Overall, treatment with rutin significantly prevented the expression of several MMPs and increased type III collagen in the gingiva around the surgical site. Additionally, rutin-treated animals showed enhanced bone formation with higher bone marrow content in the jawbone defect area compared with the control group. Taken together, these findings demonstrate that rutin gel, when added to bone grafts, quickly enhances bone formation and may serve as a suitable alternative to expensive growth factors for the same purpose.Bone graft techniques are used to compensate for bone loss in areas with deficient regeneration. However, matrix metalloproteases (MMPs) can limit bone formation by degrading extracellular matrices, which are required for bone regrowth. Noteworthily, rutin is a natural flavonoid compound that inhibits the genetic expression of various MMPs. Therefore, rutin may serve as an inexpensive and stable alternative to the growth factors used to accelerate dental bone graft healing. This study aimed to evaluate the potential of mixing rutin gel with allograft bone to accelerate the healing of bone defects in an in vivo rabbit model. Bone defects were surgically induced in New Zealand rabbits ( In periodontics, bone graft materials are used to encourage bone formation at defective sites, a process that increases the potential and predictability of bone regeneration. After the bone graft is placed at the planned site, bone formation is affected by the particle size of the bone graft, its physical and chemical properties, and the grafting procedure used . MoreoveBone healing is a complex process in which diverse cell types, signaling pathways, and extracellular matrix (ECM) components interact . In partVarious methods have been investigated for controlling MMP activity and expression, including the application of small-molecule inhibitors, gene therapy, and alteration of native MMP inhibitors . NoteworRutin is poorly soluble in aqueous solvents, which hinders its application in clinical settings due to reduced bioavailability. Therefore, we used the proniosomal gel to improve rutin solubility and bioavailability. In addition, using gel will aid in extending rutin’s pharmacological effects due to the controlled release properties of gel formulations. A further advantage of using proniosomal gel is that it can penetrate physiological barriers . Thus, aThe present study aims to provide an easy, simple, and rapid pathway to achieve desired results through bone grafting in vivo using rutin as a replacement for growth factors. Our study findings are expected to pave the way for the methodological development of practical dentistry, which will ultimately reduce the economic burden of the treatments and improve patient outcomes.The pilot study revealed that at the end of the sixth week, the bone healing process was still incomplete, whereas the healing process and new bone formation were almost completed 12 weeks after just tooth extraction (without intervention).n = 3) were randomly divided into two groups following unilateral lower first premolar tooth extraction: the control group received vehicle gel (without rutin) and the treated group received rutin gel, which was mixed with bone graft to fill the bone defect. As described in Briefly, six New Zealand rabbits . In the control and rutin treatment groups, there was no significant difference in connective regions (%). Noteworthily, the amount of connective tissue was reduced at the bone deficit site in rutin-treated animals as compared with the control group, but this difference was not statistically significant.A detailed analysis of the histological features of the bone deficit site revealed that the apical width, bone formation area, and bone marrow region were significantly increased in animals treated with rutin gel . The remHistological analysis of sliced tissue samples of the grafted bone area was performed to determine the amount of newly formed bone a and bonFurther histological analyses showed improved bone formation and distribution and higher levels of collagen fibers as well as improved angiogenesis upon rutin treatment b,d,f as MMP1, MMP3, MMP9, and MMP13 at bone formation sites, which agrees with previous reports. Consequently, rutin could significantly improve the desired functional outcomes [This study showed that treatment with rutin can significantly reduce the levels of outcomes ,19,20. Aoutcomes . Moreoveoutcomes . Accordioutcomes .COL3A1 expression, which agrees with the results of a previous study [In the present study, the rutin-treated group showed a significant increase in us study . Type IIus study . Moreoveus study .Histological findings showed that rutin treatment can enhance bone formation in the buccal region, which is most likely due to an interaction between the anti-inflammatory and osteogenic properties of rutin. This finding is consistent with previously published studies which showed that rutin administration to a rat model of osteoporosis in mandibular alveolar bone significantly increases bone formation . AdditioHerein, our histomorphometry results revealed a significant increase in the bone marrow area upon rutin treatment. In accordance with our findings, a previous in vivo study confirmed that rutin can promote the differentiation of marrow mesenchymal stem cells into osteoblasts . During Further evaluation of rutin as an adjuvant agent for bone grafts is still required to optimize the bone regeneration process. In particular, future studies should explore the most appropriate rutin dosage to use in the clinical setting and the precise molecular mechanisms underlying its therapeutic effects. The study was limited by the fact that it was conducted on healthy laboratory animals, which differed from humans in health status, diet, and environment. Therefore, clinical translation of these laboratory findings requires extensive preclinical research.The rutin gel was prepared as previously described, but with some modifications . Rutin aIn this study, six male New Zealand rabbits aged 10 months weighing between 2.75 and 3.5 kg were randomly divided into two groups: the control group received the vehicle gel, and the treated group received the rutin gel, which was combined with a demineralized freeze-dried bone allograft (DFDBA) and pericardium membrane . A sterile standard diet and free access to water were provided to all animals under pathogen-free conditions.For general anesthesia, 40 mcg/kg dexmedetomidine HCl  was combined with continuous isoflurane  inhalation. Lidocaine hydrochloride  was used to induce local anesthesia. The experimental defects were created using an extraoral approach through skin incisions. The first premolar to first molar teeth were incised, and a sulcular incision was made from the distal side of the lower second premolar to the mesial side of the first premolar. A vertical incision was made on the mesial side of the first premolar to achieve primary closure. Once the alveolar bone was exposed by raising the flap, lower anterior pediatric forceps were used to extract (atraumatic extraction) the mandibular first premolar PM-1  from the lower teeth. Subsequently, the granulation tissue in the socket was completely cured. Randomly selected defects were assigned to the control and rutin groups. The defects were filled with 80 mg DFDBA with or without rutin gel and then covered with a pericardium collagen membrane. Interrupted sutures  were used to close the flaps.Following surgery, all animals were provided with special food pellets. As part of their post-surgery care, the rabbits were intramuscularly administered analgesics  and antibiotics  every 24 h for 5 days. Two weeks after surgery, the extraoral sutures were removed and the intraoral sutures were reabsorbed .The experiment was terminated after 6 weeks based on the findings of our pilot study. In this pilot study, we examined bone formation without intervention (just tooth extraction) at different time points. At the end of the sixth week, we found that the bone healing process was still incomplete, whereas the healing process and new bone formation were almost completed 12 weeks after extraction. Therefore, we chose 6 weeks as a relevant time point for comparison as we were interested in studying whether adding rutin to the bone graft accelerates bone formation or not.The animals were euthanized with carbon dioxide after 6 weeks. Then, the jawbones were extracted from sacrificed rabbits a,b. BoneTotal RNA was extracted from tissue samples using Invitrogen TRIzol reagent , according to the manufacturer’s instructions, and was quantified by measuring the absorbance at 260 nm using a Jenway Genova Nano Micro-Volume Life Science Spectrophotometer . To determine the purity of the RNA, absorbance ratios of 260/280 nm (>1.8) were used. The respective cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit , in accordance with the manufacturer’s instructions. Briefly, 1.5 µg of total RNA obtained from each sample was mixed with 2 µL 10× of reverse transcriptase buffer, 0.8 µL of 25× dNTP mix, 2 µL of 10× reverse transcriptase random primers (100 mM of each), 1 µL of MultiScribe reverse transcriptase, and 4.2 µL of nuclease-free water. The final reaction mixture was maintained at 25 °C for 10 min, then heated to 37 °C for 120 min, heated again to 85 °C for 5 min, and then cooled down to 4 °C.The quantitative expression of specific mRNAs was assessed by real-time polymerase chain reaction (PCR). Briefly, 1.4 µL of cDNA was added to 25 µL PCR reaction mixture, which also contained 0.25 µL of forward and reverse primers , 10.6 µL nuclease-free water, and 12.5 µL SYBR Green Universal Master Mix . The primers used in the study  were syn−∆∆Ct method. The target gene levels were normalized to those of GAPDH for comparing the fold change between the treated and untreated groups based on the following equation: fold change = 2−∆(∆Ct), where ∆Ct = Cttarget − CtGAPDH and ∆(∆Ct) = ∆Cttreated − ∆Ctuntreated.As previously described , real-tiThe measuring of histomorphometric parameters was described, specified, and accomplished according to the work of Okada et al. (2019) . ApproxiEach bone sample came with a tooth, and the sample was oriented such that the tooth was posterior to the study area, apical width was measured in lower position of the study area, middle width was measured in the central position of the study area, coronal width was measured in the higher position of the study area, and bone formation area was the place where bone growth was observed within the study area.n = 3 for each group) were analyzed. Unpaired t-tests were used to determine significant differences between the groups. p < 0.05 was deemed statistically significant.The collected data were analyzed using GraphPad Prism software version 9.4.0 . A total of six samples divided into two groups (Rutin gel and bone grafts can be locally applied to bone defects without the need for specialized instruments. This mixture, which contains biocompatible, readily available, inexpensive, and safe substances, supports rapid bone production and promotes good bone health. Hence, rutin gel and bone grafts can be used together to treat a wide range of dental and bone-related conditions. These findings provide new foundations for the periodontics and medical fields as they describe an easy, simple, and cost-effective approach for achieving the desired results in bone grafting procedures. In future studies, bending, axial compression, and torsion tests could be used to assess bone mechanical strength following rutin treatment with a bone graft mixture.


