P < 0.05) compared to WD. OPP-D increased caecal digesta mass, caecal digesta concentrations of total SCFA, acetate and propionate , the numbers of mucus-producing goblet cells per colonic crypt, and caecal digesta abundance of some bacteria which may provide benefit to the host . HAMS induced similar effects but with greater potency and had a broader impact on microbe populations, whereas GT had minimal impacts. These results suggest dietary OPP may benefit the large bowel.Oil palm fruit is widely used for edible oils, but the health benefits of other components are relatively unknown. We examined if consuming a polyphenol-rich extract of the fruit, from a vegetation by-product of oil processing, which also contains fibre, has gastro-intestinal benefits in rats on a Western-type diet (WD). The oil palm preparation (OPP) was added to food (OPP-F) or drinking water (OPP-D) to provide 50 mg of gallic acid equivalents (GAE)/d and compared to effects of high amylose maize starch  in the diet or green tea extract  in drinking water over 4 wk. OPP treatments induced some significant effects ( Elaeis guineensis, is a significant global food source, being the world’s largest source of edible oils, and of high economic value to numerous countries [The fruit of the African oil palm, ountries . The genountries . The oilThere is growing evidence that the consumption of plant polyphenols can have many health benefits in humans  and oil in vivo benefits. One of the most widely recognised benefits of dietary fibre is the strong association with the reduced risk of colorectal cancer (CRC) [Dietary fibres are important components of a healthy diet and the oil palm fruit is likely to be a rich source of dietary fibres, which have yet to be characterised or examined for their er (CRC) . The bener (CRC) . Experimer (CRC) ,16,17,18Recently, a novel preparation rich in polyphenols was extracted from the vegetation liquor generated during the milling and extraction of oil from the oil palm fruit . This oin = 10–13 per group) and fed the experimental diets for 4 wk. The composition of experimental diets . To simulate a western-type diet (WD), all diets contained 25% cooked red meat (premium beef mince) and the fat used in the diets was sunflower oil  which contained approximately 11% SFA, 20% MUFA, and 69% PUFA. The basal diet is referred to as the WD diet. OPP was added to the WD diet. OPP and green tea were also added to the drinking water and maintained at 50 mg gallic acid equivalents (GAE)/d . GAE was determined by assessing total phenol and other oxidation substrates and anti-oxidant content using the Folin–Ciocalteu method. OPP contained 3.5 g/100 g of total dietary fibre (TDF), as determined by a modification of AACC Method 32-05. That is, the homogenised sample (1 g) was treated sequentially with α-amylase at 100 °C, protease at 60 °C and amyloglucosidase at 60 °C to hydrolyse all of the starch and proteins in the sample. The fibre was precipitated by the addition of ethanol to make the final mixture 80% v/v ethanol. After standing overnight, the precipitated fibre was collected by filtration through a bed of Celite. The collected fibre was determined by weight after subtractions for ash, protein and enzyme blanks. Individual body weights were monitored throughout the study. At the completion of the dietary intervention period, rats were anesthetised with 4% halothane/oxygen to allow collection and weighing of gut tissues and digesta at the time animals were killed. Faecal and caecal digesta was homogenised and divided into aliquots for various analyses and frozen at −80 °C. Experimental procedures were approved by the animal ethics committee at CSIRO Health and Biosecurity and complied with the Australian code of practice for the care and use of animals for scientific purposes.Fifty-six male Sprague-Dawley rats of ~200 g weight were obtained from the Animal Resource Centre, Murdoch University, Perth, Australia. They were housed in wire-bottomed cages in a room of controlled temperature (23 °C) and lighting (a 12 h light-dark cycle) and allowed free access to food and water. The rats were assigned randomly to 1 of 5 groups , followed by the separation of SCFA in the distillate, using gas liquid chromatography, as described previously . The totd before ,24. PoolDNA was extracted from thawed digesta by a process involving repeated bead beating in the presence of high concentrations of SDS, salt and EDTA, followed by purification using QIAamp columns . PurifieColonic length was measured and 0.5 cm of tissue at the distal and proximal end was discarded. Samples for histological assessment (2 cm) were taken at 8 cm from the distal end. Colonic tissue was opened along the section length, cleaned of digesta, placed in histology cassettes, submerged in 10% neutral buffered formalin for 24 h and then stored in 70% ethanol until processed through graded levels of ethanol and chloroform before being embedded in wax. Embedded tissues were cut into 5 μm sections and were stained for mucins using Alcian blue  and periodic acid-Schiff’s reagent (sulphomucins) in combination with previously described methods . HistoloFor wall thickness, 15–20 measurements were taken at different points along each section. Final wall thickness was reported as the average thickness (in µm) over the length of the wall. Crypts were measured from the base of the muscularis mucosa to the luminal surface. Crypt cell height was also recorded as the number of columnar epithelial cells on one side of the crypt. The total number of columnar epithelial cells (both sides of the crypt) were counted and recorded from an average of 8–10 crypts per section. Goblet cells in the crypt were counted and the total area of the goblet cells within each crypt was measured by drawing around each individual goblet cell and adding the areas together. The crypt cell area was calculated by drawing around individual crypts used for assessment. The total area of the goblet cells was expressed as a percentage of the crypt area. Goblet cells were recorded as containing either strongly sulphated mucins  or neutral mucins .P < 0.05. Statistical analysis was performed using GraphPad Prism version 6.01 for Windows, GraphPad Software, La Jolla, California, USA.All values are expressed as the mean ± standard error of mean (SEM). Comparisons were performed by one-way ANOVA followed by Tukey’s multiple-comparison post hoc test. Differences were considered to be significant at There were no significant differences in final body weight between the treatment groups (data not shown) or weights of organs . Caecal digesta bulk  was signThe pH of the caecal digesta was lowered significantly relative to the WD group by HAMS but not by other treatments. In digesta from the colon, the pH was significantly lowered by HAMS relative to WD and both OPP treatments. Colonic digesta pH was significantly higher than WD for OPP drink .Levels of individual and total SCFA in digesta  were sigp-cresol were significantly lowered by HAMS relative to WD in the caecum and in the colon.Protein fermentation products were also analysed in digesta and shown to be impacted by diet . AmmoniaFaecalibacterium prausnitzii, Akkermansia muciniphila and Ruminococcus gnavus. The positive control, HAMS, elicited the greatest number of changes in the microbiota, significantly increasing  the numbers of Ruminococcus bromii, the Clostridium leptum group, Bifidobacterium, A. muciniphila and Ruminococcus torques. The bacteria of the genus Roseburia decreased in number in response to HAMS. The numbers of Lactobacillus were increased by GT but not by OPP or by HAMS, and the numbers of Bacteroides were significantly higher for OPP drink when compared to GT.Dietary treatment had a significant impact on the composition of bacterial populations in the caecal digesta, although total numbers of bacteria/g of digesta did not differ significantly between the treatments . When raHistological analysis of colonic tissue  revealedThis study examined whether a polyphenol-rich, fibre-containing extract from a vegetation liquor generated from the processing of the oil palm can have GI benefits in rats consuming a Western-type diet typically high in meat and fat. Results show that OPP consumption had a range of effects consistent with promotion of GI health. These include increased digesta mass, increased production of SCFA, shifts in digesta microbiota populations that are suggestive of benefit, and increases in mucus-producing goblet cells within colonic crypts. These effects were in many instances similar to those induced by dietary HAMS, a source of RS known to have multiple benefits to the large