782). The lattice parameter values measured for LMO/GO composites are presented in 2O4 powders was not changed after modification with graphene oxide. Raman analysis for all LMO/1–5%wt. GO powders  [−1) [Expectedly, the crystal structure of all LiMn powders B showed 00 cm−1) ,24,25 as−1) [−1) . The cha2O4 spinel crystal structure has five optical Raman active modes  and T2g(2) are attributed to large oxygen movements and very small Li ion displacements, respectively [1g shoulder peak at 581 cm−1 and the following weaker band at 491 cm−1 are assigned to large oxygen movements and very small Li ion displacements, respectively [2g(1) phonon, which is assumed to be the vibrations of Li sublattice, is associated with the visible band at 385 cm−1 [The LiMnodes see  [23,24,2ectively ,51,52. Tectively ,51,52. T385 cm−1 ,51,52.−1 (see −1. The most intense G line was located owing to a doubly degenerate E2g phonon mode active for sp2 carbon networks [1g symmetry) of “honey-combed” carbon rings, or in a graphitic structure originated in the disorder due to limited crystallite size and defects [−1 between the D and G bands [−1), and corresponded to the 2D peak at 2696 cm−1 [−1 could be induced by disorder or assigned to the sp2 and sp3 C-H stretching vibrations [−1 [−1, decreased. This suggests that the graphene oxide covered the surface LMO particles. This was further confirmed by scanning electron (SEM) as well as transmission electron microscopy (TEM) analyses.Seven bands were registered in the graphene oxide structure at 1150, 1350, 1490, 1595, 2696, 2945, and 3180 cm−1 see  [44,53,5networks . D line  defects . The pho G bands . A secon696 cm−1 . A combibrations . The oveions [−1 . In addi2O4 powders with various contents of graphene oxide. On the powder LMO grains, crystal growth planes are clearly visible with a size distribution between 100 and 600 nm, which agglomerate into larger particles. While the morphology of LMO grains in all these samples remains practically the same, additional features in a form of folded thin coating layers (some marked with yellow arrows) can be noticed in the samples with the addition of GO flakes. Such GO flakes attached to LMO particles are better visible in TEM images obtained from these samples , oxygen (O), and carbon (C) elements was performed for sample LMO with 5%wt. GO to confirm the presence of a carbon structure on LMO surface grains. The results are depicted in 2O4 material, was observed. At 20 mA g−1 current density, in the first and second discharge process, the pristine LMO and with 5%wt. GO electrode showed a specific capacity of 98, 91, and 95, 90 mAh g−1, respectively. The specific capacities of the cells discharged at 20 mA g−1 current rate after the 10th and 50th cycles were: 93, 87, and 87, 84 mAh g−1, for LMO and LMO/5%wt. GO, respectively. After 100 cycles of charge/discharge processes, both electrodes revealed similar values of specific capacity of 84 and 83 mAhg−1 for pristine LMO and modified with 5%wt. of graphene oxide, respectively.Two materials were chosen for electrochemical performance testing, namely pristine LMO and LMO modified with 5%wt. graphene oxide. The galvanostatic charge-discharge profiles of the positive electrodes were measured using constant current mode in a potential range between 3.0 and 4.3 V at room temperature see . The sam−1 current rate) LMO/5%wt. GO material retained 91.2% of first discharge capacity, while the pristine LiMn2O4 showed only 85.7%.−1 current density are summarized in The coulombic efficiency of both analyzed electrodes was at the same level between 99% and 100%. The achieved cyclability tests at 20 mA g2O4 grains, graphene oxide (GO) material, which contained various functional groups such as C-O hydroxyl and epoxy, O-C=O carboxyl, carbonyl functional groups, as well as C=C/C-C in aromatic rings, was utilized [−1 of initial discharge capacity, with 75.6% of initial capacity remaining after 200 charging and discharging cycles [−1, respectively [2O4 with graphene using a precipitation synthesis [−1, with a capacity retention rate of 96.2% after 100 charging and discharging cycles [The surface modification with graphene oxide led to improved cyclability of LMO powder. Despite the observed slightly lower values of specific capacity in all cycles for the LMO/5%wt. GO material as compared with the pristine LMO, the decrease in capacity in each cycle was smaller. As a result, a positive effect of surface modification of LMO grains with graphene oxide flakes was observed. To modify the surface of LiMnutilized ,49. Depeutilized  used a lg cycles . S.-M. Bg cycles . At 1, 5ectively . A. Li aynthesis . The inig cycles . The obtg cycles ,44,45,46g cycles ,44,45,462O4 crystallizes in the cubic spinel structure with the Fd3m space group. The LMO structure remained intact after the surface modification by GO coatings. Raman analysis showed a typical spinel feature of LiMn2O4 and relatively sharp D and G peaks of graphene oxide flakes. The electrochemical characterization was performed using a CR2013 coin-type cell configuration. The specific discharge capacity after 100 cycles for LMO and LMO/5%wt. GO were 84 and 83 mAh g−1, respectively. The LMO material modified with 5%wt. of graphene oxide flakes retained more than 91% of its initial specific capacity, as compared with 86% measured for pristine LMO material. In the future, it is planned to modify such a series of samples to produce LMO with graphene oxide in the reduced form. It is expected that after the reduction of certain functional groups of graphene oxide, a better electrochemical performance can be achieved while maintaining a high level of coulombic efficiency.In this work, we reported a facile, wet chemical synthesis of surface modification of LMO grains with graphene oxide flakes. The XRD results showed that LiMn