The ultrasmall gold nanoparticles (AuNPs), serving as a bridge between small molecules and traditional inorganic nanoparticles, create significant opportunities to address many challenges in the health field.This review discusses the recent advances in the biological interactions and imaging of ultrasmall AuNPs.The challenges and the future development directions of the ultrasmall AuNPs are presented.Ultrasmall gold nanoparticles (AuNPs) typically includes atomically precise gold nanoclusters (AuNCs) and AuNPs with a core size below 3 nm. Serving as a bridge between small molecules and traditional inorganic nanoparticles, the ultrasmall AuNPs show the unique advantages of both small molecules  and nanoparticles . The emergence of ultrasmall AuNPs creates significant opportunities to address many challenges in the health field including disease diagnosis, monitoring and treatment. Since the nano–bio interaction dictates the overall biological applications of the ultrasmall AuNPs, this review elucidates the recent advances in the biological interactions and imaging of ultrasmall AuNPs. We begin with the introduction of the factors that influence the cellular interactions of ultrasmall AuNPs. We then discuss the organ interactions, especially focus on the interactions of the liver and kidneys. We further present the recent advances in the tumor interactions of ultrasmall AuNPs. In addition, the imaging performance of the ultrasmall AuNPs is summarized and discussed. Finally, we summarize this review and provide some perspective on the future research direction of the ultrasmall AuNPs, aiming to accelerate their clinical translation. Ultrasmall gold nanoparticles (AuNPs) consist of atomically precise gold nanoclusters (AuNCs) or AuNPs with a core size below 3 nm –4, whichCurrently, various large-sized plasmonic AuNPs –23 and sThe ultrasmall AuNPs are formed from the aggregates of small amount of gold atoms protected by surface ligands  as well as reduce toxicity, Kim et al. proposed a host–guest system that utilized supramolecular chemistry for intracellular activation . . −1) was3+, perfluorocarbon, 198Au and 64Cu) to generate multimodal imaging capabilities such as magnetic resonance imaging (MRI) AuNPs were renal clearable and exhibited molecular pharmacokinetics with a rapid t1/2α of 5.0 min and a t1/2β of 12.7 h. Wang et al. developed a 810 nm-emitting AuNCs encapsulated with a fluorinated polymer (AuNCs@PF) [44NCs (1.6 nm) with emissions at both 1080 and 1280 nm by conjugating an aromatic photoacoustic/photothermal molecules (Cy7) through a click chemistry [The ultrasmall AuNPs with strong absorption in the visible and NIR region can serve as contrast agents for photoacoustic imaging . Using tn of 1 s . This hisms Fig. b. Zheng  2.6 nm) , which wuNCs@PF) , which wuNCs@PF) . The gluhemistry , which wThe fundamental understanding on the nano–bio interactions of ultrasmall AuNPs is an important issue in the development of translatable intelligent nanomedicine with both maximum efficacy and minimum toxicity toward disease theranostics. In this review, we briefly summarize the recent advances of biological interactions and imaging of ultrasmall AuNPs at both in vitro and in vivo levels including the cellular interactions, organ interactions, tumor interactions and imaging performance. The fundamental physicochemical properties of ultrasmall AuNPs, such as surface charge, surface coverage, hydrophobicity, functionality and concentration, play important roles in governing their nano–bio interactions with cells, organs and tumors. By taking the advantages of unique quantum confinement effect, high atomic number of Au and easy functionality, the ultrasmall AuNPs with the excellent intrinsic emissions and outstanding biological behaviors show great potentials as promising multimodal probes to address many challenges in the healthcare field using the imaging techniques such as fluorescence imaging, CT imaging, photoacoustic imaging, MRI imaging, PET imaging and Raman imaging.With the above advances, the future biomedical applications of ultrasmall AuNPs hold great promise. However, the research on the nano–bio interactions of ultrasmall AuNPs is still in their early stage. An in-depth understanding of the nano–bio interactions of ultrasmall AuNPs highly relies on the development of synthetic strategies towards water-soluble different-sized atomically precise AuNCs to overcome the long-standing size heterogeneity issue of the nanoparticles, so that the precisely understanding of size-dependent nano–bio interactions of ultrasmall AuNPs will be achieved. In addition, the understanding on whether and how ultrasmall AuNPs cross the biological barriers such as blood brain barrier (BBB) , 232, th


Lonicera caerulea, which is rich in polyphenolic compounds, has been shown to exert anti-oxidative and anti–inflammatory effects. This study aimed to explore the effects and mechanisms of concentrated Lonicera caerulea juice (LCJ) on ALD in mice. ALD was established in mice via gradient alcohol feeding for 30 days. The mice in the experimental group were given LCJ by gavage. The reduction of aspartate transaminase (AST) and alanine transaminase (ALT) in the serum of mice indicated that LCJ has a liver-protective effect. LCJ improved the expression of AMPK, PPARα, and CPT1b in ALD mice to reduce the liver lipid content. Additionally, LCJ increased the expression of farnesoid X receptor (FXR), fibroblast growth factor 15 (FGF15), and fibroblast growth factor receptor 4 (FGFR4), which lowers the expression of cytochrome P450 7A1 (CYP7A1) and lessens bile acid deposition in the liver. In mice, LCJ improved the intestinal barrier by upregulating the expression of mucins and tight junction proteins in the small intestine. Moreover, it accelerated the restoration of microbial homeostasis in both the large and small intestines and increased short–chain fatty acids in the cecum. In conclusion, LCJ alleviates ALD by reducing liver and serum lipid accumulation and modulating the FXR–FGF15 signaling pathway mediated by gut microbes.Alcoholic liver disease (ALD) is a growing public health issue with high financial, social, and medical costs.  The chronic daily intake of a specified quantity of alcohol can lead to the development of alcoholic liver disease (ALD), which comprises a spectrum of ailments ranging from reversible hepatic steatosis to acute alcoholic hepatitis, chronic fibrosis, cirrhosis, and hepatocellular carcinoma . ALD is Lonicera caerulea (LC) is classified as a “homology of medicine and food” fruit due to its high concentration of bioactive compounds [Prior research has demonstrated that edible plants and their bioactive compounds, such as polyphenols, can safeguard against ALD by modulating the gut microbiota ,10. By iompounds . High leompounds . The totompounds ,16. In aompounds . LC polyompounds .Lonicera caerulea juice (LCJ) can alleviate the disease and examine potential mechanisms. In addition to promoting the thorough development and utilization of LC resources, our results may also provide a new reference for the treatment of ALD.Herein, we hypothesized that LC—which is high in polyphenols—can alleviate ALD. We developed a mouse model of ALD to determine if Lonicera caerulea were combined in a colloid mill at a ratio of 1:1 (w/v), and the pulp was processed repeatedly. The juice was collected after the pulp had been centrifuged at 3000 rpm for 30 min. Finally, the juice was evaporated by rotation into two types of LCJ, each with a total phenol content of 9 mg/mL and 18 mg/mL. The Lonicera caerulea was provided by the Heilongjiang Fengran Agricultural Group Company. The company certified that the Lonicera caerulea L. was the “Beilei” variety, belonging to the Lonicerae genus of the Caprifoliaceae family. Pure water and LCJ with a total phenolic content of 18 mg/mL was diluted by a factor of four, and then about 100 μL of the sample was transferred to an Eppendorf tube. After the addition of 300 μL of extract solution (methanol), samples were vortexed for 30 s, sonicated for 10 min in an ice–water bath, and incubated for 2 h at −20 °C to precipitate the proteins. Then, the sample was centrifuged at 12,000 rpm for 15 min at 4 °C. The resulting supernatant was transferred to a fresh glass vial for analysis. A triple quadrupole compound linear ion trap liquid-mass coupling instrument  was used to detect phenols in LCJ . ChromatIt was reported that the chronic plus––binge ethanol challenge induced a greater degree of adipose tissue inflammation and liver injury in female mice than in male mice ,21. TherIt has been proven that 200 mg/kg of blueberry polyphenol and grape leaf extract containing 250 mg/kg of polyphenol can effectively relieve ALD ,12. Sincw/v) alcohol for 3 days, 2.5% (w/v) alcohol for 3 days, and 3.3% (w/v) alcohol for 3 days. Then, they were given a Lieber-DeCarli diet containing 5% (w/v) alcohol for 16 days. On the last day, the experimental group was given a concentration of 31.5% (vol/vol) ethanol solution by gavage, and the volume of the gavage (μL) = body weight (g) × 20. The control group was administered with an isocaloric maltodextrin solution. Mice were sacrificed nine hours after intragastric administration. To establish a model of chronic intake and single binge drinking, all mice were given the Lieber-DeCarli control liquid diet for the first 5 days. The model group and the test group were fed with gradient alcohol adaptation mode for 9 days, which was 1.7% (v/v) and embedded in paraffin. Samples were cut into small sections for staining with hematoxylin and eosin (HE) and oil red O; sections were then examined using a microscope. According to Osho et al. [Specimens of the live large intestine and small intestine were collected for histological analysis. They were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS) , alanine transaminase (ALT), aspartate transaminase (AST), triglycerides (TG), and total bile acid (TBA) were evaluated using a BK–400 automatic biochemical analyzer. In addition, the kits of serum ALT, AST, TG, TC, and TBA were purchased from Nanjing Jiancheng Biological Engineering Research Institute . Lipopolysaccharides (LPS) ELISA kits were purchased from SenBeiJia Biological Technology Co., Ltd. .A liver sample weighing 0.1 g was homogenized using a high-speed homogenizer in 0.9 mL of physiological saline to produce a liver homogenate. The supernatants were collected by centrifugation at 3000 g for 15 min at 4 °C. The levels of TC, TG, and TBA in the liver were determined using commercial kits obtained from Nanjing Jiancheng Biological Engineering Research Institute . Fecal bile acid levels were detected according to a method previously described by Guo et al. .All the contents of the large and small intestines were scraped and stirred well. Samples were then divided into 6 groups: SCG, small intestine in the control group; SAG, small intestine in the alcohol group; SHG, small intestine in the high–dose LCJ group; LCG, large intestine in the control group; LAG, large intestine in the alcohol group; LHG, large intestine in the high–dose LCJ group. The total DNA of microorganisms in intestine contents was extracted using a fecal DNA extraction kit . DNA was successfully separated by agarose gel electrophoresis.The highly variable region (V3–V4) of bacterial 16S ribosomal DNA was used for polymerase chain reaction (PCR) amplification. The primers were 341F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). According to the instructions of Nanjing Vazyme Biotech Co., Ltd.  reagent kit, a PCR reaction system was prepared by taking a qualified genomic DNA sample of 30 ng and corresponding fusion primers. The PCR products were purified according to the