bowel, although OPP generally exhibited a lower potency. There were no obvious detrimental effects of OPP on gut health, although the consequences of the increase in colonic digesta pH are unknown. Consequently, there is potential for development of OPP or its constituents for use in humans to promote bowel health.Consumption of dietary fibres has long been known to be associated with a reduced risk of colorectal diseases, especially cancer, and this reduced risk occurs through multiple mechanisms which can be dependent on the type of fibre. A primary means of benefit occurs as a result of the promotion of stool bulk, which acts to dilute toxins and assist their excretion ,29,30. IAnother mechanism through which dietary fibres may protect against colorectal disease is by the production of SCFA through microbial fermentation. Highly fermentable fibres, such as RS, can generate significant amounts of the main forms of SCFA, namely acetate, butyrate and propionate, and these in turn have multiple effects and benefits . In addiLactobacillus spp. and Bifidobacterium spp.; also commonly used as probiotics) but also because of an ability to alter the numbers of other microbes with emerging roles in health [Faecalibacterium prausnitzii, a key butyrate-producer with independent anti-inflammatory activity [Akkermansia muciniphila and Ruminococcus gnavus, are low in the large bowel of individuals with inflammatory bowel disease [A. muciniphila. However, HAMS increased the numbers of a different Ruminococcus species associated with mucus, namely Ruminococcus torques [F. prausnitzii but did increase the numbers of the Clostridium leptum group, which contains many butyrate-producing bacteria. Some members of the genus Roseburia can also produce butyrate, and interestingly, the numbers of this genus were down in response to HAMS but not affected by OPP. HAMS increased the numbers of the starch degrader Ruminococcus bromii, as shown to occur previously in the stool of humans in response to dietary RS [Escherichia coli and Enterococcus faecium , and the Bacteroides genus. The numbers of potentially beneficial Lactobacillus were increased by GT but not by OPP or by HAMS. Although this suggests that the polyphenols in GT but not OPP are able to stimulate Lactobacillus growth, it also confirms that dietary polyphenols can promote the growth of beneficial bacteria. One of the most promising findings of the study relates to the effects of OPP on the composition of gut microbiota populations. There is a growing recognition of the important role that gut microbes play in GI health and also in mediating the health of tissues distant to the gut through mechanisms such as modulation of the immune system. Microbial products, such as butyrate, appear to have an important role in facilitating these effects . Consequn health . Our stuactivity . The num disease ,42 and i disease . Overalletary RS . HoweverLactobacillus, suggesting some benefit to the gut. This indicates that the observed beneficial impacts of OPP on the large bowel are unlikely to be largely a result of polyphenols, although it is possible that GT and OPP polyphenols differ substantially in their characteristics and biological effects. Polyphenols derived from OPP may have benefits for tissues or activities that were not examined in this study and this should perhaps be explored in future work.While many of the effects we have observed in response to OPP suggest that fibre plays a central role, it is not possible to eliminate significant roles for polyphenols or other components of the OPP extract. We included GT as a treatment in this study, as it is a well-known source of polyphenols and has been demonstrated to have numerous beneficial biological impacts in vivo (5–7), including anti-microbial effects against some gut pathogens . HoweverLevels of fibre in the OPP extract, and consequently in the respective treatments, are relatively low when compared to the amount of fibre given in the HAMS treatment, and this is likely to explain the lower potency of many of the beneficial effects seen with the OPP treatments. Purification of the fibre component from the OPP extract for future studies will allow a better assessment of the physical characteristics of the fibre(s) and allow more meaningful comparisons with HAMS and other beneficial fibres when examining physiological effects in vivo. The method of analysis used to determine the amount of fibre present in the OPP extract does not provide information on fibre type.The effects observed suggest the route of OPP administration (food or drink) can influence physiological outcomes. Both routes of administration were designed to deliver similar doses of OPP on the basis of calculations of intakes and levels of inclusion for drink or food. The reasons for our finding of generally greater effects with the drink form of OPP administration can only be speculated upon. One possibility is that the incorporation of OPP into the food matrix hinders its availability to microbes and tissues within the large bowel to a greater degree than occurs for the liquid form. This should be taken into consideration in developing any dietary treatments that incorporate OPP or its constituents.In conclusion, we provided evidence that the consumption of OPP can influence several markers of large bowel health that are indicative of benefits. Further studies to ascertain the constituents responsible for these benefits are warranted.


Patients on peritoneal dialysis are at risk of developing peritoneal fibrosis and angiogenesis, which can lead to dysfunction of the peritoneal membrane. Recent evidence has identified cross‐talk between transforming growth factor beta (TGFB) and the WNT/β‐catenin pathway to induce fibrosis and angiogenesis. Limited evidence exists describing the role of non‐canonical WNT signalling in peritoneal membrane injury. Non‐canonical WNT5A is suggested to have different effects depending on the receptor environment. WNT5A has been implicated in antagonizing canonical WNT/β‐catenin signalling in the presence of receptor tyrosine kinase‐like orphan receptor (Ror2). We co‐expressed TGFB and WNT5A using adenovirus and examined its role in the development of peritoneal fibrosis and angiogenesis. Treatment of mouse peritoneum with AdWNT5A decreased the submesothelial thickening and angiogenesis induced by AdTGFB. WNT5A appeared to block WNT/β‐catenin signalling by inhibiting phosphorylation of glycogen synthase kinase 3 beta (GSK3B) and reducing levels of total β‐catenin and target proteins. To examine the function of Ror2, we silenced Ror2 in a human mesothelial cell line. We treated cells with AdWNT5A and observed a significant increase in fibronectin compared with AdWNT5A alone. We also analysed fibronectin and vascular endothelial growth factor (VEGF) in a TGFB model of mesothelial cell injury. Both fibronectin and VEGF were significantly increased in response to Ror2 silencing when cells were exposed to TGFB. Our results suggest that WNT5A inhibits peritoneal injury and this is associated with a decrease in WNT/β‐catenin signalling. In human mesothelial cells, Ror2 is involved in regulating levels of fibronectin and VEGF. We examined the interaction between TGFB and WNT signalling in the development of peritoneal fibrosis and angiogenesis. We observed WNT/β‐catenin signalling to be active during TGFB‐induced peritoneal membrane injury, and our results suggest cross‐talk with the TGFB pathway during the development of injury.The majority of studies in fibrotic diseases have focused on the role of β‐catenin dependent signalling. There are few studies on the pathological role of non‐canonical WNT5A in fibrosis. Physiologically, WNT5A has been observed to regulate cell polarity and intracellular calcium levels through the planar cell polarity pathway (PCP) and calmodulin‐dependent protein kinase II pathway, respectively.In studies of tumorigenesis, WNT5A has been reported to have opposing effects. WNT5A has been shown to work as a tumour suppressor in specific types of cancer.WNT5A has been observed to mediate signalling through receptor tyrosine kinase‐like orphan receptor 2 (Ror2) and through FZD 3, 4, 5 and lipoprotein receptor‐related proteins (LRP) 5 and 6. WNT5A and Ror2 have been demonstrated to interact physically and also functionally by activating the PCP pathway to regulate the cytoskeleton during embryogenesis.The role of WNT5A and Ror2 in peritoneal membrane injury has not been explored. Aberrant WNT/β‐catenin signalling has been observed to contribute to progressive fibrosis in several different organs.22.1The construction of the adenovirus vector AdTGFB has been