The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.  Coronaviridae family in the order Nidovirales (Coronaviruses (CoVs) are single-stranded positive-sense (+) RNA viruses that constitute the ovirales . CoVs caovirales ,3. Severovirales . The preovirales  and makeCoVs have very large (approximately 30 kb) genomes that encode non-structural proteins (nsps) required for viral gene expression and replication. Upon infecting human cells, the SARS-CoV-2 RNA genome is translated to produce a long polyprotein that is cleaved into nsps 1 through 16 by the viral protease nsp5  and is t2  domain  comprising the NiRAN signature  domain . The NiRignature . The NiRovirales .Arteriviridae family of nidoviruses, RdRp self-NMPylates in vitro with a clear preference for UTP as a substrate and the Mn2+ ion as a cofactor  enzyme from the cofactor . Such ancofactor . Substitresidues . These rclusions .in vitro (i) the Asn2 residue was critical for modification; (ii) Asn1 could be substituted with Ala or Ser with a modest loss of reactivity; and (iii) the presence of even one additional N-terminal Ala residue abolished nsp9 NMPylation  studies of SARS-CoV-2 RdRp ,21 reveain vitro . A findiin vitro ,17,22, sin vitro . Consistin vitro , a smallin vitro . nsp9 anin vitro , as a siin vitro . MutatioPylation . In supplication . Furtherlication . Strikin pathway . In the 2+ and Mn2+, is largely insensitive to the identity of the natural NTP and can utilize nucleotide analogs such as remdesivir triphosphate. We also show that NMPylation is reversible in the presence of pyrophosphate. Nucleotide analogs that lack the triphosphate moiety and pyrophosphate analogs bisphosphonates inhibit the forward reaction, suggesting a starting point for identification of NiRAN inhibitors.Our findings that noncognate NTPs and nucleoside analogs modulate RdRp activity suggested that the RdRp and NiRAN active sites  could be allosterically connected . Testingnsp7/8/9/12 genes were codon-optimized for expression in Escherichia coli and synthesized by GenScript and subcloned into standard pET-derived expression vectors under control of the T7 gene 10 promoter and lac repressor, as described previously (Plasmids used in this study are shown in eviously . The derE. coli BL21 (DE3) cells . Cells were grown in lysogenic broth (LB) with kanamycin (50 μg/ml). Cells were cultured at 37°C to an OD600 of 0.6–0.8 and the temperature was lowered to 16°C. Expression was induced with 0.1 mM isopropyl-1-thio-β-d-galactopyranoside  for 18 hours. Induced cells were harvested by centrifugation (6000 × g), resuspended in lysis buffer , 1 mM Phenylmethylsulfonyl fluoride , 5 mM β-ME, 10 mM imidazole), and lysed by sonication. The lysate was cleared by centrifugation (10 000 × g). The soluble protein was purified by absorption to Ni2+-NTA resin , washed with Ni-buffer A , and eluted with Ni-buffer B . The eluted protein was further loaded onto a desalting column  in desalting buffer  to remove imidazole. The fusion protein was treated with TEV protease (for pIA1364) or SUMO protease (for pIA1414) at 4°C overnight. The sample was supplemented with 20 mM imidazole and passed through Ni2+-NTA resin. The untagged protein was loaded onto a Sephacryl S-100 HR column  in desalting buffer. Peak fractions were assessed by SDS-PAGE and Coomassie staining. Purified protein was dialyzed into storage buffer , aliquoted and stored at −80°C.The expression and purification of nsp7/8/12 and nsp12 mutants are described in our previous study . All purA total of 75 viral genomes, representing alpha-, beta-, gamma- and deltacoronaviruses, were fetched from Uniprot reference Proteomes (version 2021_02) . Multipl2, 2 mM DTT) for 5 min, then 25 μM GTP and 10 μCi [α32P]-GTP  were added to start the reaction. After a further 10 min incubation, the reaction was mixed with 4× NuPAGE™ LDS Sample Buffer .All NMPylation assays were carried out at 37°C. For standard NMPylation assay, 0.5 μM nsp12 and 5 μM nsp9 were incubated in NMPylation buffer , 25 μM GTP, and 10 μCi [α32P]-GTP were added to start the reaction. For nucleotide competition assays, NTPs, NTP analogs, and pyrophosphate were used at 0.5 mM. After incubation of nsp12/9 for 5 min in NMPylation buffer, the competitor NTPs , GDP , GMP , Inosine-5′-Triphosphate , ppGpp , GpCpp , Remdesivir triphosphate , and pyrophosphate  was added together with 25 μM GTP and 10 μCi [α32P]-GTP to start the reaction. After a further 10 min incubation, the reactions were stopped with LDS Sample Buffer as above.To assess the competition of i can reverse the NMPylation reaction, 0.5 μM nsp12, 5 μM nsp9, 25 μM GTP and 10 μCi [α32P]-GTP were incubated in NMPylation buffer for 15 min, then 0.5 mM PPi was added. To determine which active site of nsp12 is responsible for the de-NMPylation activity, 0.5 μM His-tagged nsp12, 20 μM nsp9, 50 μM GTP, and 10 μCi [α32P]-GTP were incubated in NMPylation buffer (2 mM DTT in the buffer was replaced by 2 mM β-mercaptoethanol) for 20 min. Dynabeads  were added to remove the His-tagged nsp12, followed by adding 0.5 μM nsp12 variants and 0.5 mM PPi. Samples were quenched at indicated time points and analyzed by electrophoresis.To determine if PP32P]-GTP were added to start the reaction. Reactions were performed for 10 min.0.5 μM nsp12 and 5 μM nsp9 were incubated with different concentrations of Risedronate  or Foscarnet  in the NMPylation buffer for 5 min, then 25 μM GTP and 10 μCi -GTP, which supports efficient NMPylation  and an extended nsp9 with two additional residues at –1 and –2 (GSNnsp9), identical to that used in (Nnsp9 but not to GSNnsp9 by the wild-type (WT) nsp12  increased from 0.01% at 0.2 μM GTP to 1% at 200 μM GTP , approximately 25% of nsp9 was GMPylated . This co0 μM GTP .et al. , we repeated our assays in the presence of nsp7, nsp8, and an RNA scaffold under conditions that support robust RNA extension by SARS-CoV-RdRp  of nsp9 in the viral life cycle remains to be elucidated. Substitutions of nsp9 residues that interact with nsp12  abolish Nidovirales  or the GTP analog AT-9010 that binds to AS2  or to nsp9 modification by an NTP analog (e.g. by ITP or RTP). To evaluate the second possibility, we used mass spectrometry. Our results show that, as reported by Slanina et al. , or hydrolysis assisted by the PPi-bound Mg2+ ion. In cellular RNA polymerases, diverse small molecules and accessory proteins can deliver Mg2+ to the active site to stimulate the cleavage of the nascent RNA . In the presence of 0.5 mM PPi, we observed rapid disappearance of the labeled nsp9 , necessitating a more detailed analysis with additional variants of AS1 and AS2 residues.In nsp12, two active sites mediate NMP transfer. A model in which NMPylated nsp9 serves as a primer for RNA synthesis implies that nsp9 binds to AS1 and positions the NMP for extension . Thus, bo Figure . We founo Figure , ruling o Figure , it was i-induced de-GMPylation of Nsp9 using thin layer chromatography  and Risedronate (Ris), as representative non-nitrogenous and nitrogenous bisphosphonates, respectively. Fos inhibits viral DNA polymerases, including HIV reverse transcriptase . VSV encodes a giant 2100-residue L protein, which contains RdRp, nucleotidyl transferase, and methyl transferase modules (ϵ2)-pRNA intermediate, L transfers the pRNA moiety to GDP to yield GpppA-RNA  and clearly communicate aspects of research methods that might be expected to restrict the applicability of their results.All data that support the findings of this study are available from the corresponding author upon request.gkab677_Supplemental_FileClick here for additional data file.