instructions of the AxyPrep DNA Gel Extraction Kit  reagent kit. The Agencourt AMPure XP  magnetic beads were used to dissolve and purify the PCR amplification products before labeling them to finish the library construction. An Agilent 2100 bioanalyzer  was used to analyze libraries for fragment range and concentration. Libraries that passed the test were selected for sequencing on a HiSeq platform  according to insert sizes. The data were filtered, and the remaining high-quality, clean data were used in post-analyses. Sequence splicing was performed using FLASH software , which assembles pairs of reads from double-end sequences into a single sequence using overlap relationships to obtain tags for high–variation regions. Amplicon sequence variants (ASVs) were obtained, compared with the database, and annotated with the species using the DADA2  method in QIIME2. Sample species complexity analysis, intergroup species variation analysis, association analysis, and functional prediction were carried out based on the results of OTU and annotation. The BGI platform made the bioinformatics analytic techniques available for free online.v/v) sulfuric acid solution precooled at 4 °C was added to extract the fatty acids, followed by the addition of 1 mL of anhydrous ether  precooled at 4 °C. The whirlpool was then shaken for 30 s. Samples were centrifuged at 3000 rpm for 15 min at 4 °C. Finally, SCFAs in the supernatant were analyzed using gas chromatography–mass spectrometry. [About 0.09 g of fecal content homogenate was added to 0.5 mL of saturated sodium chloride solution precooled to 4 °C. Then, 10 µL of a 10% . The expression level of the liver gene was normalized to the β-actin gene, while the expression level of the small intestine gene was normalized to the GAPDH gene and calculated utilizing the 2Three mice in each group were selected for the Western blot assay. The tissues were lysed with RIPA buffer (1 mL) containing proteinase inhibitors . The homogenate was centrifuged at 3000 rpm for 5 min at 4 °C. The protein content was determined by the BCA protein concentration assay kit  and diluted to a suitable concentration for the experiment.The protein sample from each mouse was separated by electrophoresis in SDS-PAGE  and transferred onto a polyvinylidene difluoride (PVDF) membrane . The membrane was incubated with only one primary antibody every time, then incubated with the secondary antibody and detected. Then, all the detection reagents from the blot on the same membrane are removed and reprobed with the other primary antibody for the detection of the second target protein. Specific operations are as follows: Equal quantities of protein were separated by electrophoresis in SDS–PAGE and then deposited onto PVDF membranes. After the membranes had been blocked with 5% nonfat dry milk, the corresponding primary antibodies were incubated on the membranes overnight at 4 °C. After being washed with Tris–buffered saline with Tween, followed by incubation with secondary antibodies for 1 h at room temperature. Both a luminescence imaging workstation  and an ECL chemiluminescence kit  were utilized to create the target protein. When multiple exposures of the same PVDF membrane were required, Western blot stripping buffer was shaken at room temperature for 45 min and washed with TBST 3 times. The subsequent steps were the same as before. Image Pro Plus 6.0 software was used to analyze the optical density value . The following antibodies were used: MUC4 , CYP7A1 , FXR , MUC2 , FGFR4 , ZO-1 , Claudin-1 , FGF15 , GAPDH , β-actin , AMPK , CPT1b , PPARα .p < 0.05.Statistical analysis was performed using GraphPad Prism 8.0 software and SPSS 25.0 software. All the data in this study were expressed as the mean ± standard deviation. The statistical significance of the results was analyzed by one-way analysis of variance (ANOVA) based on Duncan’s multiple range test using SPSS. The significance level in the analyses was considered The content of 16 phenolic substances in LCJ was determined , which wp > 0.05). These results indicated that LCJ could increase the BW of ALD mice. Furthermore, alcohol consumption did not induce any notable alteration in liver weight, and there was no significant difference in liver weight observed among any of the groups  staining showed that the crypt structure of the small intestinal tissue in the AG group was severely damaged, with a large number of cell deaths and reduced mucin content. Alcohol had little effect on the mucin content and large intestine anatomy. This may be because alcohol is mainly absorbed and metabolized in the small intestine . The effThe results showed that high-dose LCJ increased the expression of tight junction proteins  and mucins (MUC2 and MUC4) in the small intestinal tract of ALD mice d–f. The Based on the biochemical indicators of the liver and serum of mice, as well as the intestinal barrier, intestinal mucin, and bile acid indicators of secondary metabolites of the microbiota, we believe that only high–dose LCJ can improve ALD in mice. Therefore, our study is limited to the CG, AG, and HG groups regarding the effect of LCJ on the intestinal microbiota and its metabolites, short–chain fatty acids, in ALD mice.SCFAs are acidic byproducts of the fermentation of difficult-to-digest carbohydrates by bacteria in the gut, which strengthen the intestinal barrier . Ethyl aAs shown in The Sobs, Chao, and Ace indexes describe community richness. The Shannon and Simpson indices describe community α diversity c–g. Our β diversity analysis was used to show differences in the microbial community composition among different groups of samples a–e. The Bacteroidetes, Verrucomicrobia, Actinobacteria, Proteobacteria, and Firmicutes were the dominant phyla in the small and large intestinal contents of normal mice  effect size (LEfSe) analysis and an LDA analysis score greater than 4. Results are shown in ALD is associated with high morbidity and mortality worldwide, which calls for investigations into its pathogenesis to find better treatments. Alcohol consumption can disrupt the composition of the gut microbiota, and resolving the disruption of the gut microbiota caused by alcohol can alleviate ALD. Previous studies have shown that plant natural products and polyphenolic compounds exert beneficial effects on several metabolic disorders associated with ALD via the gut–liver axis . LC is rThe changes in mouse liver index and serum ALT, AST, and AST/ALT ratio indicated that this alcohol feeding mode affected the normal growth of mice and damaged their liver cells. However, high–dose LCJ can effectively slow down alcohol–induced liver cell damage. The damage to ALD mouse liver cells may be due to the accumulation of liver lipids or inflammatory cell infiltration caused by alcohol metabolism, or bile acid accumulation and peroxidation . This inIn our study, we observed that LCJ treatment significantly reduced the increase in serum TG and TC as well as the accretion of fat granules in the liver while increasing the protein expression of AMPK, PPARα, and CPT1b in the liver in ALD mice . AMPK isLong–term ethanol feeding has been found to reduce intestinal FXR activity. This may be because prolonged alcohol exposure increases FXR acetylation and interferes with FXR’s ability to bind with the retinoid X receptor alpha (RXR), leading to the inactivation of FXR . FXR is Due to the fact that more than 90% of alcohol is absorbed in the gut after ingestion, excessive alcohol consumption can cause damage to the intestinal barrier. Intestinal barrier damage increases intestinal permeability, which allows the blood to carry LPS, a byproduct of bacteria found in the intestine, into the liver . After LVerrucomicrobia and Akkermansia in the large intestine of ALD mice but decreased the relative abundance of Parabacteroides, Oscillospira, and Proteus. In the small intestine of ALD mice, LCJ treatment increased the relative abundance of Firmicutes, Bacteroidetes, Allobaculum, and Akkermansia while reducing the relative abundance of Proteobacteria, Staphylococcus, and Proteus. A previous study reported that Proteobacteria contributes to inflammation [Proteobacteria, was reduced in the HG group, which was consistent with the reduction in the Gram–negative bacteria product LPS. Furthermore, impairment of the intestinal barrier increases the likelihood of LPS entering the bloodstream, while LCJ has been shown to enhance intestinal barrier function. This suggests that LCJ can improve the intestinal barrier and regulate intestinal flora to decrease the production of LPS, thereby reducing blood LPS to improve alcohol-induced liver injury.A high dose of LCJ had different effects on the microbial composition between the large and small intestines. Notably, LCJ increased the relative abundance of ammation . In our Akkermansia [Allobaculum [Akkermansia and Allobaculum, the mucin content, and intestinal barrier function in the small intestine of the AG group were significantly reduced, but treatment with LCJ reversed these changes. Moreover, the cecal levels of SCFAs, including acetic acid, propionic acid, butyric acid, and valeric acid, significantly increased in mice with ALD following LCJ intervention. Notably, acetic acid is the most abundant SCFA produced by the gut microbiota and has been shown to improve the function of the intestinal mucosal immune barrier [Akkermansia can produce propionic acid by increasing mucin degradation [Allobaculum is physiologically capable of generating butyric acid from carbohydrates [Akkermansia and Allobaculum), resulting in high intestinal SCFA production. In this way, it strengthens the intestinal barrier by increasing the expression of intestinal compact junction protein and mucin content to alleviate alcohol–induced liver injury. Oscillospira was strongly correlated with the proportion of secondary bile acids in the stool, suggesting that it may contribute to the formation of secondary bile acids in the stool [Parabacteroides hydrolyzes a variety of binding cholic acids into secondary cholic acids  through multiple pathways such as bile brine hydrolysase (BSH) [Oscillospira and Parabacteroides in the intestine. These results demonstrate that LCJ can reduce liver bile acid levels and further ameliorate alcohol-induced liver injury by regulating gut flora via the FXR–FGF15 axis. ermansia  and Alloobaculum  play imp barrier . Butyrat barrier . These fradation . Allobachydrates . In conche stool . In the se (BSH) . In lighCombined with this study, human treatment of ALD needs to pay attention not only to how to reduce the damage to liver cells but also to the changes in intestinal flora and the intestinal barrier. Plant extracts and probiotics with the impact of enhancing the intestinal barrier and improving intestinal flora are substances worthy of further investigation in the treatment of ALD in the future. This is because ALD can also be relieved by improving the intestinal flora. Additionally, LCJ has no side effects, which helps to maintain a healthy intestinal structure and qualifies it as an ingredient in functional foods for relieving alcoholic liver disease. It also raises public awareness of the health advantages and economic value of LC.In conclusion, LCJ can reduce the accumulation of lipids and bile acids in the liver by reducing the levels of serum AST and ALT, thus producing hepatoprotective effects. Moreover, LCJ significantly improved the harm to the intestinal barrier caused by alcohol as well as the imbalance of short–chain fatty acids in the cecum. LCJ was found to reduce alcohol–induced liver injury by regulating gut microbes and its mediated FXR–FGF15 signaling pathway, based on intestinal flora analysis and related gene expression measurements.