previously described. AdTGFB expresses constitutively active TGFB1.2.28 plaque‐forming units (pfu) of first generation AdDL , AdWNT5A , AdTGFB + AdDL  or AdWNT5A + AdTGFB . AdTGFB or AdWNT5A was diluted to 100 mL in phosphate‐buffered saline. Animals were infused with a peritoneal dwell of 4.25% glucose dialysis solution an hour prior to kill. Animals were anesthetized with isoflurane  and killed at day 10 using anaesthesia and exsanguination.All animal studies were performed according to the Canadian Council on Animal Care guidelines. All animal studies were approved by McMaster University's Animal Research Ethics Board (AREB). Animals were housed under specific pathogen‐free conditions and were administered treatments in their home cage. Mice  were injected intrapertioneally with 1 × 10At sacrifice, the entire anterior abdominal wall was then removed and divided into two sections. The lower section was placed in 10% formalin for histological analysis, and the upper section was placed in 1 mL Easy‐blue™ reagent (Froggabio) for RNA isolation. Omental tissue was taken and frozen in liquid nitrogen for protein analysis.2.3Human mesothelial cells (MET5A)  were cultured in M199 media (Sigma‐Aldrich Canada Ltd). In the first experiment, human MET5A cells were transfected with siRNA against Ror2  or a non‐targetting siRNA (Fisher Scientific) for 16 hours. Following this, serum‐starved cells were treated with AdWNT5A (10 MOI) for 24 hours. In the following experiment, human MET5A cells were transfected with small interfering RNA (siRNA) against Ror2  or non‐targetting siRNA (Fisher Scientific). Twenty‐four hours after transfection, serum‐starved cells were treated with rTGFB (R&D Systems) at 2.5 ng/mL or PBS for 8 hours. Cells were harvested to collect supernatant, protein and RNA.2.4Tissue samples were fixed in a sufficient amount of 10% neutral‐buffered formalin for 48 hours, followed by 70% ethanol. The tissue samples were processed, paraffin embedded and cut in 5‐mm sections. Sections were stained with Masson's trichrome. Sections were also stained using vonWillebrand Factor antibody/Factor VIII (Dako) to measure blood vessel density. Five images were taken per stained section from each animal. Image J was used to quantify blood vessel density and thickness of the submesothelial zone.2.5Immunofluorescence was performed by using a monoclonal mouse anti‐human α smooth muscle actin (α‐SMA) antibody (1:50) (Dako) with a Texas Red goat antimouse secondary antibody (1:100)  followed by a FITC‐labelled monoclonal mouse antibody against pan‐cytokeratin (1:50) (Sigma‐Aldrich). Slides were mounted with nuclear Dapi (Vector Laboratories). Quantification of dual‐labelled cells in the mesothelial and submesothelial zones was completed using ImageJ software . Cells co‐expressing α‐SMA and pan‐cytokeratin were classified as dual labelled. Dual‐labelled cells were identified in the mesothelial cell layer and in the submesothelial cell layer from five high‐power fields from each section using a blinded approach.2.6Protein was extracted from frozen omental tissue by homogenization in a standard lysis buffer with proteinase inhibitors. Equal amounts of the protein were run on a 8% or 10% SDS‐PAGE gel, transferred to a membrane, and probed with VEGF , pGSK3β , GSK3β , total b‐catenin , WNT5A , Ror2 , Fibronectin , β‐actin  and α‐Tubulin . Western blot analysis was completed in Image J using the Gel Analyzer Program.Concentration of human VEGF was measured in cell culture supernatant using a standard ELISA kit (Sigma‐Aldrich Canada Ltd) according to the manufacturer's instructions.2.7RNA was extracted from MET5A cells according to manufacturer's instructions (Invitrogen). The concentration of RNA obtained was measured by nanodrop. 1 μg of RNA obtained was reverse transcribed by RT‐PCR. Gene expression of VEGF was determined by 7500 Real Time PCR (Applied Biosystems). Samples were run in duplicate and normalized to 18s housekeeping gene. A standard curve generated from pooled mRNA samples was used for comparative quantification. Results are reported relative to 18s RNA.2.8t test. Differences between multiple groups were assessed using ANOVA with Tukey's post hoc analysis. Analysis was carried out using SPSS v 22.0 (IBM).Differences between two groups were evaluated using 33.1P = .001) compared with AdTGFB alone      Figure G‐I,M. Co3.3P = .008) , and this effect was attenuated with the co‐expression of AdTGFB and AdWNT5A (P = .025)  , and levels were reduced after treatment with both AdTGFB and AdWNT5A (P = .008)  Figure A,B. AdTG) Figure C,D. To f) Figure E,F.3.4P = .022)   Figure A,B. Ror2) Figure C,D.3.5P = .005).We observed that WNT5A inhibits angiogenesis and submesothelial thickening in the presence of Ror2 in this model of peritoneal membrane injury. Existing evidence suggests that WNT5A has differential roles in fibrogenesis based on the receptor context and inhibits WNT/β‐catenin signalling in the presence of Ror2.3.6Our observations suggested that WNT5A and Ror2 work together to regulate levels of fibronectin in human mesothelial cells. Thus, we sought to understand the role of Ror2 in the context of peritoneal membrane injury including fibrosis and angiogenesis. We expanded our investigations using a TGFB model of mesothelial cell injury. We exposed MET5A cells to recombinant TGFB to induce mesothelial cell injury and used an siRNA to silence Ror2. We examined fibronectin and VEGF levels as markers of fibrosis and angiogenesis.P = .002), and this effect was amplified with Ror2 silencing (P < .01)  , and this effect was exacerbated in response to Ror2 silencing (P = .008)  Figure A,B. We a) Figure C. Using ) Figure D.4Patients undergoing peritoneal dialysis begin to develop a change in peritoneal membrane structure including fibrosis and angiogenesis. Peritoneal fibrosis has been associated with a decline in ultrafiltration capacity by altering hydraulic conductance of the membrane observed in many biopsy studies.In this study, we assessed the role of WNT5A, in the development of both fibrosis and angiogenesis and observed that it reduces both elements of peritoneal membrane injury. Addition of WNT5A to the mouse peritoneum resulted in reduced submesothelial thickness and blood vessel density in TGFB‐induced peritoneal fibrosis. Accumulating evidence has shown that WNT5A exhibits distinct effects depending on the cell surface receptor present. Studies of angiogenesis report that WNT5A can either stimulateWe further examined the function of this receptor in injured mesothelial cells by silencing Ror2. With the addition of AdWNT5A, we observed an increase in levels of fibronectin when Ror2 was inhibited. We expanded our investigation to examine a TGFB model of mesothelial cell injury. Overexpression of TGFB induced an increase in fibronectin and VEGF. Interestingly when Ror2 was silenced, increases in fibronectin and VEGF protein were observed. This suggests that Ror2 potentially has some protective capacity in injured mesothelial cells by regulating fibrotic and angiogenic factors.In contrast to our observations, WNT5A and Ror2 were shown to contribute to injury in a recent study of renal fibrosis.It has become apparent that WNT5A can also regulate β‐catenin dependent signalling. Mikels and colleagues, observed the dual nature of WNT5A where it could both activate and inhibit the canonical WNT/β‐catenin signalling pathway depending on the cell surface receptor that was present. In the presence of FZD4, WNT5A increased β‐catenin levels. Paradoxically, in the presence of Ror2, WNT5A suppressed β‐catenin activity in vitro.The peritoneal membrane is a lifeline for patients with end‐stage kidney disease on peritoneal dialysis therapy. We know that injury to the peritoneum increases the risk of technique failure and mortality. This current research expands potential therapeutic options for preservation of the peritoneal membrane in PD patients. Based on our findings, we suggest that WNT5A blocks the canonical WNT pathway to reduce both fibrosis and angiogenesis in the peritoneum and Ror2 is a potential candidate for mediating these effects. Although further investigation is necessary, the dual nature of WNT5A may be used as a method of targeting and blocking aberrant WNT/β‐catenin signalling to prevent progression of injury.No conflicts of interest, financial or otherwise, are declared by the authors.MP and PJM conceived and designed research, analysed data, interpreted results of experiments, prepared figures, edited and revised manuscript, and approved final version of manuscript; MP and LL performed experiments; and MP drafted manuscript. Click here for additional data file.