Metformin is an antidiabetic drug used for the treatment of diabetes and metabolic diseases. Imbalance in the autonomic nervous system (ANS) is associated with metabolic diseases. This study aimed to test whether metformin could improve ANS function in obese rats. Obesity was induced by neonatal treatment with monosodium L-glutamate (MSG). During 21–100 days of age, MSG-rats were treated with metformin 250 mg/kg body weight/day or saline solution. Rats were euthanized to evaluate biometric and biochemical parameters. ANS electrical activity was recorded and analyzed. Metformin normalized the hypervagal response in MSG-rats. Glucose-stimulated insulin secretion in isolated pancreatic islets increased in MSG-rats, while the cholinergic response decreased. Metformin treatment normalized the cholinergic response, which involved mostly the M3 muscarinic acetylcholine receptor (M3 mAChR) in pancreatic beta-cells. Protein expression of M3 mAChRs increased in MSG-obesity rats, while metformin treatment decreased the protein expression by 25%. In conclusion, chronic metformin treatment was effective in normalizing ANS activity and alleviating obesity in MSG-rats. There iMonosodium L-glutamate (MSG) treatment during neonatal life produces cardiometabolic dysfunction in rodents. Rats and mice experience obesity, dyslipidemia, glucose intolerance, insulin resistance, hyperinsulinemia, hypertension, and ANS impairment, among other hallmarks, when they reach adulthood , 8. Thes®, Curitiba, PR, Brazil). Experiments were approved by the Ethics Committee for Animal Experimentation (CEUA) of the State University of Maringá.During all experimental protocols, male Wistar rats were kept at a temperature of 23 ± 2°C, a light/dark cycle of 12 h, and had free access to water and a standard rodent chow diet ; Sigma-Aldrich, Germany] in the cervical area during the first five days of life .To evaluate the effect of chronic metformin treatment in the metabolism of MSG-rats, the animals were divided into two groups during weaning. The MSG-metformin group received metformin hydrochloride  dissolved in water at a dose of 250 mg/kg of bw using a gavage once a day from 21 to 100 days of age. The MSG-water group (control) received water. Compared with other studies, the metformin dose used in the present work had no detectable toxic effects in the animals , 14. TweUnder ketamine and xylazine anesthesia , a silicone cannula was implanted into the right jugular vein of the animals from both groups and stabilized on the dorsal region of the neck. The cannula was previously treated with heparin diluted in saline solution  to avoid blood clots. Twenty-four hours after the surgery and after 12 h of fasting without anesthesia, a glucose load (1 g/kg bw) was infused into the animals’ vein through the cannula. Blood samples (350–400 µl) were collected immediately before the glucose load (time 0) and at 5, 15, 30, and 45 min after glucose administration.To study the role of the mAChR on glycemic levels, 5 min before glucose infusion, an intraperitoneal injection of acetylcholine (ACh), a cholinergic physiological agonist , or atropine, a cholinergic non-selective antagonist , was administered to another batch of animals from both groups.®, Belo Horizonte, MG, Brazil). The measurements were performed in a single assay.To verify whether the cholinergic response was related to M3 mAChR function, the effects of a selective M3 mAChR antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide , were assessed in both experimental groups. The untreated rats received saline solution (0.9% of NaCl). Plasma obtained from blood samples was stored at −20°C for subsequent determination of glycemia and insulinemia. Glucose concentrations were measured by the glucose oxidase method, using a commercial kit   15 and /10 min, pH 7.4)] containing -N′-(2-ethanesulphonic acid)], 0.6%, Sigma-Aldrich®, St. Louis, MO, USA), was injected into the common bile duct. The pancreas swelled rapidly due to the collagenase solution and was excised and incubated in a glass beaker for 17–18 min at 37°C. The suspension was then discarded and washed with HBSS in three continuous washes. Islets were collected with the aid of a stereomicroscope. At least three rats from three different litters were used for each experimental procedure in each group.Islets were isolated by collagenase digestion of the pancreas, as previously described . At 101 −1) for 60 min in 1 ml of normal Krebs–Ringer solution  at pH 7.4 containing 5.6 mmol l−1 glucose. The Krebs–Ringer solution was gassed with  to maintain a pH of 7.4. To study the insulinotropic effect of glucose, the islets were incubated for an additional 60 min in Krebs–Ringer solution containing glucose at concentrations 5.6 and 8.3 mmol l−1 after pre-incubation.Isolated islets were pre-incubated with baseline glucose concentration (5.6 mmol l−1 glucose or 8.3 mmol l−1 glucose plus 10 μmol l−1 ACh in the presence of neostigmine (10 μmol l−1). Neostigmine is an inhibitor of acetylcholinesterase and avoids ACh degradation in the isolated pancreatic islets, ensuring a cholinergic effect.To study mAChR function, the islets were incubated for 60 min in Krebs–Ringer solution containing 8.3 mmol l−1), ACh (10 μmol l−1), and neostigmine (10 μmol l−1), we used either Atr  or one of the selective antagonists for the M1 (pirenzepine-PZP: 100 mmol l−1), M2 (methoctramine MTT: 1 mmol l−1), and M3 (4-DAMP: 100 mmol l−1) mAChR subtypes.Additionally, to study the function of the insulinostatic and insulinotropic mAChR subtypes, another batch of isolated islets was incubated. In addition to glucose (8.3 mmol l−1), neostigmine (10 μmol l−1), and ACh at different concentrations: 1; 10; 100, and 1,000 μmol l−1. Insulin secretion was determined based on the concentration in the supernatant of the incubated solution and insulin levels were measured by radioimmunoassay (RIA) with a gamma counter . Human insulin was used as a standard, and an anti-rat insulin antibody (Sigma-Aldrich) and 125I-labeled recombinant human insulin (PerkinElmer) were used in detection of insulin. The intraassay and inter assay coefficients of variation were 12.2 and 9.8%, respectively, for insulin. The limit of detection was 0.006 ng/ml. The measurements were performed in a single assay.To study the insulinotropic response of pancreatic islets to high cholinergic signal stimulation, another batch of isolated islets was incubated with glucose .At 101 days of age and after a 12-hour fasting period, rats from each experimental group were anesthetized with thiopental (45 mg/kg bw) in a batch, and a longitudinal surgical incision was performed on the anterior cervical region. Under a dissection