The NEK7 protein is required for spindle formation, cell division, and the activation of the NLRP3 inflammasome receptor. The aberrant expression of NEK7 has been implicated to the growth of metastasis and severe inflammatory conditions like rheumatoid arthritis, liver cirrhosis, and gout. An emergent target for the development of anti-cancer drugs is NEK7. In this context, the PubChem database was used to retrieve the 675 compound library and FDA-approved protein kinase inhibitors, which were then thoroughly examined via in-silico experiments. Computational studies investigated the binding orientation, electronic, and thermodynamic characteristics of drug candidates related to target protein. Drugs were investigated using density functional theory and molecular docking to find binding interactions with NEK7. Molecular dynamic simulations assessed interactions and stability of protein–ligand complex. DFT analyses showed that selected compounds maintained a significant amount of chemical reactivity in both liquid and gaseous states. Alectinib, Crizotinib, and compound 146476703 all displayed promising molecular interactions, according to molecular docking studies, with docking scores of − 32.76, − 30.54, and − 34.34 kJ/mol, respectively. Additionally, MD simulations determined the stability and dynamic characteristics of the complex over a 200 ns production run. The current study’s findings indicate that the drugs Alectinib, Crizotinib, and compound 146476703 can successfully inhibit the overexpression of the NEK7 protein. To discover more potent drugs against NEK7, it is recommended to synthesize the derivatives of Alectinib and Crizotinib and carry out additional in-vitro and in-vivo studies at the molecular level. Despite notable advancements in the development of innovative cancer treatments, the demand for medical care continues to rise as our comprehension of cancer biology expands2. Cancer cells develop from an uncontrolled growth of cells that leads to genetic instability and abnormal cell proliferation3. Various therapeutic strategies have been devised to target cancer cells, but none have yet been successful in inhibiting the dysregulated enzymes that contribute to their growth4.Cancer is a leading contributor to mortality worldwide, ranking as the second highest cause of death5. While most protein kinases have been extensively studied as potential targets for anti-cancer drugs, the NEK kinases have received comparatively limited attention, except for NEK7. NEK7 has been identified as a significant player in the development of cancer and continues to be a promising target for the development of anticancer drugs. It is a unique member of the NEK family, with 85% structural similarity to NEK6 and a distinct role during cell division6. NEK7 has been shown to promote the formation of spindles and is involved in the regulation of intracellular protein trafficking, cell division, and inflammation7. Its aberrant expression has been linked to various types of cancer, including breast cancer, colon cancer, and non-small cell lung cancer9. There are 518 protein kinases in the human genome, 478 of which have highly conserved sequences in their catalytic domains. The protein kinases that regulate cell cycle control include the NEK kinases, Aurora and Polo-Like Kinases (PLKs), as well as Cyclin-Dependent Kinases (CDKs). The K/E/D/D (Lys/Glu/Asp/Asp) signature was found in all of these protein kinases, which is required for the placement of adenosine triphosphate (ATP) and the stability of the active conformation. In the catalytic domain, the site where phosphorylation occurs and transforms the protein into its active form, kinase proteins possess a crucial ATP-binding site which triggers critical processes during cell division, such as cytokinesis, centrosome separation, and spindle formation10. Currently, there are 53 FDA-approved drugs that inhibit protein kinases, including Alectinib and Crizotinib (carbonitrile and pyridine derivatives for breast cancer) and Erlotinib and Gefitinib 11. Despite the advances in protein kinase-targeted anticancer drugs, the search for a selective inhibitor of NEK7 remains a challenge. To date, only Erlotinib and Gefitinib have been found to inhibit NEK7, but the extent of its inhibition and the mechanism of binding interaction are not yet known12. These findings underscore the ongoing need for the development of specific and effective drugs targeting NEK7.The development of drugs targeting protein kinases holds great promise for anticancer therapy, as these drugs have been shown to possess anti-proliferative and anti-mitotic properties13. Presently, 30% of newly launched medications in US markets are the result of pharmacological repurposing. Aspirin, mifepristone, and topiramate are just a few examples of repurposed medications that are currently on the market and have been given FDA approval14. According to estimates, 6.7% of innovative anti-cancer medications developed between 2003 and 2011 were successful in getting FDA market approval after completing phase 1 trials16. This process took an average of 8.3 years. So, there is a need around the world for fast approval of effective cancer treatments, which can only be done by reusing drugs that have already been approved17. Figure Instead of creating novel pharmacological molecules from scratch, computational repurposing of medications is a cutting-edge tool for the development of pharmaceuticals20. After acquiring these improved drug structures, molecular docking studies with the NEK7 target were carried out to anticipate the free binding energies and binding orientation of a subset of medicines, which may help to comprehend the inhibitory mechanism. Then, using molecular dynamic (MD) simulations, the conformational flexibility and dynamic character of molecular interactions were determined. Additionally, 675 structurally related Alectinib analogues were virtually tested against the target protein after being downloaded from the PubChem database. The compound exhibited a higher docking score than standard Alectinib and was considered a hit and subjected to in-silico ADMET prediction and MD simulation studies. Figure This study assessed the in-silico inhibitory capability of FDA-approved medications that were effective against different protein kinases. The initial goal of DFT investigations was to foretell molecular characteristics that sufficiently explained the chemical reactivity, bioactivity, and stability of drug candidates in gas and liquid phasesIn the present work, structural geometry optimizations, the energetic parameters, FMOs analysis, and global reactivity descriptors of Alectinib, Crizotinib, Erlotinib, and Gefitinib were calculated using Gaussian 09W. Gauss View 6 was used for the visualization of output files. Investigations were conducted using the DFT/B3LYP functional and the SVP basis set.In two phases (gas and water), the geometry of selected FDA drugs was completely optimized. After optimizing the geometry, no imaginary frequencies were found, indicating that the current geometries are real local minima. It was discovered that Alectinib and Erlotinib have gas phase optimization energies of − 1532.70 and − 1316.84 Hz, respectively, showing their space stability. Only Alectinib showed a high polarizability value in both phases, i.e., the gas and the solvent phases (370.94 and 496.71 a.u respectively). In addition, the comprehensive geometric parameters of optimized drugs is provided in Supplementary Files Tables –S7. The 21. Molecules with large E gaps (ELUMO–EHOMO) are non-polarizable and considered the least reactive chemical entities. The computed values for the HOMO/LUMO and E gap (ELUMO–EHOMO) are shown in Table The FMOs are necessary because they affect the molecule’s reactivity and stability. The outermost electrons actively engage in the interaction between the ligand and the target protein. The highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) provide sufficient insight into the chemical reactivity of the compounds. The HOMO reflects a compound’s capacity to give electrons, while the LUMO represents its ability to take or remove electrons. A very small energy gap E (ELUMO–EHOMO) between HOMO and LUMO indicates effective charge transfer and increases molecule polarizability22. Compounds with a large ΔE (ELUMO–EHOMO) energy gap refer to the molecule’s least reactivity and more stability, while compounds with a small energy gap are often more reactive. In the solvent phase, Alectinib exhibited a chemical softness value of 7.77 whereas Crizotinib exhibited a softness indices of 6.55, indicating that Alectinib is highly reactive drug than Crizotinib. Likewise, Erlotinib and Gefitinib exhibited good value for chemical softness in solvent phase i.e., 5.50 and 7.50 respectively. They are tending to be more polarizable compounds. The Koopmans’s theorem was used to calculate the electron affinity and ionization energies of selected drugs23. The calculated values for hardness and softness are given in Table The energy band gap between (ELUMO–EHOMO) for compound Alectinib and crizotinib was 0.138 eV and 0.144 eV in gas whereas it was 0.128 eV and 0.152 eV in solvent phase, respectively. Whereas, energy gap for Erlotinib and Gefitinib was 0.155 and 0.128 in gas phase and 0.153 and 0.133 eV in the solvent phase, respectively. It was discovered that a small energy gap corresponded to the compound’s increased reactivity. In addition, two important parameters i.e., chemical softness and hardness is measure of reactivity of a compoundElectrophilicity index: ω = µ/2η, chemical hardness: η = 1/2 (ELUMO − EHOMO); chemical potential: µ =  − χ; chemical softness: S = 1/2η; electronegativity =  − 1/2 (ELUMO + EHOMO).It is necessary to determine the electron density of the lowest unoccupied molecular orbital and the highest occupied molecular orbital because valance electrons are involved in the interaction and chemical reactivity of the compound. The current study has investigated the electron density of FDA drugs. The electron density of HOMO orbital in the gas phase for Alectinib was found to be confined to the morpholine and the piperdinyl ring, whereas the electron density of LUMO orbitals was restricted to the carbo-nitrile and the benzocarbazole moiety. The HOMO and LUMO electron densities of Alectinib in the solvent phase were substantially identical to the gas phase, albeit it was more limited toward the carbonitrile ring. Similarly, the electron density of HOMO orbitals of the Crizotinib molecule were confined to the pyrazole and pyridine rings, while the LUMO orbitals were confined to the trifluorophenyl ring. The FMOs of both drugs are shown in Fig. The FMOs analysis of Erlotinib and Gefitinib revealed that electron density of HOMO orbitals for Erlotinib was concentrated to quinazoline ring which indicated that reactive character of Erlotinib was due to quinazoline moiety. It was observed that electron density of LUMO orbitals for Erlotinib was majorily confined toward benzene ring and ethyl group substituted on the benzene ring which is responsible for electron accepting character of the compound. The concentration of HOMO/LUMO orbitals along with hydrophobic and hydrophilic interactions contribute toward accumulative chemical reactivity of the compound. In terms of Gefitinib, electron density of HOMO orbitals were confined toward morpholine ring and propoxy group but LUMO orbitals were concentrated toward quinazoline ring of the compound. The HOMO/LUMO orbitals of both drugs are shown in Fig. 24 and PyMol25 was used for visualization of top ranked poses. Only four FDA-approved drug exhibited excellent binding energies and formed a stable protein–ligand conformation. The FDA drugs i.e., Alectinib, Crizotinib, Erlotinib and Gefitinib showed comparatively good docking scores as shown in Table Molecular docking studies provide significant insight into static interactions between compound and targeted protein. Two docking software were used in current study to assess the binding orientation of selected drugs with targeted protein. Both docking programs were assessed for their reliability in predicting the docking scores. It was observed that the AutoDock produced more consistent and better docking scores than MOE. Therefore, the current study further explores the molecular interaction of top ranked poses obtained from AutoDock. The binding affinities of the best pose were also assessed through predicted inhibitory constants (ki) obtained through the AutoDock. The Discovery Studio Visualizer 17.2The detailed bonding and nonbonding interactions of Alectinib, Crizotinib, Erlotinib and Gefitinib are tabulated in Table The docked conformation of NEK7-Alectinib complex produced substantial molecular interactions with docking score of − 32.76 kJ/mol and a predicted inhibition constant (ki) of 2.97 µM. The odds-on amino acids involved in bonding interactions with Alectinib were as follows; ASP179, LYS63, VAL48, PHE168, ALA114, ASP115, ILE40, ARG50, LYS38, ASP118, GLY117, ALA165, ASN166, LEU180, ASP161, and LYS163. Alectinib exhibited strong molecular interactions, comprising hydrogen bonding, alkyl, pi-alkyl, carbon-hydrogen bonding, and van der Waals interactions. All of these interactions contributed to the stabilization of the protein–ligand complex. In brief, it was discovered that the 4-morpholine ring and the piperidinyl ring of Alectinib were interacting with ILE40 through an pi-alkyl bond. Carbon hydrogen bonds were also formed between the morpholine and piperidinyl rings and ASP115 and ALA114, respectively. In addition, benzocarbazole and the 3-carbonitrile ring generated significant molecular interactions, such as T-shaped, alkyl, conventional hydrogen bonds, and carbon hydrogen bonds with PHE168, VAL48, ASP179, and LEU180, respectively. In addition, van der Waals interactions were observed with GLY117, ASP118, ALA165, LYS163, ASP161, ASN166, LYS63, and ARG50. Figure 26. Only single molecule (146476703) exhibited powerful and persistent interactions, and its docking score of − 34.72 kJ/mol was better to that of Alectinib , root mean square deviation (RMSD) and