Epoxy resins are widely used for different commercial applications, particularly in the aerospace industry as matrix carbon fibre reinforced polymers composite. This is due to their excellent properties, i.e., ease of processing, low cost, superior mechanical, thermal and electrical properties. However, a pure epoxy system possesses some inherent shortcomings, such as brittleness and low elongation after cure, limiting performance of the composite. Several approaches to toughen epoxy systems have been explored, of which formation of the interpenetrating polymer network (IPN) has gained increasing attention. This methodology usually results in better mechanical properties  of the modified epoxy system. Ideally, IPNs result in a synergistic combination of desirable properties of two different polymers, i.e., improved toughness comes from the toughener while thermosets are responsible for high service temperature. Three main parameters influence the mechanical response of IPN toughened systems: (i) the chemical structure of the constituents, (ii) the toughener content and finally and (iii) the type and scale of the resulting morphology. Various synthesis routes exist for the creation of IPN giving different means of control of the IPN structure and also offering different processing routes for making composites. The aim of this review is to provide an overview of the current state-of-the-art on toughening of epoxy matrix system through formation of IPN structure, either by using thermoplastics or thermosets. Moreover, the potential of IPN based epoxy systems is explored for the formation of composites particularly for aerospace applications. C or critical strain energy release rate GC . L. L1/2) w1/2, respectively, at this concentration . Above this content of SiMPU, the enhancement in the fracture toughness and impact strength of epoxy system was negligible ..−1) [1/2nco) being slightly over- or under-stoichiometric, 0.95 resp. 1.03, of polyurethane prepolymer on the mechanical properties of modified epoxy systems has also been investigated [nco, hence being terminated with a isocyanate group (opposed to a hydroxyl group termination for a low Inco). The impact strength was maximally enhanced by adding 20% of polyurethane with highest isocyanate index while the addition of 15% of the toughener was enough to obtain the best tensile strength and elongation at break [In addition to the molecular weight of components, the effect of isocyanate index  and poly(m-BAB/SPB)) have been prepared through azide–alkyne polymerization reaction . The in-Tg while the phase separated one and the two Tg of its constituents. The Fracture toughness of the homogeneous morphology was higher at low PES contents (up to10%) and become more effective in the phase separated case for higher PES contents only. The processing viscosity of the uncured systems increases a lot due to the high molecular weight of the PES.In addition to the above mentioned parameters, curing temperature can also affect the mechanical properties  of the toughened epoxy resin. For instance, exploiting the phase behavior, Mimura et al.  investigIn summary, the effect of several parameters on the mechanical properties  of the toughened epoxy system has been investigated in literature. The increase in toughener content typically lead towards the gradual increase in fracture toughness of epoxy resin, which is attributed to the formation of IPN structure and phase separated morphologies ,38,44. HAs seen in the previous chapter, the incorporation of a ductile thermoplastic phase in a brittle epoxy resin usually results in a viscosity increment, which in turn affects the process ability of the epoxy system . FurtherFor example, Fu et al.  reportedTg of 303–312 °C, tensile strength of 87–95 MPa and impact strength of 27–37 kJ·m−2. These enhancements were attributed to the presence of high fluorine content, low crosslink density, high chain rigidity, and strong intra- and intermolecular interactions. This study provides some useful knowledge about developing modified epoxy resins having higher toughness without sacrificing their excellent mechanical and thermal features [Wen et al.  investigfeatures . Similarfeatures .oil (ESO) and poly (PFA) . SEM imaWu et al.  reportedIn conclusion, apart from toughening of epoxy resin with thermoplastics, different thermosetting polymeric systems have also been examined to toughen epoxies. The incorporation of thermosetting toughener usually results in enhanced fracture toughness and impact strength of epoxy system ,68, whicThe enhancement in the damage tolerance of epoxy matrices of fibre reinforced composites has become a serious concern particularly for the aerospace industry, and consequently, toughening of epoxy resin has received significant attention. As already discussed, the formation of (semi-)IPN structures can lead to the systems having higher fracture toughness and other mechanical properties i.e., elongation at break, tensile strength and modulus. Furthermore, this increase in mechanical properties can further be tuned by playing with different parameters such as toughener content, structure and molecular weight of toughener components, curing agent, synthesis parameters, etc. Therefore, in this section, this enhancement in fracture toughness of epoxy-based fibre-reinforced composites commonly used for aerospace applications by forming (semi-)IPN structure will be discussed. Furthermore, the change in morphology by incorporating fibre into toughened epoxy will be explained.Likewise, glass fibre reinforced composites were prepared by using a mixture of PEN and BA-ph as a matrix . The resThe efficient joining of different parts, in order to prepare a complete component, is very crucial for aerospace applications. Therefore, the mechanical properties of adhesives, used to join steel and fibre-reinforced polymeric components, have also been modified by using the approach of IPN structure. The authors reported the formation of micro-phase separated IPN using DGEBA/PDCPD and investigated their adhesive performance as compared to the neat epoxy adhesive . The blePolyetherimide (PEI) belongs to the class of amorphous engineering thermoplastic and has an interesting set of properties including high glass transition temperature of 217 °C, higher strength and rigidity at elevated temperatures, lower shrinkage, intrinsic flame retardancy, higher moisture resistance, higher dielectric resistivity, lower coefficient of thermal expansion, etc. . MoreoveThe fracture toughness of high performance epoxy resin can be enhanced by incorporating PEI or a modified form of PEI. Curing of an epoxy resin in the presence of PEI, can lead to local dissolution of the PEI in the epoxy, followed by a reaction-induced phase separation. This inter-diffusion process results in the formation of semi-IPN structure at the epoxy-PEI interface. The diffusion of PEI into the epoxy resin increases its plasticity and also induces stress relaxation via local shear deformation. The increment in toughness is mainly controlled by the phase separation between PEI and epoxy during curing. Moreover, the fracture energy is dissipated by the ductile deformation of dispersed PEI phase, which occurs by the stress transfer through semi-IPN structure .The effect of PEI content on the phase morphology and mechanical properties of toughened epoxy system has been investigated in literature ,33,79,80The analysis of mechanical properties of epoxy/PEI system showed that maximum toughening was obtained with 20 wt % of PEI b, which In addition to the toughening of epoxy resin, toughened polymeric composites have also been reported in literature. For instance, Turmel & Partridge  preparedTg, higher strength and rigidity at elevated temperatures, good compatibility with epoxy, etc. The incorporation of PEI in epoxy resin usually results in the formation of a semi-IPN structure, which eventually provides the improved mechanical characteristics, i.e., fracture toughness of toughened epoxy system. For example, elongation at break and fracture toughness of BADCy/PEI system are plotted as a function of toughener content  IPN formation proved to be a useful alternative for the toughening of epoxy resin for aerospace applications. In addition to the enhancement in mechanical properties , the development of (semi-)IPN structure in epoxy based adhesives can also facilitate