microscope, the left superior branch of the vagus nerve, close to the trachea, was isolated and placed over a silver electrode inside a Faraday cage to avoid any electromagnetic interference, as previously described .After 12 min of vagus nerve electrical activity recordings, the sympathetic branch nerve of the superior cervical ganglia was dissected, and the electrode was placed under the sympathetic branch nerve. Firing rates were obtained as described for the vagus nerve.2, 1; and EDTA, 10; plus, Triton X, 1%, v/v) containing a protease inhibitor cocktail (Roche®). The islets were then centrifuged at 12,000 rpm for 5 min at 4°C. The total protein content was determined using a BCATM Protein Assay Kit  and a microplate reader . The samples were treated with Laemmli sample buffer , 40%; and 0.5 mol l−1 Tris, pH 6.8, 0.5%; plus, deionized water and bromophenol blue). Total protein extracts (40 µg) from the pancreatic islets were separated using 10% SDS-PAGE at 90 V for 120 min. The proteins were transferred from the gel to a nitrocellulose membrane using the TransBlot® Semi-Dry Electrophoretic Transfer Cell  and blocked with 5% skim milk in Tween-Tris-buffered saline  for 90 min under continuous shaking. The blotted membranes were incubated overnight at 4°C with rabbit anti-M3mAChR  polyclonal primary antibody at 1:1,000 dilution, followed by incubation with peroxidase-conjugated anti-rabbit antibodies at 1:5,000 dilution . The antibodies were diluted in buffer . Immunoreactive proteins were visualized with ECL  and a scanner . The bands were quantified by densitometry using Image J 1.4 software . β-Actin protein content  was utilized for normalization.The M3mAChR protein content in the isolated pancreatic islets from 101-day-old rats was determined by western blotting. In total, 300 islets from both experimental groups were frozen at −80°C and subjected to posterior sonication  in lysis buffer , followed by Bonferroni test and Student’s t-test. Data are presented as mean ± SEM, and p values of less than 0.05 were considered statistically significant. Analyses were carried out using GraphPad Prism, version 6.0, for Windows .As shown in The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) and Homeostasis Model Assessment of β-cell function (HOMA-β) in the MSG-metformin rats decreased  when compared to that in the MSG-water rats. Chronic metformin treatment significantly reduced insulin levels , thereby preventing the development of insulin resistance.in vivo results of intraperitoneal administration of mAChR agonist and antagonist in metformin-treated and untreated animals. ACh caused a decrease of 18.8% in the glycemic area under the curve (AUC) values in the MSG-water rats during ivGTT, compared to animals that did not receive ACh (p <0.0003). The MSG-metformin rats presented a reduction of 14.6% in the glycemia AUC values compared to MSG-metformin-ACh , as shown in As shown in As expected, chronic metformin treatment did not change glucose-induced insulin secretion (GIIS) (Atr inhibited the ACh insulinotropic effect on GIIS in the islets of both groups. The decrease in insulin secretion was 58.8% in MSG-water and in 44.5% in MSG-metformin rats (p <0.0001). Likewise, 4-DAMP and PZP decreased insulin secretion by 61.5 and 53.7% (p <0.0001) in MSG-water rats, respectively, and by 49.5 and 49.3% in MSG-metformin rats, respectively (p <0.002). In contrast, MTT enhanced the insulinotropic effect of ACh by 24.0% in the islets from MSG-water rats and induced a 7% enhancement of the potentiating effect of ACh in the islets from MSG-metformin rats , the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Paraná Science Foundation (Fundação Araucária) and Science without Borders Program (Programa Ciencias Sem Fronteiras). This study was funded by grant number 475765/2013-3 (CNPq) and AUX-PE-PNPD 2396/2009 (CAPES).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.


In vivo, suppressing the MALT1 expression or its proteasome activity effectively reduced the size of the subcutaneous tumor in nude mice. Mechanistically, miR-375 and miR-365a-3p were identified to inhibit NF-κB activation via targeting MALT1. Overall, our results highlight that a novel regulatory axis, miRNA-MALT1-NF-κB, plays a vital role in the progression of CRC and provides novel and hopeful therapeutic targets for clinical treatment.Colorectal cancer (CRC) is a malignant tumor with the second highest morbidity and the third highest mortality in the world, while the therapeutic options of targeted agents remain limited. Here, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), known as the upstream of the NF-κB signaling pathway, was identified to be highly upregulated in CRC tumors and cell lines. Furthermore, the downregulation of MALT1 or inhibition of its proteolytic function by MI-2 suppressed the cell proliferation and migration of CRC cells.  Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths worldwide. In 2020, there were surprisingly about 1.9 million new cases of CRC and more than 935,000 deaths all over the world. Moreover, the incidence rate of early-onset CRC is rising by 1%–4% per year , which iThe NF-κB pathway has a broad role in various cellular responses involving immunity, inflammation, and cancer. Its activation is closely associated with all cancer hallmarks, especially in enhancing cell proliferation and metastasis. Targeting inhibitors, such as proteasome inhibitors and non-steroidal anti-inflammatory drugs (NSAIDs), have been applied in CRC for chemotherapeutic resistance and the prevention of recurrence , whereasMicroRNAs (miRNAs) are a kind of small non-coding RNAs with 18–25 nucleotides in length. They function as post-transcriptional repressors by binding to the 3’ untranslated region (UTR) of target mRNAs. In various types of cancers, miRNA expressions are altered to promote or suppress tumorigenesis and tumor development . So far,via epigenetic regulation or its activity.In this study, we discovered that MALT1 was highly expressed in CRC and promoted its malignant progression. Our study revealed that downregulated MALT1 or the inhibition of its protease activity with MI-2 inhibited cell proliferation and migration through activating the NF-κB pathway. Meanwhile, miR-365a-3p and miR-375 were identified as the upstream regulators of MALT1. Taken together, these results demonstrated that MALT1 acted as an oncogene in CRC, which would be a promising therapeutic target The CRC tissue samples were obtained from 58 patients who were diagnosed and underwent surgery from January 2010 to December 2011 in Jiangmen Central Hospital, Affiliated Jiangmen Hospital of Sun Yat-sen University. A written informed