protein–ligand interactions were calculated through MD trajectory analysis. The Root Mean Square Deviation (RMSD) is a statistic used to calculate the average change in atom displacement in comparison to a reference frame. Examination of RMSD over simulated time provides sufficient insight into structural deviation of protein–ligand complex. RMSD demonstrate the fluctuation of complex with respect to reference frame.The MD simulation for the NEK7-Alectinib complex was performed in triplicate to validate the docking results. Initially, the main MD simulation run was performed for 200 ns, followed by another two production runs each for 50 ns. Longer MD simulations provided deep insight into the stability of protein–ligand complexes. The average RMSD values obtained from 200 ns, 50 ns and 50 ns production runs were 4.9, 4 and 4.6 Å respectively. The analysis of main production run (NEK7-Alectinib complex) is discussed below and detailed analysis of other two production runs is provided in the supplementary file Figs. , S2. In This section display the RMSD value of C alpha atoms of protein with respect to time. The RMSD plot Fig.  is depicThe stability of protein-146476703 complex was investigated via MD simulation studies. The Desmond software was utilized for the production run of 100 ns. The resulting RMSD trajectory was inspected for determining the structural deviations and nature of molecular interactions. The evolution of RMSD pattern indicates the protein–ligand complex was equilibrated well as the fluctuations remained below 3 Å. The average RMSD value was calculated to be 2.9 Å which is considered as stable and deemed to be equilibrated. The Fig. The RMSF is helpful for characterizing local modifications in the protein chain. Low RMSF values of binding site residues demonstrate the stability of ligand binding to the protein. Figure During the course of simulation time, different types of ligand interactions were observed with targeted protein. This interaction included hydrogen bonding, hydrophobic, ionic bonds and water bridges. Figure The trajectory analysis of Alectinib-NEK7 complex revealed that ligand remained attached to active pocket and ddidnt deviate from activation loop. The super aligned structures of liganded protein obtained at different intervals is illustrated in Fig. 29.The molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) and molecular mechanics generalized born surface area (MM/GBSA) are promising methods for determination of free Binding energies during MD simulations. Since they are modular and do not require training set calculations. They have been used effectively to reproduce and justify experimental data, as well as to enhance the outcomes of virtual screening and docking. But they have a number of wrong and unlikely calculations, such as the lack of information on conformational entropy and the number and free energy of water molecules at the binding siteThe MMGBSA energies for all the four complexes were determined through Thermal_mmgbsa script of Schrodinger. MMGBSA Energies are tabulated in Table The visual depiction of binding affinities of docked conformations was illustrated through SeeSAR analysis. SeeSAR analysis was performed using SeeSAR version 12.0.1. Coronas represented in green and red colors were used to depict the structural features of compounds contributing toward binding affinity. Structural characteristics that improve binding affinity are represented as green colored coronas, while those features that reduce binding affinity are represented as red colored coronas. Structural component of the compounds having no contribution toward binding affinity remained colorless. It was observed that benzene ring was contributing favorably toward binding affinity of Alectinib with Hydrogen bond and Dehydration (HYDE) energy of − 2.8 kJ/mol. Similarly halogen substituted benzene ring was also contributing toward binding affinity of compound. SeeSAR analysis of FDA approved drugs is shown in Fig. 30. The findings of NMA investigations revealed some notable differences in the deformability of these systems. The main-chain deformability is a property that describes the flexibility of a molecule, specifically the ability of its main chain to change its shape at each residue (amino acid) along the chain. The main chain refers to the backbone of a protein molecule, which is composed of repeating units of nitrogen and carbon atoms. The deformability of a molecule is an important factor that determines its function and stability. The apo NEK7 protein was found to be highly deformable with a deformability score of 0.9. This is indicated by the presence of large peaks in the deformability profile, suggesting that the protein is able to undergo significant conformational changes at these sites. This high deformability of the apo protein is likely due to the lack of any stabilizing interactions with other molecules, such as ligands or inhibitors. In the absence of these interactions, the protein is free to adopt a wide range of conformations, resulting in its high deformability.NMA  is a computational technique used to study the conformational changes in proteins. The NMA analysis performed on the apo protein and protein-alectinib complex, as well as the Nek7-compound 146476703 complex via IMODS webserverThe protein-alectinib complex was found to be stiff and showed no significant peaks of deformability Fig. . This suCovariance plots are a useful tool for visualizing the structural interactions between different residues in a protein. In this study, covariance plots were calculated for the apo NEK7 protein, the NEK7-alectinib complex, and the NEK7-compound 146476703 complex. The covariance plot of the apo NEK7 protein showed a mix of red, blue, and white regions. The red regions indicate high positive covariance, meaning that the residues in these regions tend to move together in a coordinated manner. On the other hand, the blue regions indicate high negative covariance, meaning that the residues in these regions tend to move in opposite directions. The white regions indicate low covariance, meaning that the residues in these regions do not tend to move together or in opposite directions.30 showed the highest concentration of red regions, indicating high levels of positive covariance between residues in this complex. This suggests that the alectinib molecule is forming strong interactions with the protein, leading to coordinated movements of residues in specific regions. The blue regions were also significant, indicating that the formation of these interactions may also lead to the restriction of certain movements in other regions of the protein.The covariance plot of the NEK7-alectinib complexThe covariance plot of the NEK7-compound 146476703 complex showed a similar pattern to that of the NEK7-alectinib complex, with high levels of positive covariance indicated by the red regions Fig. . However27 for prediction of the pharmacokinetic profile of the compound. MolDesigner is an interactive tool for the production of efficacious drugs with a deep learning model, i.e., MPNN. The solubility and lipophilicity of compound 146476703 were predicted to be − 4.36 log mol/L and 3.37 (log-ratio) respectively. The detailed ADMET profile is tabulated below  for appropriate assumptions on drugs’ electronic characteristics and attributes. The B3LYP functional is a hybrid approach that combines the Slater exchange functional, Becke gradient correction, and LYP correlation. It provides a good balance of accuracy and computational efficiency for a wide range of molecular systems. It is widely used due to its ease of implementation and good results for molecular geometries, exchange–correlation energy, and is faster than most post-Hartree–Fock techniques. B3LYP is a robust DFT method with 3 parameters compared to other hybrid functionals with up to 26. In addition, the split valance polarized (SVP) set is a [3s2p] contraction of a [7s4p] set of primitive functions reliable for attianig the stable configuration of compounds. The SVP basis set promisingly covers 1st and 2nd row elements of periodic table however the coverage of elements depend upon level of contraction applied to a basis set. Furthermore, using the same level of theory, the frontier molecular orbital (FMO) analysis, reactivity descriptors and chemical reactivity was also determined. Gauss View 631 was used to analyze the output files.The Gaussian 09W programme32 and AutoDock 4.233. The present study has employed two docking software in order to improve the accuracy and reliability of docking results. A crystallographic structure of NEK7  was retrieved from RCSB protein data bank (https://www.rcsb.org/)34. The 2D structures of all FDA approved drugs were generated using IUPAC names obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). In addition, 675 compounds library were also retrieved form PubChem database and subjected to virtual screening against NEK7 utilizing the proposed approach. All compounds were subjected to preliminary energy minimization process in order to remove any steric clashes. The prepared compounds library was saved to required database for docking against targeted protein35. Protein preparation was carried out using MOE protein preparation utility. Protein preparation included removal of het and water molecules. Missing residues were repaired by 3D protonating the protein structure and correcting the identified issues at pH 7 and 300 K temperature. Furthermore, polar hydrogens were added with standard 3D geometry and gasteiger partial charges were assigned using MMFF94x forcefield36. Afterwards, the active site residues were identified using the dimensions of co-crystal ligand (ADP) utility and dummies atoms were generated at respective alpha spheres. Selected amino acid residues of active site were as follows; LYS63, ARG160, LEU180, ASP179, LEU113, ILE40, GLY41, GLY117, ALA61, ALA116, ALA165, ASP118, GLY43, LEU111, PHE168, ASP115, ASN166, SER46, GLU82 ARG121, GLY112, ALA114, PRO200, ARG207, TYR213, SER204, MET203, TYR202, ILE109, PHE45, CYS79, ILE83 and VAL48. Finally, docking was performed using the MOE default parameters, with a triangular matcher as placement method and refinement was set to induce fit. The scoring function was set to London dG and total 100 poses were generated for each compound37. The docking protocol was validated by re-docking co-crystal ligand, and the conformation with RMSD value less than 2 angstrom was selected for further analysis39.Molecular docking is an effective approach for determining the binding orientation and binding affinities of compounds against targeted protein. Molecular docking studies were performed using Molecular Operating Environment (MOE) 2015.1037. Both software’s were accessed for reliability of docking scores. The docking output from both software’s are tabulated in Table Protein preparation process was also undertaken in AutoDock including addition of polar hydrogen, removal of water and hetero atoms. A grid box centered at dimensions  of co-crystal ligand (ADP) was generated and molecular docking was performed using Lamarckian genetic algorithm40 package was used to simulate protein–ligand complexes over 100 ns. Molecular docking facilitates the generation of initial protein–ligand complexes in a static state41, whereas MD simulations gravitate to figure out atoms evolutions over time. MD simulations revealed about the orientation of ligand binding in a physiological environment43.Schrodinger LLC’s Desmond44. The system was neutralized by the addition of NaCl ions at a concentration of 0.15 M. The system was equilibrated for 1 ns in the NVT ensemble at 300 K temperature and 1 atmospheric pressure. The NPT ensemble was then equilibrated for additional 1 ns. Temperature and pressure were maintained using a Martyna-Tobias-Klein and Noose-Hoover thermostat throughout simulation. The production run was performed for 200 ns45.Maestro protein preparation wizard was utilized for optimization of initial protein–ligand complex. The system was developed using a system builder tool. The system was submerged in a water model (TIP3P) containing an orthorhombic box. For production run, the OPLS 2005 force field was employed46. In SeeSAR software 12.0.147, the top-ranked conformation acquired from molecular docking was subjected to pose generation. Each protein–ligand complex was submitted to the SeeSAR docking mode, which produced many orientations based on binding affinity. Green colored coronas represent structural properties of drugs that contribute positively to binding affinity, whereas red colored coronas represent structural aspects that contribute adversely. The structural properties of the drugs that have no effect on their binding affinity were left colorless.SeeSAR analysis was used to assess the persuasive rationale for binding affinities and Hydrogen bond and Dehydration energies (HYDE) of FDA drugs with NEK7The present work has utilized a comprehensive in-silico approach for the repurposing of FDA drugs against the NEK7 protein. The selected drugs, including Alectinib, Crizotinib, Erlotinib, and Gefitinib, were optimized and in-depth electronic and reactivity parameters were estimated using DFT studies. All drugs exhibited reactive potential. Moreover, molecular docking studies revealed the formation of stable protein ligand complexes with all drugs, but Alectinib and Crizotinib produced relatively stronger molecular interactions and produced stable protein–ligand complexes. Moreover, the top-ranked conformation of Alectinib and Crizotinib was subjected to molecular dynamic simulations, which further supported the stability of the protein–ligand complex under experimental conditions. Furthermore, 675 structural analogs of Alectinib were subjected to virtual screening against NEK7, resulting in the identification of compound 146476703 as having potential drug-like properties. It is recommended to further investigate compound 146476703 at the molecular level, with the goal of synthesizing and developing an effective therapeutic approach for NEK7-related cancers and associated malignancies.Supplementary Information.