the joining of different composite parts with better mechanical properties, which is a crucial requirement for the aerospace industry. Furthermore, PEI appears to be a potential candidate for the toughening of epoxy resin due to its higher e., PEI) . Three rEpoxy resins are attractive matrix systems for fibre reinforced polymer composites in the aerospace sector due to excellent properties such as ease of processing, low cost, superior mechanical, thermal and electrical properties. However, the brittleness and low elongation after cure of pure epoxy system restricts its potential. In literature, several strategies have been adopted to solve this problem. Amongst all those methodologies, formation of interpenetrating polymer network (IPN), by incorporating either thermoplastic or thermoset in brittle epoxy, is an attractive approach to improve the fracture toughness of modified system. This methodology usually results in better mechanical properties  of the modified epoxy system. Toughener content, chain mobility, chemical functionalization and morphology seem to be the main drivers affecting the fracture toughness of such systems.Several parameters have been investigated in literature to tune the mechanical properties  of the toughened epoxy system. For instance, the toughener content (either thermoplastic or thermoset) has been observed to be a crucial parameter in controlling the mechanical properties of resultant epoxy system. The increase in toughener content typically leads toward the gradual increase in fracture toughness of epoxy resin, which is attributed to the formation of IPN structure. However, after a certain level of concentration of toughener, the fracture toughness of modified epoxy system approaches a plateau behavior, which is associated with the fact that there are no further interactions between the epoxy and toughener. In the case of impact and tensile strength, after certain concentration of toughener, a decreasing trend is usually observed which is linked with the formation of toughener rich domains, which eventually inhibits the development of IPN structure. However, this decrease in impact strength of epoxy system can be tackled by playing with the branching and structure of the thermosetting toughener. In addition to the toughener content, other parameters have also been observed to affect the mechanical properties of toughened epoxy resin such as molecular weight of toughener components, different curing agents, etc. Hence, the final mechanical properties including fracture toughness of the modified epoxy system can be finely tuned by controlling the phase morphology and/or creating the IPN structure.Tg, higher strength and rigidity at elevated temperatures, good compatibility with epoxy, etc. This IPN formation approach can further facilitate the joining of different composite parts with superior mechanical properties, which is an essential process in the aerospace industry. In addition to the pure epoxy modification, the preparation of polymer composites from toughened epoxy resin was also performed. In the case of modified epoxy system based on thermosetting toughener, polymer composites display better mechanical properties  as compared to the un-modified epoxy while the thermoplastic toughening approach usually reduces the fracture toughness of the toughened polymer composite. Therefore, further research is still needed to efficiently utilize the thermoplastic modification approach to prepare the aerospace grade polymeric composites. For instance, the formation of (semi-) IPN using thermoplastic multilayers  for epoxy toughening and the utilization of this toughened epoxy system for producing aerospace grade polymeric composites would be an interesting research area for future work.Moreover, the concept of (semi-)IPN formation using PEI proved to be an interesting alternative for the toughening of epoxy resin for aerospace applications, due to its higher


Murraya koenigii (M. koenigii) belongs to the Rutaceae family, which is commonly used as a medicinally important herb of Indian origin in the Ayurvedic system of medicine. Previous reports have demonstrated that the leaves, roots, and bark of this plant are rich sources of carbazole alkaloids, which produce potent biological activities and pharmacological effects. These include antioxidant, antidiabetic, anti-inflammatory, antitumor, and neuroprotective activities. The present review provides insight into the major components of M. koenigii and their pharmacological activities against different pathological conditions. The review also emphasizes the need for more research on the molecular basis of such activity in various cellular and animal models to validate the efficacy of M. koenigii and its derivatives as potent therapeutic agents.The discovery of several revitalizing molecules that can stop or reduce the pathology of a wide range of diseases will be considered a major breakthrough of the present time. Available synthetic compounds may provoke side effects and health issues, which heightens the need for molecules from plants and other natural resources under discovery as potential methods of replacing synthetic compounds. In traditional medicinal therapies, several plant extracts and phytochemicals have been reported to impart remedial effects as better alternatives.  Presently, huge numbers of people in developing countries depend on medicinal plants for healthcare, skin care, economic benefits, and cultural development. For centuries, medicinal plants have been widely used in traditional medicine in countries like India, China, Germany, Thailand, etc. . The WorMurraya koenigii (M. koenigii) (L) Spreng (Family: Rutaceae) is usually known as “curry leaves”. The tropical and subtropical regions in the world have large distributions of M. koenigii [Murraya, only two, M. koenigii and M. paniculate, are available in India. M. koenigii is more important due to its huge spectrum of traditional medicinal properties. For centuries, this plant has been used in diverse forms and holds a place of pride in Indian Ayurvedic medicine, known as “krishnanimba” [M. koenigii, such as its leaves, root, bark, and fruit, are known to promote various biological activities. Aromatic bioactive constituents in the leaves of M. koenigii retain their flavor and other qualities, even after drying [M. koenigii leaves are slightly bitter in taste, pungent in smell, and weakly acidic. They are used as antihelminthics, analgesics, digestives, and appetizers in Indian cookery [M. koenigii are used in treating piles, inflammation, itching, fresh cuts, dysentery, bruises, and edema. The roots are purgative to some extent. They are stimulating and used for common body aches. The bark is helpful in treating snakebites [M. koenigii leaves is reported to possess anti-oxidative, hepatoprotective [M. koenigii have been accredited to several chemical constituents of different carbazole alkaloids and other important metabolites, like terpenoids, flavonoids, phenolics, carbohydrates, carotenoids, vitamins, and nicotinic acid from different parts of the M. koenigii plant.koenigii . Among tnanimba” . Differer drying ,12,13,14 cookery ,16. The akebites ,18,19,20otective ,22,23,24otective ,26,27, aotective ,29,30. TM. koenigii in traditional medicines and home remedies. On the other hand, limited studies have been conducted for evaluating the pharmacological and medicinal efficacy of M. koenigii in promoting health benefits and curing disease [M. koenigii and its bioactive compounds, highlighting their pharmacological effects. This review aims to present a well-managed summary and possible recommendation on existing studies to provide information regarding the current reports that can direct future research. Therefore, instead of discussing a few selected studies in a specific time interval, the present review will discuss and cover previous and existing major studies on M. koenigii related to the topics chosen. The details, like phytochemical screening, identification, and pharmacological activities, will be systematically categorized, compared, and summarized. We hypothesized that, through all of these efforts, a good summary on pharmacological activities that could initiate future perspectives with the utmost clarity could be produced.In recent years, greater attention has been paid to the use of  disease ,34,35,36M. koenigii are discussed