consent to use the surgical samples has been signed by all the patients, and the study was approved by the Institutional Review Board of Nanfang Hospital affiliated to Southern Medical University. Collected samples were fixed by paraformaldehyde and embedded in paraffin.pH = 6.0) and quenched for peroxidase activity with 3% hydrogen peroxide for 20 min to retrieve antigens. After being treated with 10% goat serum, the slides were incubated overnight with antibodies at 4°C. PBS washing was followed by incubation with goat anti-rabbit IgG for 1 h and then stained with 3,3-diaminobenzidine. When all sections were dehydrated and sealed, we selected images with an inverted microscope (ZEISS). Immunohistochemistry was performed with the following antibodies: Ki67 , MALT1 , p-p65 , fibronectin , N-cadherin , E-cadherin , vimentin  Snail1 , and Snail2 .Paraffin-embedded tissue was cut into 5 μm thick slices, dewaxed with xylene, and hydrated with ethanol. Then, the slides were heated at 95°C in a 0.01 M citrate buffer (http://www.ncbi.nlm.nih.gov/geo/). In GSE21510, patients were divided into the normal group and tumor group according to the type of tissue resected in the surgeries. The expression of MALT1 is compared by the content of corresponding mRNA. Meanwhile, in GSE17536, patients were divided into high expression group (85%) and low expression group (15%) depending on the protein expression of MALT1, so that we were able to judge the relationship between MALT1 and prognosis by comparing the time of disease-free survival.The mRNA microarray datasets  and miRNA microarray datasets  were obtained from the Gene Expression Omnibus (GEO) database . All cell lines were cultured in Dulbecco’s modified eagle medium  supplemented with 10% fetal bovine serum , 100 μg/ml streptomycin, and 100 units/ml penicillin. They were all placed in a humidified incubator with 5% COTotal RNA was extracted with TRIzol Reagent . After gDNA was removed, 500 ng of total RNA was reversed with Evo M-MLV RT Kit with gDNA Clean for qPCR II AG11711 (Accurate Biology) according to the manufacturer’s instructions. Real-time PCR was performed using PerfectStart Green qPCR SuperMix (TransGen Biotech) on a LightCycler 96 Detection System (Roche). GAPDH was served as an internal reference gene, and the primers used for qPCR in this study were as follows: MALT1: F, TGGAAGCCCTATTCCTCACTACC; R, CATGACACCAG-TAGGTTCCTTGG, GAPDH: F, GATATTGTTGCCATCAATGACC; R, AGCC-TTCTCCATGGTGGTGAAGA, miR-218-5p: F, TTGTGCTTGATCTAACCATGT; miR-338-3p: F, TCCAGCATCAGTGATTTTGTTG; miR-365-3p: F, TAATG-CCCCTAAAAATCCTTAT; miR-375-3p: F, TTTGTTCGTTCGGCTCGCGTGA; and U6: F, CGTTCACGAATTTGCGTGTCAT. The reverse primer used in the qPCR of miRNA was the mRQ 3′ primer supplied with the microRNA first-strand synthesis and miRNA quantitation kits (Takara).β-actin , GAPDH , p-p65 , MALT1 , A20 , RELB , Fibronectin , N-cadherin , E-cadherin , Vimentin  Snail , secondary antibodies’ anti-mouse IgG-HRP , and anti-rabbit IgG-HRP . The data were acquired by Tanon 5200 system (Tanon Science) with Luminata Forte Western HRP substrate (Millipore).Total protein was extracted using a cell lysis buffer  mixed with a protease inhibitor cocktail on ice, and protein concentration was determined by the Bradford method. SDS-polyacrylamide gel electrophoresis was use to separate the desired proteins. Subsequently, proteins were transferred to PVDE membranes and blocked with 10% milk powder solution. Membranes were placed in a primary antibody overnight at 4°C and then incubated with a secondary antibody at room temperature for 2 h. Immunoblotting was performed with the following antibodies: 6 cells per well, and the cells were transfected with plasmid  and siRNA for 6 h. After transfection, cells were seeded into 96-well plates at 1,000 cells per well. CCK-8 was added to each well according to the instructions, and after waiting for 2 h of reaction, the OD value at 450 nm was measured with a microplate reader.HCT116 and SW480 were seeded into six-well plates at a number of 1 × 10For the colony formation assay, transfected HCT116 were seeded into 6-well plates at 500 cells per well and transfected SW480 were seeded into 6-well plates at 500 cells per well. All cells were cultured in DMEM supplemented with 10% FBS for 14 days. After 14 days, the colonies were fixed with 3.7% formaldehyde permeabilized with pure methanol and stained with 0.1% crystal violet. The ability of colony formation was determined by counting the number of stained colonies.The siRNA (RIBOBIO) used in this study were as follows: siMALT1_1: sense, CCGGAGAUAAUAAUGUGUGdTdT; antisense, CACACAUUAUUAUCUCCG-GdTdT, siMALT1_2: sense, CUACGAUGAUACCAUUCCAdTdT; and antisense, UG-GAAUGGUAUCAUCGUAGdTdT. The miRNA mimic and inhibitor (RIBOBIO) used in this study were as follows: miRNA-365-3p mimic: sense, AGGGACUUUUGGGGGCA-GAUGUG; antisense, CACAUCUGCCCCCAAAAGUCCCU, miRNA-365-3p inhibitor: sense, CACAUCUGCCCCCAAAAGUCCCU, miRNA-375-3p mimic: sense, UUUGUUCGUUCGGCUCGCGUGA; and antisense, UCACGCGAGCCGAACG-AACAAA; miRNA-375-3p inhibitor: sense, UCACGCGAGCCGAACGAACAAA.6) transfected with MALT1 or siMALT1 were seeded to the top migration chambers (Millipore) with 200 μl FBS-free DMEM, and 750 μl of DMEM with 10% FBS was added to the lower chamber. After 30 h, the upper chambers were washed with PBS, fixed with 3.7% formaldehyde, permeabilized with pure methanol, and stained with 0.1% crystal violet. Photographs were recorded under an inverted microscope (ZEISS), and the number of cells was then counted with ImageJ.HCT116 and SW480 cells , glass coverslips were placed in primary antibody dissolved in 3% BSA overnight at 4°C and then incubated for 1 h with specific fluorescence-conjugated secondary antibodies in the dark. 4′,6-diamidino-2-phenylindole was used to stain the cells on glass coverslips. Photographs were collected under an inverted microscope (ZEISS).For the immunocytochemistry assay, transfected HCT116 were seeded on glass coverslips in 12-well plates at 2.4 × 104) was seeded into 24-well plates, co-transfected with NF-κB-Luc reporter vector , MALT1 and siMALT1. About 48 h later, luciferase intensity was measured using a dual-luciferase reporter assay kit , and the expression of each component was analyzed according to the instructions.HCT116 and SW480 (8 × 106 cells in 50 μl DMEM) transfected with siNC or siMALT1 were subcutaneously inoculated into the scapular region of nude mice. The body weight and tumor volume [largest diameter × 2/2] were measured daily, and after 7 days, a mixture of DMEM (50 μl) with siNC or siMALT1 (12 μl) and LIPO 2000 (12 μl) was injected into the tumor location every 2 days. Tumors were removed immediately after