A series of bifunctional hexagonal MOFs have been successfully constructed by the introduction of various amine functional groups within the unsaturated Cu-based MOF, HKUST, to access amino-modified frameworks. The prepared compounds are cost-effective and display high chemical and thermal stability. They were effectually exploited as efficacious and superb heterogeneous catalysts in rapid and facile Knoevenagel condensation reactions for a variety of substrates containing different electron-donating and electron-withdrawing substituents with very high conversion, good reusability under mild conditions, and very short reaction time. The contaminant presence of Lewis acid and basic sites resulted in efficient condensation reactions by the prepared catalysts. The fundamental condensation reactions took place between aromatic carbonyl compounds and activated methylene groups to construct carbon–carbon bonds6.Knoevenagel condensation is one of the most widely employed reactions in the industry which is used to produce imperative intermediates for pharmaceuticals, fine chemicals, functional polymers, and biologically active materials9. However, homogeneous systems deficiencies and limitations such as catalyst recovery, difficult and costly product separation make researchers develop strategies to form efficient heterogeneous catalysts10. Therefore, different heterogeneous catalysts such as zeolites, mesoporous silica, metal oxides, and ionic liquids were ascertained to employ in condensation reactions11. Although developing appropriate catalysts is still a challenging issue for scientists.Various homogeneous catalysts were utilized in the condensation reactions such as ammonium salts, diverse amine groups and complexes, and organometallic catalysts14. Among different categories of porous crystalline structure, the newly developed metal–organic frameworks (MOFs) which are constructed from metal ion clusters bridged by multidentate organic linkers, are exclusively exploited in various valuable applications such as catalysis, drug delivery, adsorption, separation and etc.17. These compounds are considered a new generation of catalysts with widespread research interest in recent decades due to their distinctive properties such as tunable pore size, high thermal stability, designable channels, high specific surface area, recoverability, tunable structures, etc. which make them exceptional heterogeneous catalysts20.For this purpose, porous crystalline compounds have attracted Scientific attention toward their research owing to their unique characterization as high surface area and well-ordered structures and porosity23. Amine functional groups are one of the good electrondonating groups that utilize in diverse reactions where basic sites are required26. Thus, MOFs with basic functional groups have been widely investigated for condensation reactions in recent years29. On the other hand, MOFs have organic ligands and unsaturated metal center sites which can undergo post-synthetic modification (PSM). Therefore, designing and applying ligands with appropriate functional groups is a crucial strategy for the fabrication of modified MOFs with novel structural characteristics and desired applications.The presence of basic sites within the structure can promote and accelerate the Knoevenagel condensation reactions and is vital for reaction progressWith the above consideration in mind, in this work, HKUST was synthesized by solvothermal method and various linear amine functional groups were introduced into the unsaturated metal centers of the framework with refluxing method Fig. . The preMaterials and instrumentation are explained in supporting information.30. The obtained solutions were mixed together and stirred for 5 min at room temperature and transferred to a 50-ml Teflon-lined steel autoclave, heated at 120 °C for 24 h. The obtained blue powder was filtered by centrifuging and washed with a mixture of ethanol and deionized water in the same proportion and then activated by drying at 150 °C for 24 h.According to the previously reported procedure, for synthesis, 3.6 mmol cupper (II) nitrate trihydrate (865 mg) and 2 mmol of trimesic acid (420 mg) were separately dissolved in 12 ml of deionized water and ethanol, respectivelyIn a typical procedure, 0.1 g of activated HKUST was suspended in 20 ml toluene following by the addition of 50 µl (0.7 mmol) ethylene diamine. The suspension refluxed for 16 h, cooled to room temperature, isolated by filtration. The resulting participate was washed with dichloromethane to eliminate unreacted ligands and dried at 100 °C for 3h.This compound was synthesized under reflux conditions by treating HKUST and 1,2-phenylene diamine. A solution of 1,2-phenylene diamine  in 30 ml dichloromethane was prepared and added to a suspension containing 0.1 g of activated HKUST in 10 ml toluene. The reaction mixture was refluxed for 16 h and the resulting powder was collected by centrifuging, washed with dichloromethane, and dried at 100 °C for 3h.The synthesis procedure for HKUST-DA is completely similar to HKUST-PhDA synthesis method. The difference is that 70 mg of 1,4-diaminobutane (0.8 mmol) is used instead of 1,2-phenylene diamine.In a typical procedure, 86 µl (0.8 mmol) of diethylene triamine was added to a suspension containing 0.1 g of activated HKUST in 30 ml. The light-blue powder formed was separated by filtration, washed with dichloromethane several times to purify. Therefore, the final product was dried at 100 °C for 3h.To a 25 ml of round-bottomed flask containing 10 ml ethanol, malononitrile  was added. Then 10 mg of the prepared catalyst and 1mmol of benzaldehyde substrate was introduce to the aforementioned solution. The reaction mixture was stirred at room temperature for 5 min. The catalyst was isolated after the completion of the catalytic process and the ultimate filtered solution was characterized by gas chromatography.−1 were related to the out-of-plane deformation vibration of C‒H groups in the phenyl rings31. The emerged bands at 1378 and 1642 cm−1 can be attributed to the symmetric and asymmetric stretching vibration of carboxylate functional groups32. In addition, the broad stretching vibration band of the hydroxyl group of water molecules appeared at 3450 cm−1. The FT-IR spectra of HKUST-ED, HKUST-PhDA, HKUST-DA, and HKUST-DiT were represented in Figs. S1−1. On the other hand, it can be observed that the amount of amine functional groups loaded within the structure is very low  was investigated in Knoevenagel condensation reactions in terms of activity, selectivity, and reusability. For this purpose, benzaldehyde and malononitrile were chosen as substrate models in order to perform Knoevenagel condensation reactions. Different parameters such as time, amount of catalyst, temperature, and solvent were optimized and the results are displayed in Figs S4-7, respectively. According to the results, the condensation reaction was carried out within 5 min and at ambient temperature, in the presence of 10 mg of catalyst with an excellent conversion rate of 100%. In addition, Ethanol was designed as the optimized solvent of the reaction. Based on the results, protic solvents act better than aprotic solvents due to their polarity and the ability to stabilize intermediates Fig. . Both et2 > Cl > Br. Also, the presence of substitution in the ortho position compared to the para position causes more activity of the catalyst , the electron pairs of the amine group are involved in resonance with the aromatic ring. With this regard, despite the proper orientation, a weaker performance was observed compared to ethylenediamine functional groups. In the case of using catalyst 3, the farther distance of amine groups from metal ion centers, and sterically hindrance increment resulted in the worse performance of the catalyst and lower results were observed. Catalyst 4 is the least efficacious catalyst among the utilized catalysts. Although this catalyst has more basic sites, it exhibits weaker activity compared to others due to increased steric hindrance.Knoevenagel condensation reactions were performed in the presence of catalysts with various amine functional groups. The trend of catalytic performance of utilized catalysts is as the following process; In order to investigate the catalytic performance of the synthesized frameworks, a comparison was made between the prepared catalyst and other previously reported heterogeneous catalysts based on MOFs applied in Knoevenagel condensation reaction. The mentioned compounds are compared in terms of activity with each other and the results are listed in Table The recyclability of the prepared catalysts was carried out via consecutive Knoevenagel condensation reactions under the optimized reaction conditions and the results are presented in Figs. In summary, an unsaturated Cu-based MOF, HKUST, was constructed and successfully modified with diverse amine functional groups. The prepared heterogeneous catalysts were applied as efficient catalysts for rapid and facile Knoevenagel condensation reactions. The Knoevenagel products were achieved with high conversion under mild conditions and very short reaction time compared to other previously reported catalysts. Other unique characteristics of the prepared catalysts include recyclability, the cost-effectiveness of the synthesized catalysts, and performing reactions at room temperature.Supplementary Information.