in detail in The pharmacological activities of M. koenigii are useful in seasoning food items and preparing ready-to-eat foods. Owing to the higher antimicrobial activities of the essential oil from leaf extracts [Essential oils and fresh leaf powder of extracts ,38, thisextracts ,40,41.M. koenigii has numerous disease remedial activities, for instance, different parts of the plant, such as the leaves, roots, and bark, can be prepared as tonics for inducing digestion and flatulence or as antiemetics [iemetics ,42. Afteiemetics . The leaiemetics .M. koenigii. M. koenigii extracts of leaves, root, stem bark, fruits, and seeds have yielded alkaloids, flavonoids, terpenoids, and polyphenols, as shown in M. koenigii and its pharmacological activities.A wide range of phytochemicals have been isolated from the leaves, roots, and stem bark of M. koenigii—in mice through blood serum estimations based on high-performance liquid chromatography (HPLC) analysis. Mahanine, at a dose of 100 mg/kg of body weight, was found to take 60 min to reach the maximum concentration in blood serum [An in vivo pharmacokinetic study revealed that after oral administration of the bioactive compounds at the rate of 0.1 gm/kg body weight (b.w.), the maximum systemic concentration (Cmax) of koenimbine was 1.81 ± 0.55 μM and koenidine was 2.82 ± 0.53 μM, and the time required to reach the maximum concentration was 49.8 ± 8.4 min and 240 ± 0.00 min, respectively . Bhattacod serum .2), hydrogen peroxide (H2O2), the superoxide anion (O2•−), and the hydroxyl radical (•OH), are often generated as byproducts of cellular metabolic reactions and exogenous induction. These ROS create homeostatic imbalances, which lead to the generation of oxidative stress, which in turn, induces cell death and tissue injury [M. koenigii and exhibit remarkable antioxidant properties [Reactive oxygen species (ROS), such as singlet oxygen  free radical scavenging assay, with quercetin as a positive control. The ethanolic extract of M. koenigii showed an 80% scavenging activity, which was similar to the activities exhibited by the control antioxidant compound quercetin [M. koenigii by the DPPH free radical scavenging assay, with ascorbic acid as a positive control. The extracts of M. koenigii exhibited activities with an half-maximum effective concentration (EC50) value of acetone of 81.81 ± 19.92 at 4.7 µg/mL, alcohol of 79.80 ± 18.68 at 4.1 µg/mL, and aqueous extract of 62.82 ± 13.62 at 4.4 µg/mL, which was comparable to the EC50 value exhibited by ascorbic acid (the positive control), which was 97.13 ± 12.64 at 2.69 µg/mL [M. koenigii through DPPH radical scavenging assay, cupric reducing antioxidant capacity (CUPRAC), and ferric reducing antioxidant power (FRAP) assays, with ascorbic acid as a positive control. The benzene fraction of M. koenigii was found to be the most active free radical scavenger, exhibiting an 88.3% decrease at a concentration of 100 μg/mL, followed by ethyl acetate at 79.5% and petroleum ether at 78.7%, while positive controls of ascorbic acid and butylated hydroxytoluene (BHT) at a concentration of 100 μg/mL inhibited 93.1% and 86.5% DPPH absorption, respectively. Similarly, the antioxidant activity created by reducing activity and CUPRAC assays indicated the highest reducing potential in the benzene fraction, followed by petroleum ether and ethyl acetate. The activity was greater than that of ascorbic acid and on par with that of BHT [The leaf extracts of tivities . Rao et uercetin . Gupta e69 µg/mL . Zahin eM. koenigii by DPPH free radical scavenging activity and reducing power assays. The results indicated that an M. koenigii berry extract is a powerful free radical scavenger compared to known antioxidants, such as butylated hydroxytoluene, ascorbic acid, and tannic acid [M. koenigii leaves by estimating the H2O2 scavenging activity. The acetone extract of old leaves was found to have a maximum activity at 151.58%, and for young leaves in petroleum ether, the value was 72.23% [M. koenigii with DPPH free radical scavenging activity, inhibition of nitric oxide radical (NO) and thiobarbituric acid reactive substances (TBARS) activity, and reducing power assays, and •OH was also estimated, with vitamin C as a positive control. The fruit extract of M. koenigii exhibited antioxidant activities, and the EC50 value of the extracts for the DPPH assay was 2.6 mg/mL; for the NO radical, was 2.9 mg/mL; for TBARS, was 3.1 mg/mL; for the reducing power assay, was 2.7 mg/mL; and for H2O2, was 3.3 mg/mL, which were comparable to the EC50 value of 5 mg/mL exhibited by the vitamin C positive control [M. koenigii were probably due to the presence of flavonoids and phenolic derivatives. The above studies revealed that various extracts of M. koenigii display high antioxidant activity. The studies also indicated the potential for this plant to be a natural source of strong antioxidant substances that can be used in therapy for human diseases induced by ROS.Yogesh et al. evaluated the antioxidant activity of berry extracts of nic acid . Tomar es 72.23% . Waghmar control . The ant2•−, H2O2, and •OH [3−), NO, and S-nitrosothiols, also contribute to the generation of oxidative stress. Both ROS and RNS arise as intermediates in several metabolic processes and are specifically produced as part of the cellular defense against invasive pathogens. Free radicals also regulate many processes, including cellular growth, glucose metabolism, and proliferation [Chemical species with one or more unpaired electrons are called free radicals. In biological systems, the term “free radicals” refers to reactive oxygen species (ROS). Major ROS include O and •OH . In addiferation .S-nitrosylation [Apart from certain beneficial effects, free radicals induce various deleterious effects. In a non-specific manner, ROS can react on significant biomolecules, which leads to deleterious effects like a loss of enzyme activity, genetic mutations and permeability alterations in the cell membrane, and RNS-induced protein sylation . Becausesylation .M. koenigii has recently been shown to possess potential antioxidant activity and protection against oxidative stress induced in diabetes [M. koenigii has been shown to reduce lipid peroxidation and decrease cellular damage, thereby protecting the liver from ethanol-induced toxicity [M. koenigii leaf extract. They found that there was a significant decrease in the tissue cadmium level when the rats were pre-treated with the M. koenigii leaf extract before cadmium administration [An increase in the free radical concentration in the body can cause subsequent oxidative and cellular damage to lipids, proteins, RNA, and DNA . The leadiabetes . The aqutoxicity . Khan ettoxicity . Mitra estration .2) into the electron transport chain (ETC). Mitochondrial dysfunction is a characteristic of all chronic diseases and aging. It is characterized by a loss of efficiency in the ETC, as well as reductions in the synthesis of high-energy molecules [Mitochondria are the primary source of high-energy metabolism within the cell. Mitochondria are known as the powerhouse of the living cell. Mitochondria also regulate calcium homeostasis and play a role in scavenging free radicals and controlling programmed cell death and/or the apoptosis-signaling pathway . Mitochoolecules . These dolecules ,84.M. koenigii. By using different in vitro assays, a study reported that the above bioactive compounds exhibited the ability to restore the MMP levels and repair the mitochondrial damage [Mitochondria, as essential organelles, have a noteworthy role in the viability of neuronal cells. Excess ROS formation is due to complex I inhibition inducing impairments in the mitochondrial membrane potential (MMP) and the pro-apoptotic members are believed to permeabilize the outer mitochondrial membrane due to the formation of oligomeric pores, which permits the release of apoptogenic molecules from the intermembrane space. Recent studies have evaluated the neuroprotective activities of isolongifolene and structurally similar compounds, such as girinimbine, murrayazoline, and O-methylmurrayamine A isolated from l damage ,86,87.M. koenigii leaves showed significant analgesic and anti-inflammatory activity when explored using carrageenan-induced hind paw edema in albino rats [M. koenigii leaf extract in carrageenan-induced paw edema [M. koenigii leaf extracts effectively