the mice were sacrificed on Day 21 and frozen at −80°C to prepare for the following experiments.For the xenograft experiments, BALB/c nude mice  were adopted from Guangdong Medical Laboratory Animal Center. As mentioned earlier, HCT116 cells  were subcutaneously inoculated into the scapular region of nude mice . Three days later, MI-2 and dimethyl sulfoxide (DMSO) dissolved in PBS were injected intraperitoneally at a dose of 25 mg/kg body weight in a volume of 10 μl/g body weight every 2 days. Mice were killed and the tumors were removed after 14 days. All procedures were approved by the Southern Medical University Animal Care and Use Committee. All animal studies were conducted in accordance with institutional guidelines.For the t-test, paired t-test, log-rank test, and one-way ANOVA were used for the analysis of statistical difference between two groups in IBM SPSS statistics 25. p < 0.05 was considered statistically significant.The experimental results were expressed as mean values ± SD. A two-tailed Student’s n = 123) compared with adjacent tissues (n = 25) (n = 22) had a worse prognosis compared with those with low MALT expression (n = 123) (p < 0.001)  database (GSE21510). The data demonstrated that MALT1 expression was significantly increased in CRC tissues ((n = 25) . Anothern = 123) , which r< 0.001) . The pro< 0.001) . In lighin vitro.In order to identify the role of MALT1 in CRC malignancy, we transiently transfected HCT116 or SW480 cells by siRNA and an expression plasmid, and the protein levels of MALT1 were determined by immunoblotting . The CCKMetastasis is a vital hallmark of carcinomas progressing to higher pathological grades of malignancy. Therefore, to further investigate the effect of MALT1 on the malignancy of CRC cells, we performed transwell assays to test the migration ability of CRC cells after transfection. The results showed that the migration abilities in both HCT116 and SW480 cells were significantly suppressed after MALT1 was silenced by siRNA transfection . In addiin vivo, we established a subcutaneous tumor model. HCT116 cells were transfected with two different siRNAs targeting MALT1, and 1 × 107 cells with MALT1 knockdown or negative control were injected subcutaneously into the two flanks of nude mice, respectively. Compared with the negative control group, the tumor volumes in the MALT1 knockdown group were significantly decreased in both two siRNAs (in vitro observations, the staining intensity of the proliferation marker Ki67 was much lower in xenografts derived from the MALT1 knockdown group than those in the control group (in vitro results (To evaluate the pro-oncogenic function of MALT1 o siRNAs . Tumors o siRNAs . In accool group . As for  results .in vivo. Three days after injection, mice were randomly divided into two groups, the DMSO and MI-2 groups. The results showed that the tumor volumes in the MI-2 group were significantly reduced compared to the DMSO group (in vivo.Meanwhile, HCT116 cells were injected subcutaneously to identify the effect of MI-2 SO group . ConsistSO group . Taken tIn vivo experiments also demonstrated that MALT1 knockdown or its protease activity inhibition led to a decrease of phosphorylated p65 in mice subcutaneous tumor tissues (To investigate the downstream molecular mechanism of MALT1 promoting CRC malignancy, we detected the activation of the NF-κB pathway, which has been reported as the downstream. The phosphorylation of p65 is a hallmark of NF-κB activation, which results in p65 nuclear translocation and further transcriptionally activates the target genes. Given that, we examined the expression of phosphorylated p65 by immunoblotting, and the results showed that the downregulation of MALT1 inhibited the phosphorylation of p65, whereas the upregulation of MALT1 increased it . In vivo tissues . To veri tissues .To gain a better understanding of the complete regulatory axis of MALT1-NF-κB, miRcode and GEO databases were carried out to identify a number of miRNAs targeting MALT1. Through miRcode database prediction, 97 miRNAs probably targeted MALT1 . Among tvia the NF-κB pathway (CRC is the third commonly diagnosed cancer and the world’s second most deadly cancer in 2020 . Althoug pathway . Indeed, pathway . Here, iNF-κB has been found over 30 years as a rapidly inducible transcription factor, which has been confirmed a crucial role in tumor malignancy. NF-κB is directly bound to the receptors of TNF or IL-1, leading to the upregulation of anti-apoptosis genes, such as BCL-XL . Besidesin vivo and inhibited MALT1 proteolytic activity and NF-κB activation in vitro (in vitro, which put up a promising target agent for CRC.It is explicit that MALT1 activates the NF-κB pathway through the CBM complex in lymphoma. After the activation of TCR or BCR, CARMA1 is recruited to the plasma membrane, followed by BCL10 and MALT1; then, the CBM complex is assembled. This complex activates the IKK complex through ubiquitination, which further phosphorylates IκB, and its degradation activates the NF-κB pathway. MI-2 is a small molecule inhibitor that directly binds to MALT1 and suppresses the protease function, which is irreversible . DSS-indin vitro . In our via targeting MALT1, which provided a new insight of CRC therapy.Nowadays, more and more miRNAs have been identified as biomarkers and potential therapeutic targets of cancers. The five miRNAs in our study have also been identified as crucial mediators of CRC, and all of them were downregulated in CRC, while only miR-365a-3p and miR-375 have been confirmed to directly suppress NF-κB activation . miR-365via NF-κB activation. The inhibition of MALT1 proteolytic function or silencing its expression suppressed the proliferation and metastasis of CRC cells through NF-κB activation. Moreover, miR-375 and miR-365a-3p inhibited NF-κB activation by targeting MALT1 in CRC. Our research unveiled that MALT1 acted as an oncogene in the progression of CRC and provided a novel therapeutic target for CRC clinical treatment.Taken together, our study demonstrated that MALT1 promotes CRC progression