The long-term association between preconception maternal hemoglobin (Hb) concentrations and child health and development is unclear.1) birth outcomes ; 2) child Hb at 3 mo, 6 mo, 12 mo, and 24 mo; and 3) motor and mental development at 12 mo and 24 mo  and cognitive functioning at 6–7 y .We examined associations between maternal preconception Hb concentrations and anemia with n = 1599) through 6–7 y (n = 1318). Multivariable linear and logistic regressions were used to assess the association between preconception Hb or anemia (Hb < 12g/dL) on child health and development outcomes, adjusted by supplementation group (tested for interactions) and confounding at maternal, child, and household levels.We used data from a randomized controlled trial (PRECONCEPT) conducted in Vietnam. Over 5000 women who were intending to conceive were recruited, and offspring were prospectively followed from birth , 6 mo , 12 mo , and 24 mo . Likewise, maternal preconception Hb was associated with reduced risk of child anemia at 6 mo , 12 mo , and 24 mo . Maternal preconception anemia was negatively associated with cognition  and language development  at 24 mo. Preconception Hb was not associated with birth outcomes or cognitive outcomes at 6–7 y.Maternal preconception Hb was associated with child Hb across the first 1000 d of life. However, preconception Hb was not a significant predictor of birth outcomes or cognitive outcomes at 6–7 y in this cohort from Vietnam.PRECONCEPT study (NCT: 01665378). Globally anemia impacts over 571 million women of reproductive age, 32 million pregnant women, and 269 million children . It is aEmerging evidence indicates the timing of maternal anemia during pregnancy may have differential implications for the infant ; however1) birth outcomes ; 2) child Hb at 3 mo, 6 mo, 12 mo, and 24 mo; and 3) motor and mental development at 12 mo and 24 mo and cognitive functioning at 6–7 y. We hypothesize that maternal preconception Hb concentrations will be positively associated with child health and development outcomes across early childhood.We are in a unique position to address this important research gap, using data from a large micronutrient supplementation trial  that was conducted in Vietnam . Women wChildren in this study are offspring of women who participated in the PRECONCEPT study (NCT: 01665378). Details of the PRECONCEPT study have been published previously . Briefly1) birth outcomes, 2) offspring Hb concentration during the first 1000 d, and 3) child development at 12 mo, 24 mo, and 6–7 y.The key outcomes of interest include Birth weight was measured as early as possible, within 7 d after birth, using electronic weighing scales precise to 10 g. Birth length was also measured using standard procedures  with colOffspring Hb concentration was measured from finger prick capillary blood samples at 3 mo, 6 mo, 12 mo, and 24 mo of age using a portable field B-Hb Analyzer (HemoCue 301) (23). Child anemia was defined as a Hb value <11 g/dL .Child development at 12 mo and 24 mo of age was assessed using the Bayley Scales of Infant Development (BSID) III , which iChild intellectual development at 6–7 y was assessed using the Wechsler Intelligence Scale for Children—Fourth Edition (WISC-IV) . The WISThe primary exposure variable was preconception Hb concentration, which was measured at recruitment and prior to receiving supplements by HemoCue 301 from a capillary blood sample obtained by finger prick  using stConfounding variables were considered at child, maternal, and household levels. These included child age and sex, infant and young child feeding practices, childhood illnesses , maternal age, ethnicity, education, intelligence, and depression, intervention group, and duration of intervention prior to conception. Infant and young child feeding practices included early initiation of breastfeeding, exclusive breastfeeding at 6 mo, and dietary diversity at 12 mo and 24 mo using the standard WHO/UNICEF indicators . MaternaP value of 0.01 for birth outcomes (0.05/5 outcomes), 0.013 for Hb concentration (0.05/4 outcomes), 0.017 for development at 12 and 24 mo (0.05/3 outcomes), and 0.01 at 6–7 y (0.05/5 outcomes).The Kolmogorov-Smirnov test was used for testing the normality of the continuous outcome variables. Descriptive statistics were used to report the characteristics of the study population, with frequencies and percentages to describe categorical variables, means, and SDs to describe quantitative variables. Multivariable linear regressions (for continuous outcome) and logistic regression (for binary outcomes) were used to assess the associations between preconception Hb concentration or anemia on birth outcomes, offspring Hb concentration during the first 1000 d, and child development at 12 mo, 24 mo and 6–7 y. Associations were examined as follows: (1) unadjusted; and (2) adjusted for maternal age, ethnicity, education, SES, infant age and sex, intervention group and duration of time prior to conception, infant and young child feeding practices and child illness. Further adjustment for home environment, mother’s IQ, and depression were made in the models with child development at 12 mo and 24 mo and child intellectual development at 6–7 y as the outcomes. Effect modification by the intervention group was evaluated by including interaction terms in all models. In addition, effect modification by home environment was also evaluated by including interaction terms with maternal Hb concentration for models with child development outcomes. All analyses were conducted using Stata v17 . The significance level was adjusted for multiple testing using a Bonferroni adjustment at each domain level, including a The study was approved by the Ethical Committee of Institute of Social and Medicine Studies in Vietnam and Emory University's Institutional Review Board, Atlanta, Georgia, USA. The trial was registered in the US Clinical Trials registry (identification number NCT01665378). Written informed consent was obtained from all study participants.The final analytical sample included 1579 mother-infant pairs women with singleton, live births, and preconception Hb concentration data . SelecteP value that accounted for multiple testing except for child Hb concentration at 3 mo and child anemia at 6 mo.There were no significant interactions with the intervention group, so pooled findings for all models are presented. Neither preconception Hb concentration nor anemia were significantly associated with birth outcomes . HoweverP value was utilized to take into account multiple testing. Although there were no significant interactions with home environment and child cognitive development, stronger associations with maternal preconception anemia and language development were noted in high home environments at 24 mo  compared with those in a low home environment . Maternal preconception Hb and anemia were not significantly associated with child cognition at age 6–7 y , with significant attenuation in the fully adjusted model , ge 6–7 y .TABLE 5Ast 1000 d were weak or mixed. Preconception Hb concentration was also not a significant predictor of birth outcomes or cognition at 6–7 y after adjustment for maternal and sociodemographic variables in our study population.We find that maternal preconception Hb concentration was positively associated with offspring Hb concentrations during the first 2 y of life. In contrast, associations between maternal preconception Hb and anemia and child development during 1Our null findings on maternal preconception Hb concentration and birth outcomes contrast with earlier findings in a systematic review where preconception anemia was associated with an increased risk of low birthweight  and SGA births  , and recIn our cohort, we present novel data on the association of maternal preconception Hb concentration and child Hb concentration across the first 2 y of life. Women who are anemic and/or iron deficient before conception are also at increased risk of getting worse during pregnancy because of the high physiologic demands of pregnancy . MaternaWe report significant associations with maternal preconception Hb concentration on child motor development in the first y of life, particularly in low home environments, but no long-term associations at 24 mo. We also report associations between maternal preconception anemia and child cognition and language development at 24 mo, which were stronger in high home environments for the latter. The rationale for mixed findings on home environment is unclear and merits further examination. It is possible this is because of different mechanisms, as the role of stimulation may vary by child age and outcomes, but it could also be due to chance and recommend caution in interpterion given multiple testing. Periconceptional nutrition may influence early childhood development by affecting the growth and development of key organs, such as the brain, liver, and pancreas, during the first few weeks of pregnancy . HoweverOur study provides novel insight into the importance of maternal anemia during the preconception period and child health and development. This is one the largest prospective studies to date to examine the role of maternal preconception nutrition on long-term child health outcomes. A key strength of this work is the prospective design and the low rates of attrition (<10%) over the past decade. Our design and high-quality data collection allowed for accurate estimation of gestational age and, thus, calculation of key birth outcome variables such as preterm birth and SGA . HoweverIn summary, maternal preconception Hb concentration was associated with child Hb concentrations across the first 1000 d. Modest associations with maternal preconception Hb concentration and anemia and child development in early life merit further examination. However, in this cohort from Vietnam, preconception Hb concentration was not a significant predictor of birth outcomes or long-term child development. Our findings support the need to revisit the use of measuring Hb concentration in women of reproductive age to predict later risk of adverse outcomes, and more research is needed in settings with a greater burden of anemia and iron deficiency as we support programs and policies to expand maternal anemia reduction programs to the adolescent and preconception periods.