attenuate the pain which is induced by an intraperitoneal injection of acetic acid and subplantar injection of formalin in mice, and the analgesic effect was elucidated with the writhing responses and pain responses in the late phase. Furthermore, it was reported that higher concentrations ) reduced the early-phase inflammatory responses induced by formalin [Tissue injury, cell damage, infections due to pathogens, and alterations in biochemicals lead to a biological response called inflammation. In neurological disorders, the important components involved in inflammatory processes are believed to be mast cells, ependymal cells, microglia, astrocytes, and macrophages . Microglino rats . Anotheraw edema . Additioformalin .M. koenigii curry leaves rich in carbazole alkaloids against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells. The activity of inflammatory cytokines interleukin 1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and p65-NFκB was significantly reduced by the hydroalcoholic extract of M. koenigii. In addition, the hydroalcoholic extract of M. koenigii reduced the expression of nitrotyrosine (NT), myeloperoxidase (MPO), IL-1β, intercellular adhesion molecule 1 (ICAM-1), and cyclooxygenase (COX-2), and increased the expression of Nrf2 [M. koenigii and its bioactive compound girinimbine against lipopolysaccharide/interferon-gamma-induced RAW 264.7 cells. The girinimbine showed reduced levels of NO overproduction and pro-inflammatory cytokine levels IL-1β and TNF-α in the peritoneal fluid. These findings strongly suggest that girinimbine could act as an anti-inflammatory agent by suppressing inflammation [M. koenigii leaf extract. Bioactive compounds like murrayakonine A, O-methylmurrayamine A, and mukolidine were reported for their efficiency in inhibiting TNF-α and IL-6 release in LPS-induced inflammation in human peripheral blood mononuclear cells (PBMCs) [M. koenigii leaves and their active compounds, which are comparable to nonsteroidal anti-inflammatory drugs (NSAIDs).Khurana et al. evaluated the in vitro and in vivo efficacy of a hydroalcoholic extract of  of Nrf2 . Iman etammation . Another (PBMCs) . The aboApoptosis is a physiological programmed cell death mechanism which is essential for the flawless growth and development of organisms. Furthermore, it is a lively physiological course causing the self-destruction of cells that comprises lethal biochemical and morphological changes in the nucleus and cytoplasm. During cellular strain like oxidative stress and DNA damage, the process of apoptosis can arise, particularly in cells with high proliferation rates and a high expression of pro-apoptotic genes . IntrinsM. koenigii extract and its primary active compounds regulate multiple signaling pathways, including phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT), mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK). M. koenigii and its primary active compounds exert complementary effects on oxidative stress and the alteration of proteins [M. koenigii leaves exert anticancer effects on oral squamous cell carcinoma cells via the induction of microtubule-associated protein 1 light chain 3, type II (LC3B-II), and cleaved caspase-3, suggesting the inhibition of autophagic flux [M. koenigii, inhibited the growth of and induced apoptosis in human hepatocellular carcinoma cells (HepG2) [Previous findings have demonstrated that an proteins ,95. Theyproteins . Mahaningic flux . In humagic flux . Recentl (HepG2) . In addi (HepG2) .M. koenigii was shown to extend pro-apoptotic activities in MCF-7 cancer cells by inhibiting glycogen synthase kinase-3 beta (GSK-3β). Koenimbin induces apoptotic cell death, phosphorylation, the accumulation of β-catenin, and the activation of nuclear factor-κB (NF-kB) in cancers. Moreover, koenimbin suppresses the expression of various anti-apoptotic genes involved in the regulation of cell proliferation and apoptosis [M. koenigii could induce apoptosis in HL-60 cells. The same study also induced the loss of mitochondrial membrane potential and the subsequent activation of caspase-9/caspase-3, leading to the activation of apoptotic pathways [The alkaloid koenimbin found in poptosis . Koenimbpoptosis . Anotherpathways .M. koenigii has been reported in various studies. For example, the essential oil of the leaves was reported to possess antifungal activity [M. koenigii appreciably hold the ability of mycelial growth inhibition and thereby promote antifungal activity. The antifungal activity of M. koenigii against a wide range of pathogenic fungi has been studied. Penicillium notatum, Aspergillus flavus, Aspergillus niger, Fusarium moniliforme, Mucor mucedo, Penicillium funiculosum etc., were isolated from infected saplings and spoiled foods based on alterations of their growth characteristics, mycelial morphology, and spore morphology , Staphylococcus, Streptococcus, and Proteus. Hence, M. koenigii leaves could be efficiently used as a natural remedy in everyday meals for the prevention of several bacterial infections [M. koenigii exhibited antibacterial activity on bacterial strains of Staphylococcus aureus and Klebsiella pneumonia [M. koenigii exhibited therapeutic efficacy against multidrug resistant MDR bacteria [M. koenigii essential oil showed antibiofilm activity against Pseudomonas aeruginosa and it was reported that M. koenigii essential oil treatment revealed an 80% reduction in biofilm formation by P. aeruginosa. Microscopic analyses confirmed the drop in biofilm formation in Pseudomonas aeruginosa when treated with M. koenigii essential oil. The presence of antibiofilm substances like spathulenol (5.85%), cinnamaldehyde (0.37%), and linalool (0.04%) was reported in gas chromatography-mass spectrometry (GCMS) studies [M. koenigii counteraction on uropathogenic bacteria isolated from clinical samples was reported in a different study [M. koenigii was tested for its antibiotic action against Mycobacterium species, which was appreciable, like first-line anti-tuberculosis drugs. M. koenigii half maximal inhibitory concentration ((IC50) 400 μg/mL) was found to be more effective against Mycobacterium smegmatis compared to water extracts and petroleum ether. An M. koenigii ethanol extract exhibited significant synergistic antibacterial activity against Mycobacterium smegmatis and Mycobacterium bovis bacillus calmette-guerin (BCG) in combination with the anti-tuberculosis drug rifampicin [Combating microbial infections without side effects is always a tedious process. In this regard, in addition to classical antibiotics and synthetic drugs, there is an ongoing hunt for potent molecules from natural herbal medicines . M. koenfections . Pyranocneumonia . Green sbacteria . M. koen studies . M. koennt study . M. koenfampicin .M. koenigii extended hepatoprotective activity when crude aqueous extracts were investigated against ethanol-induced hepatotoxicity in experimental animals. M. koenigii was reported to extend a protective effect in liver impairments in chronic alcoholism and was proved to be effective in maintaining the enzymatic oxidant status [M. koenigii were explored for their hepatoprotective activity against ethanol-induced hepatotoxicity in a HepG2 cell line model. They exhibited excellent hepatoprotective activity, maintaining the enzymatic and non-enzymatic antioxidant level at a near normal value and also maintaining the integrity of the cells [M. koenigii hydro-ethanolic leaf extract was reported to attenuate the CCl4 hepatotoxic effects in rats. M. koenigii-pretreated rats showed a significant decrement in activity levels of hepatic markers and also maintained the level of enzymatic antioxidants [Liver diseases are a worldwide concern, and accessible medical treatments have an inadequate efficacy. Since ancient times, herbs have been used when treating various disease conditions; plant extracts and natural compounds have significant applications as hepatoprotective agents. The liver is the site of drug metabolism and the detoxification site of toxic products, and so it is the organ most exposed to xenobiotics . M. koent status . Water ehe cells . An M. koxidants .M. koenigii leaves was evaluated using a carbon clearance test. A considerable increase in the NO production indicated the increased phagocytic activity of macrophages. The M. koenigii extract holds promise as an immunomodulatory agent, which acts by