Combretum indicum leaf extract (CILEx), and explored its in vitro antioxidant and in vivo antidiabetic effects in a Long–Evans rat model. After a one-week intervention, the animals’ blood glucose, lipid profile, and pancreatic architectures were evaluated. UPLC-QTOF/ESI-MS fragmentation of CILEx and its eight docking-guided compounds were further dissected to evaluate their roles using bioinformatics-based network pharmacological tools. Results showed a very promising antioxidative effect of CILEx. Both doses of CILEx were found to significantly (p < 0.05) reduce blood glucose, low-density lipoprotein (LDL), and total cholesterol (TC), and increase high-density lipoprotein (HDL). Pancreatic tissue architectures were much improved compared to the diabetic control group. A computational approach revealed that schizonepetoside E, melianol, leucodelphinidin, and arbutin were highly suitable for further therapeutic assessment. Arbutin, in a Gene Ontology and PPI network study, evolved as the most prospective constituent for 203 target proteins of 48 KEGG pathways regulating immune modulation and insulin secretion to control diabetes. The fragmentation mechanisms of the compounds are consistent with the obtained effects for CILEx. Results show that the natural compounds from CILEx could exert potential antidiabetic effects through in vivo and computational study.This research investigated a UPLC-QTOF/ESI-MS-based phytochemical profiling of  Diabetes mellitus (DM) is a major public health concern associated with many debilitating health conditions. It is a complex and chronic metabolic disease in which the body’s ability to produce or respond to insulin is impaired. It was reported that about 451 million people aged 18–99 years were afflicted by diabetes in 2017, with 5 million deaths recorded worldwide. The prevalence is expected to rise to 693 million by 2045  in 2–C14H22O] at m/z 353.16 by removal of a fragment C14H22O (Path 2). The compound leucodelphinidin did not show any molecular ion peak corresponding to m/z 322.07. However, the molecular ion produced an ion [M–H–2H2O] in m/z 285.03 upon the expulsion of one H radical and successive removal of two H2O molecules. Furthermore, the molecular ion resulted in a daughter ion [M–C2H2O2–H-2OH] (Path 2) at m/z 229.02 upon the removal of a C2H2O2 fragment by homolytic fission, followed by successive expulsion of one H radical and two OH radicals. Azedarachin C did not give any peak at m/z corresponding to molecular ion M+. However, it produced a fragment [M–CH3–2OH–3H2]  radicals, followed by the expulsion of three H2 molecules. The other fragment [M–C16H17O4–OH–3CH3∙] in 16H17O4, and the successive removal of one OH radical and three CH3 radicals at m/z 251. On the other hand, the molecular ion underwent cleavage to afford a daughter ion [M–C16H24O6–C4H4O–H–OH–CH3] in m/z 173 by removing two large fragments C16H24O6 and C4H4O, followed by the expulsion of one H radical, one OH radical, and one CH3 radical. The compound 3-o-benzoyl-2-o-deoxyingenol shows (M + 1) and (M + 2) peaks at m/z 437.23 and 438.23, respectively. A fragment [M–OH–2H2] 6D (Path 1) at m/z 415.20 was observed due to the successive removal of one OH radical and two molecules of H2. Further double cleavage (Path 2) of the compound -dihydrocostic acid methyl ester-1-o-β-d glucopyranoside revealed (M + 1) and (M + 2) peaks at m/z 461.23 and 462.23, respectively. Furthermore, the molecular ion cleaved to create a fragment ion [M–C4H5O2] at m/z 375.19 in 4H5O2 fragment. m/z 472.70, even when a fragment for this compound was identified. Importantly, the LCMS produced molecular weight does not befit with the molecular formula which makes the current fragmentation proposal ambiguous for future research. Finally, arbutin displays an (M + 1) peak at m/z 273.09, which upon successive removal of 4OH radicals can result in a daughter ion [M + 1 − 4OH]  peak at 568.4235, which undergoes a fragmentation to give a peak at 2OH–3H2] C  with 205 target proteins . The top 10 significantly enriched terms in the biological process (BP), molecular function (MF), and cellular component (CC) categories were selected, according to Benjamini–Hochberg corrected p-values < 0.05. A total of 61 significant BPs are listed in GO enrichment analysis of interacted target proteins  that act with arbutin was performed using DAVID  level is higher in both type 1 and type 2 diabetes, playing prominent role in the development and progression of diabetic complications. The administration of alloxan causes sudden and drastic insulin secretion in the presence or absence of glucose. As a consequence, total suppression of beta cells occurs by inhibiting the glucose-sensing glucokinase enzyme. Thus, beta cells fail to recognize the glucose levels in the blood. Alloxan also produces ROS and superoxide free radicals, leading to the rapid damage of the islets of beta cells . ElevatiStudying with a plant extract, plant-based product, or any formulation thereof requires the evaluation of their safety profiles; this is particularly important for unknown plant extracts or compounds. The acute oral toxicity test in animal models helps select the safe dose that could be extrapolated in human clinical trials. However, different pharmacokinetic behavior could still be observed in humans than in animals. It has been reported that different pathophysiological disorders related to the gastrointestinal tract and blood in animals have similarities with those of humans . Thus, tC. sericeum and C. acutum  × 100, where A is the absorbance of the control (DPPH solution without the sample), and B is the absorbance of the DPPH solution in the presence of the sample (extract/ascorbic acid). Then, % scavenging was plotted against concentration, and ICAcute oral toxicity testing of CILEx was performed on Long–Evans rats, according to OECD-423 guidelines (acute toxic class method), with slight modifications. The animals were overnight fasted, providing only water. Two groups of three rats each were used for this study. Group I received a single oral dose of CILEx (500 mg/kg body weight), and Group II received a single oral dose of CILEx (1000 mg/kg body weight). After the oral administration of CILEx, animals were observed individually at least once in the first 30 min, and periodically over the first 24 h, with special attention given during the first 4 h, for 10 consecutive days. All observations were systematically recorded for each animal. The animals were observed for gross behavioral, neurological, and autonomic effects. Additional conditions such as tremors, convulsions, salivation, diarrhea, lethargy, sleep, coma, and lethality were also observed. The effective therapeutic dose was calculated as one-tenth of the median lethal dose using the arithmetic method of Karber G in association with the Hodge–Sterner scale (LD50 > 2.0 g/kg) (58).Normal control (I): Normal rats received saline water only.Diabetic control (II): Non-treated diabetic rats .Positive control (III): Alloxan-treated diabetic rats  + glibenclamide Treatment group (IV): Alloxan  + CILEx Treatment group (V): Alloxan  + CILEx Sixteen Long–Evans rats (average body weight 92 ± 9 g) of both sexes were used for the induction of diabetes. Diabetes was induced in overnight-fasted rats by a single intraperitoneal (IP) injection of alloxan monohydrate (150 mg/kg). Two days after alloxan injection, fasting blood glucose levels of all of the animals were recorded from tail vein blood using a portable glucometer , and rats with plasma glucose levels of >7.5 mmol/L were confirmed for the study. Treatment with CILEx was started after 48 h of alloxan injection. The plant sample CILEx, standard glibenclamide, and saline were administered with the help of feeding cannulas. Fasting blood glucose estimation was carried out on days 3, 5, and 7 of the study. The animals were grouped as follows:After 7 days of treatment, animals were fasted for 12 h and their blood glucose levels were measured. The animals were then anaesthetized using diethyl ether and euthanized by decapitation. Blood was collected in a dry test tube from cardiac vessels using a disposable syringe via the heart puncture method , centrifAfter euthanizing the animals, pancreases of two animals from each group were excised and stored in 10% buffered formalin solution after washing with normal saline water. The pancreas was washed, dehydrated with alcohol, and cleared with xylene, and then paraffin blocks were made. Serial sections of 4–5 μm in thickness were cut using a microtome . Then, the sections were deparaffinized with xylene and hydrated in descending grades of alcohol. The slides were then transferred to hematoxylin for 10 min, followed by rinsing with water. These were examined and later stained with eosin, rinsed with water, dehydrated with ascending grades of alcohol, cleared with xylene, and mounted. Different parameters of pancreatic cellular condition were observed under a compound microscope, and the histopathological images were taken with the help of an Optica DP20 system .m/z 50 to 1000 was performed with a source temperature of 120 °C. Solvent A was water with 0.1% formic acid, while solvent B was acetonitrile with 0.1% formic acid. A gradient elution was performed, starting with 99% solvent A and 1% solvent B for the first 15 min, and then 65% solvent A and 35% solvent B for 1 min, followed by a gradual increase in solvent A to 100% over 2 min, and finally a slow increase in solvent B to 99% and solvent A 1% over 2 min. Highly purified nitrogen (N2) and ultra-high-purity helium (He) were used as a nebulizing gas and collision gas, respectively. In terms of positive electrospray mode, the capillary voltage was set at 2.0 kV. Other instrument conditions implied were: source offset, 100 V; desolvation temperature at 550 °C; 50 L/h cone gas flow with temperature 120 °C; and desolvation gas flow, 800 L/h.The phytochemical profiling of the CILEx was determined using UPLC-MS. UPLC-MS analysis was performed using Waters ACQUITY UPLC IClass/Xevo in line with a Waters Xevo G2 Q-TOF mass spectrometer . Extract samples were prepared by dissolving 100 mg of CILEx in 1 mL of methanol. Separation was conducted on a Zorbax Eclipse plus Acquity UPLC BEH C18 (1.7 μm particle size) 2.1 mm × 50 mm. The UPLC was interfaced with a Q-TOF mass spectrometer integrated with positive and negative electrospray ionization (ESI) sources. Full-scan mode from LigPrep  was used in this regard to prepare the ligands, and yielded 3D structures with accurate chiralities . It geneProtein Preparation Wizard was used to modify the crystallographic structures of the proteins that were taken from the PDB (Protein Data Bank) . The proThe proteins 1XU9, 1XU7, and 6R4F had multiple binding sites, while 3A5J did not have any binding sites for ligand–protein interaction. To look for the possible binding sites, we used SiteMap from Schrödinger, 2018-4 , so thatThe sites visualized by SiteMap were used as the entry, and Glide v8.1.12  was used to discover the suitable interaction between a ligand and a protein . Van derIn ligand–receptor interaction, Van der Waals radii were fixed at a scaling factor of 0.80, and partial charge cutoff was scaled at 0.15 for the non-polar parts of the ligands. SP (standard precision) was set for ligand screening and sampling. The energy window for ring sampling to generate conformers was 2.5 kcal/mol. Initial poses for docking were kept at 5000 poses per ligand, and the scoring window was 100–400 poses for energy optimization. Post-docking minimization was performed for 5 poses per ligand, the strain-correcting threshold was 4.00 Kcal/mol, and excess strain energy was scaled at 0.25. The parameters used were defaults, as provided by Maestro 11.8 ,74,75.http://stitch.embl.de/, ver. 5.0, accessed on 3 August 2020) was used to identify target proteins related to the bioactive phytochemicals that were identified in CILEx [o-Benzoyl-20-deoxyingenol, leucodelphinidin, schizonepetoside E, 1β,3β,6α-Trihydroxy-4α(15)-dihydrocostic acid methyl ester-1-o-β-d-glucopyranoside, melianol, and arbutin) were put into STITCH 5 individually to match their potential targets, with the organism selected as “Homo sapiens” and the medium required interaction score being ≥0.4.We predicted 205 target proteins with medium confidence score for arbutin, which was confirmed. The compound targets with no relationship with the compound–protein interactions were not considered for further analysis. The obtained compound–protein interaction data of the top 20 target proteins were imported into Cytoscape 3.6.1 software to construct a compound–protein interaction network.On the basis of network pharmacology-based prediction, STITCH 5  [We constructed a PPI network of the predicted genes by using the search tool for the retrieval of interacting genes (STRING) database (st 2020) . The ranst 2020) . The obthttps://david.ncifcrf.gov/, accessed on 3 August 2020) v6.8 was employed [p-value < 0.05, calculated by the Benjamini–Hochberg method, was considered to be significant [To identify the role of target proteins that interact with the active ingredients of CILEx in gene function and signaling pathways, the Database for Annotation, Visualization, and Integrated Discovery ). p-values less than 0.05 were considered to be significant.The data on fasting blood sugar and biochemical estimations were expressed as mean ± standard deviation (SD), and statistical comparisons were performed by one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, using GraphPad Prism (version 6 for Windows, GraphPad Software, San Diego, CA, USA, C. indicum leaves have potential benefits for the treatment of diabetes and its associated complications, by protecting the pancreases through the normalization of damaged beta islets and improvement of the lipid profile. However, more studies are recommended in order to understand the mechanism and to isolate the bioactive compounds.The current study results indicate that