Numerous studies have provided evidence that diets rich in anthocyanins show a broad spectrum of health benefits. Anthocyanins in nature are usually found in the form of glycosides. Their aglycone forms are called anthocyanidins. The chemical structure of anthocyanins is based on the flavylium cation, but they differ in the position and number of substituents. However, the bioactives and foods that contain them are frequently treated as a uniform group of compounds exhibiting the same biological activity, without paying attention to the structural differences between individual anthocyanidins. The aim of this study was to find out how structural differences impact the biological activity of the six most common dietary anthocyanidins, i.e., delphinidin (Dp), petunidin (Pt), cyanidin (Cd), malvidin (Mv), pelargonidin (Pg) and peonidin (Po). The study concentrated on redox-related phenomena and compared the following parameters: antioxidant activity , ORAC assay and CAA test (cellular antioxidant activity)), the ability to inhibit growth of human colon cancer cells , and the ability of studied compounds to protect DNA from oxidative damage (comet assay). Based on the obtained results, the relationship between the structure of studied anthocyanidins and their biological activity was assessed. The obtained results revealed that the number and position of the hydroxyl and methoxy groups in the anthocyanidin structure strongly influenced not only the color of anthocyanidins but most of all their antioxidant and biological activities. Anthocyanins are natural pigments found in plants, especially in flowers, tubers, fruits, and leaves . They giThe aglycone forms of anthocyanins are called anthocyanidins. To date, over 1000 structurally distinct anthocyanin molecules and approximately 27 structurally distinct anthocyanidins have been identified . HoweverNumerous studies, mostly carried out in cell culture models, have shown that anthocyanins exhibit a wide range of health-promoting properties, including anti-inflammatory, antioxidant, antimicrobial, and anticarcinogenic activities ,5,6. TheDespite the fact that there is much evidence on the health-promoting activities of anthocyanins, a closer look at the methods used in this research reveals that the conclusions about the anthocyanin’s effects are usually based on whole fruit/vegetable extracts containing these phytochemicals rather than their standard solutions. As shown in our previous studies ,13,14,15The objective of this study was to determine the relationships between chemical structure, antioxidant properties and biological effects for structurally different anthocyanidins. The starting point was the determination of antioxidant activities using popular chemical tests . All these tests were performed under physiologically relevant temperature—37 °C. Subsequently, biological activities, such as cytotoxicity, cellular antioxidant activity (CAA), genotoxicity, and ability to protect DNA from oxidative damage, were assessed in HT29 cells. To the best of our knowledge, no such comprehensive redox-related evaluation has been undertaken before for this set of compounds.10) value was used [10), the higher the antioxidant activity. The results indicate the same trend for the two applied assays, where antioxidant activity of tested compounds increased in the following order: Pn < Pg < Mv < Cd < Pt < Dp.ABTS and DPPH assays have been widely used to determine the antioxidant activity of both plant extracts and pure compound solutions. In most of the published studies, the results of these tests are expressed as EC50 values  or as TEAC values . In this study, a stoichiometric . The replacement of a hydroxyl group with a methoxy group decreased the expected antioxidant activity (Mv < Pt < Dp) and (Pn < Cd). It has been determined that the 3′ and 4′ hydroxyl groups in the structure of anthocyanidins are crucial for the antioxidant capacity of these compounds. These groups can be easily oxidized by donating one or two electrons ,18.Our results are in agreement with those obtained by De Lima , who alsAnother assay employed in our study to compare antioxidant activity of pure anthocyanidins was the ORAC  assay. The results obtained using the ORAC assay confirmed the results obtained by spectrophotometric tests (ABTS and DPPH). According to the assay results, antioxidant activity of tested anthocyanidins increased in the following order: Pn < Pg < Mv < Cd < Pt < Dp .Wang et al.  also tesThe cellular antioxidant activity (CAA) assay was applied to compare the antioxidant properties of tested anthocyanidins in the cell model. This assay is more biologically relevant than other popular assays used for determination of antioxidant activity because it simulates the redox conditions existing in the human body, including physiological temperature and pH conditions, uptake, transport, and cellular metabolism of antioxidants . The humAs shown in Chen et al.  also stuDetermination of cell growth inhibition using the MTT (thiazolyl blue tetrazolium bromide) test is a standard first step in the assessment of biological potential of tested substances. The human colon adenocarcinoma HT29 cell line in our study played a dual role: firstly, as a model of frequent human cancer to compare the postulated ability of different anthocyanidins to inhibit cancer cell growth; and secondly, as mentioned earlier as a model of the alimentary tract, a tissue being in direct contact with ingested food components, including phytochemicals such as anthocyanidins.The obtained results showed that three of the tested anthocyanidins  in a shorter incubation period (6 h) caused significant cell growth stimulation . The comYoshino et al.  studied 2O2-mediated damage; however, some may escalate oxidative damage caused by H2O2, or may even be genotoxic themselves [It has been shown that antioxidants may protect DNA from Hemselves ,11. The 2O2 , phosphate-buffered saline  and dimethyl sulfoxide  ≥ 99.7%), McCoys’ 5A medium , fetal bovine serum , penicillin–streptomycin , low-melting-point agarose , sodium chloride , sodium hydroxide , ethylenediaminetetraacetic acid , 2-amino-2-(hydroxymethyl)-1,3-propanediol , Sybr Green I nucleic acid gel stain . An ORAC assay kit was obtained from Abcam (UK). HPLC-grade methanol was from Merck . Normal-melting-point agarose  was from Bioline . The OxiSelectTM Cellular Antioxidant Assay Kit was from Cell Biolabs, Inc. . Water was purified with a QPLUS185 system from Millipore .The following anthocyanidins were used in the study: pelargonidin chloride  ≥ 97%); cyanidin chloride  ≥ 96%), peonidin chloride  ≥ 97%), delphinidin chloride  ≥ 97%), petunidin chloride  ≥ 95%) and malvidin chloride  ≥ 97%) from Extrasynthese . We purchased from Sigma-Aldrich : 1-diphenyl-2-picrylhydrazyl ), 2,2-azinobis- diammonium salt ), sodium thiosulfate, thiazolyl blue tetrazolium bromide ), hydrogen peroxide  as described before [HT29 (human colon adenocarcinoma) cells purchased from the ATCC were grown in McCoy’s medium supplemented with L-glutamine (2 mol/L), sodium pyruvate (200 g/L), fetal bovine serum (100 mL/L), and antibiotics (100 U/mL penicillin and 100 g/L streptomycin). Cells were maintained at 37 °C under a humidified atmosphere with 5% COd before . Culture10), as described by Kusznierewicz et al. [10). The concentration of radicals scavenged by the tested antioxidants in reaction media was calculated with the use of the Beer–Lambert–Bouguer law (Beer’s law).The colorimetric determination of antioxidant activity was carried out by standard SET mechanism assays employing ABTS and DPPH radicals as described previously ,28 with z et al. . This paex = 485 nm and λem = 528 nm using the Tecan Infinite M200 spectrophotometer . The plate reader generated the plot of fluorescence change in time for each sample. To determine the antioxidant capacity of the tested compounds, the area under the curve (AUC) for the obtained plots was calculated for tested samples and for standard solutions of trolox. To calculate the antioxidant activity of the tested samples, standard curves for trolox were plotted and antioxidant activity of the tested anthocyanidins was expressed as trolox equivalents (uM).The ORAC  test is a fluorescent method using the HAT mechanism. In this method, a fluorescent probe is oxidized in the reaction with reactive oxygen species (ROS) to a form that does not emit fluorescence. Antioxidants present in the sample are able to inhibit the decrease in probe fluorescence. . For the4 cells per well in 0.15 mL of medium). The cells were allowed to settle for 24 h at 37 °C. Then different concentrations of tested anthocyanidins were added to the cell culture medium and incubated for 6 or 24 h. Final concentrations of compounds in culture medium ranged from 100 nM to 1 mM. In the cases of shorter exposure, the medium was aspirated from the wells, replaced with 0.2 mL of fresh medium, and the cells were incubated at 37 °C until 24 h of the total incubation time. After 24 h of incubation, MTT solution (4 g/L) was added (0.05 mL per well) and the multiwell plate was incubated for another 4 h at 37 °C. Finally, medium was carefully removed from wells and formazan crystals formed by metabolically active cells were dissolved in 0.05 mL of DMSO. The absorbance of resultant solutions was measured at 540 nm with the Tecan Infinite M200 plate reader . Three independent replicates of each treatment were performed. Cytotoxicity was expressed as percentage growth inhibition of cells exposed to tested antioxidants compared to control cells treated with the appropriate volume of solvent only, whose growth was regarded as 100%.The MTT test was performed to assess the ability of tested anthocyanidins to inhibit the growth of HT29 cells, as described before . Briefly4 cells per well in 0.2 mL of medium). The cells were allowed to settle for 24 h at 37 °C and then were treated with 0.05 mL of different concentrations of anthocyanidins for 1 h. Final concentrations of investigated compounds ranged from 1 to 1000 μM. Control cells were treated with the corresponding solvent. Emission of fluorescence at 538 nm was measured every 5 min for 1 h after excitation at 485 nm using the Tecan Infinite M200 spectrophotometer . Three independent replicates of each treatment were performed.The cellular antioxidant activity (CAA) of investigated compounds in HT29 cells was studied by CAA assay using the OxiSelectTM Cellular Antioxidant Assay Kit  according to the producer’s manual, as described before . Briefly5 cells per well in 1.8 mL of McCoy’s medium) and left to settle for 24 h at 37 °C. The next step involved the treatment of cell cultures with different concentrations (from 1 to 100 μM) of tested anthocyanidins for 24 h. In the case of negative controls, the cells were treated with the solvent only, while positive control cells were treated with 150 μM H2O2 solution for 1 h. For the experiments in which the protective effect against oxidative stress was assessed, an additional step was to treat the cells with 150 μM H2O2 solution for 1 h. After treatment, the cells were detached using 0.2 mL of trypsin (0.5 g/L) solution. The cell suspensions were centrifuged . The cell pellets were washed with 1 mL of PBS and centrifuged  again. After centrifugation, approximately 40 μL of the supernatant was left in the tube to resuspend cells. The cell suspension (30 μL) was mixed with 150 μL of 0.5% LMP (low melting point) agarose. Finally, 30 μL of this mixture was placed as two spots on a microscope slide precoated with 1% normal-melting-point agarose (NMP agarose) and left to set on an ice-cold tray. Three slides with two repetitions on each were prepared for every concentration of the tested anthocyanidins. After overnight lysis in a high-salt alkaline buffer (pH 10), the slides were left in electrophoresis buffer for 20 min before electrophoresis. Electrophoresis was conducted at 26 V and 300 mA for 30 min in darkness at 4–8 °C. After electrophoresis, the slides were transferred to neutralizing buffer (for 5 min). This step was repeated twice. Then, slides were washed using distilled water and fixed in 70% ethanol. Next, the DNA was stained with SybrGreen in TE buffer for 20 min. After staining, the slides were washed with distilled water for 5 min. Finally, DNA “comets” were examined under a fluorescence microscope  connected to a computerized slide scanning system . Comet analysis involved counting 200 consecutive nuclei per sample. The mean percentage DNA in the comet tail was a measure of genotoxic potency of compounds tested. As for the protection from oxidative damage experiments, results were recalculated and expressed as percentage control, where control cells were treated only with H2O2.Genotoxic properties of tested anthocyanidins and their ability to protect DNA from oxidative stress were determined using a comet assay according to the procedure described before ,11. Briep ≤ 0.05.All values are expressed as means ± SD of three independent determinations. The statistical significance of determinations was examined by one-way ANOVA with Tukey–Kramer test or one-way ANOVA with Dunnett’s test. All statistical analyses were performed using Prism 4.0 software package . The level of statistical significance was set at This study aimed to determine the relationship between structure and biological activity for the six most common anthocyanidins. The tested compounds included delphinidin chloride, petunidin chloride, malvidin chloride, peonidin chloride, cyanidin chloride and pelargonidin chloride. Antioxidant activity tests results obtained by three different methods  showed the same trend, where antioxidant activity increased in the following order: Pn < Pg < Mv < Cd < Pt < Dp. The relationship between the structure of tested anthocyanidins and their activity could be observed. The presence of hydroxyl groups increased antioxidant activity, while methoxy groups had an opposite effect. In the CAA test conducted on HT29 cells, the antioxidant activity was at the same level for the concentration of 1000 μM. For concentrations below 10 μM, anthocyanins showed a pro-oxidative character. Delphinidin, malvidin and petunidin at 1000 μM concentration showed the greatest ability to inhibit the growth of HT29 cells in the MTT test. These anthocyanins have substituents in the 3′, 4′, and 5′ positions, which makes them the most cytotoxic for cells after entering them. Results obtained by comet assay showed that tested anthocyanidins did not have genotoxic potential, and only Pt showed significant ability to protect DNA from oxidative damage in tested conditions. The chemical structure did not show significant influence on this activity. This study proved that the presence of hydroxyl and methoxy groups has a significant influence on biological activities of tested anthocyanidins. The hypothesis that this group of compounds should not be treated as they all have the same biological potential has been confirmed. This knowledge can be used by producers of functional food and dietary supplements while designing products containing anthocyanins. Future research should confirm these findings in more in vitro and then in vivo models to verify whether different biological activities of anthocyanidins can also be observed in different models.