stimulating humoral immunity and the phagocytic function [M. koenigii leaf extracts were reported to have certain effects in regulating mice immunology related to oxidative stress metabolism. An M. koenigii leaf extract can exhibit an immunomodulatory effect through which it can regulate the oxidative stress metabolism in diabetic mice [The immune system makes a network and regulates processes important for maintaining the health of an organism by hindering the entry and invasion of microbes. Impairments in the immune system lead to conditions from chronic inflammation to cancer . In an ifunction . The M. tic mice .M. koenigii has been used as a nephroprotective agent in a diabetic-induced rat model [M. koenigii leaf extract was found to be efficient in maintaining normal levels of serum creatinine, blood urea nitrogen, total serum protein, serum Na+, urine output, urinary creatinine, urinary urea, total urinary protein, and urinary Na+. Furthermore, the M. koenigii extract maintained the standard pattern in in vivo antioxidants, renal myeloperoxidase (MPO) activity, and histopathology of kidneys against unilateral renal ischemia reperfusion injury. Therefore, the extract of M. koenigii was clearly demonstrated to be useful in treating kidney disorders in rats [M. koenigii was elucidated in experimental investigations, which showed decreased levels of blood urea nitrogen (BUN), serum creatinine (Cr), and lipid peroxidation (LPO). An M. koenigii extract is efficient against cyclophosphamide-induced nephrotoxicity, which was clearly revealed through the maintenance of high levels of glutathione (GSH) and superoxide dismutase (SOD) compared to the cyclophosphamide-treated group [M. koenigii protective activity has been shown to induce significant dose-dependent decreases in serum urea and creatinine levels, as well as marked increases in the levels of plasma antioxidant capacity, in diabetic rats, compared to controls. More noteworthy is the histological integrity of kidneys, which showed comparable tissue regeneration induced by the aqueous extract [at model . The M.  in rats . The neped group . M. koen extract .M. koenigii have been explored and reported to have inhibitory effects on the aldose reductase enzyme, glucose utilization, and other enzyme systems for extending anti-diabetic effects [M. koenigii was tested for the α-glucosidase inhibitory property and was found to inhibit α glycosidase. Alpha-glucosidase inhibitors are widely used in the treatment of patients with type 2 diabetes [M. koenigii showed a significant reduction in blood glucose levels, and this effect of reducing blood glucose by M. koenigii is mediated by antioxidant properties and insulin mimetic effects. In addition, M. koenigii exhibited a profound antioxidant effect by reducing the malondialdehyde (MDA) level, increasing the GSH level, and significantly decreasing the homeostatic model assessment (HOMA)-insulin resistance index. On the whole, it is evident that M. koenigii possesses antidiabetic activity and has antioxidant effects in rats [Most prominently in developing countries, medicinal plants play a helpful role in managing diabetes mellitus due to their cost effectiveness. Diabetes mellitus, a metabolic disorder, is becoming a serious threat to human health. During the past few years, many phytochemicals responsible for anti-diabetic effects have been isolated from plants. Alkaloids present in the leaves of  effects . M. koendiabetes . A study in rats .M. koenigii possesses potential secondary metabolites that could be developed as anticancer agents. In one study, the cytotoxic activity was evaluated for three extracts: hexane, ethyl acetate, and methanol of M. koenigii leaves against the HeLa cell line. The extracts were reported as being potently cytotoxic in nature in HeLa cancer cells. These results established the potential of M. koenigii as an anticancer agent in vitro [M. koenigii has been obtained from rodent cancer cell lines, as well as different in vivo cancer models [M. koenigii extract treatment generated a decline in neoplasms in the colon [M. koenigii was reported to have the ability to reduce proliferation in breast cancer cell lines [M. koenigii leaves has been shown to have promising cytotoxic activity in breast cancer cells, with an IC50 of 14.4 µg/mL [in vitro . Additior models ,114,115.he colon . A methall lines . The tot.4 µg/mL . The ant.4 µg/mL .M. koenigii-derived carbazole alkaloid, showed growth inhibitory activity in human hepatocellular carcinoma and lung cancer cells in vitro [M. koenigii, showed the dose-dependent inhibition of endogenous 26S proteasome activity in MDA-MB-231 cells [M. koenigii contains remarkable anticancer compounds, especially mahanine, which has been reported to show anticancer activity targeting different signaling pathways [M. koenigii against different cancer cell lines are shown in Girinimbine, another in vitro . Rutin, 31 cells . Therefopathways . GirinimM. koenigii leaf extracts has been reported in the management of a wide spectrum of neurodegenerative diseases, like AD, PD, and others [M. koenigii promotes neuroprotective potential against orofacial dyskinesia induced by resperine. Additionally, it stabilizes the levels of protective antioxidant enzymes like SOD, catalase (CAT), and GSH, and inhibits LPO in the forebrain regions of reserpine-treated animals. Furthermore, it has been shown to significantly inhibit reserpine-induced abnormalities in behavior. Similarly, treatment with M. koenigii significantly restored the levels of protective antioxidant enzymes  and inhibited LPO in the forebrain region when compared with reserpine, and it also inhibited catalepsy induced by haloperidol [M. koenigii, has been reported to render neuroprotective effects against rotenone-induced mitochondrial dysfunction, oxidative stress, and apoptosis in a cellular model. Cytotoxicity, oxidative stress, and mitochondrial dysfunction were also attenuated by ILF in SH-SY5Y cells, which down-regulated Bax and caspases-3, -6, -8, and -9 expression, and up-regulated Bcl-2 expression. IFL was proved to regulate p-P13K, p-AKT, and p-GSK-3 beta expressions [M. koenigii leaves could enhance memory in rats [M. koenigii leaves was exhibited in a two-vessel occlusion (2VO) rat model of partial global cerebral ischaemia. The Morris water maze test was implemented to assess the rats’ cognitive function postoperatively. Brain samples were histopathologically examined for viable neurons within the CA1 hippocampal region. Test findings showed that M. koenigii leaves positively improved memory and learning impairments. M. koenigii leaf extracts modestly improved memory in rats with chronic partial global cerebral ischemia [M. koenigii against neurotoxicity are shown in Supplementation with d others ,34,35,41operidol . Isolongressions . Preclin in rats . The posischemia . The varM. koenigii was demonstrated to render protection in chromosomal damage against radiation and cyclophasphamide in vivo. Radiation leads to a rise in all types of aberrations, like the fragmentation of chromatids and breakages in chromosomes, rings, and dicentrics. Treatment with a methanolic extract of M. koenigii before radiation significantly reduced the aberrations. M. koenigii can significantly exert bone marrow protection against radiation and cyclophasphamide [A methanolic extract of sphamide .M. koenigii leaves extend wound healing in male albino rats through significantly increased wound contraction and reduced epithelialization, supporting the collagen synthesis which was evident in histopathological studies [Wound healing is a complex and multifactor process involving numerous biochemical and cellular processes which helps in the restoration of functional and anatomical continuity.  studies .M. koenigii. M. koenigii is a source of several bioactive compounds, including alkaloids, polyphenol, terpenoids, and flavonoids. M. koenigii and its derivatives appear to exhibit appreciable pharmacological activities, like anticarcinogenic, proapoptotic, antiangiogenic, antimetastatic, immunomodulatory, and antioxidant properties. The molecular mechanisms underlying these activities of M. koenigii and its derivatives are due to their diversified role in combinations of cell signaling pathways at multiple levels in various diseases. M. koenigii and its derivatives mitigate oxidative stress, neurotoxicity, neuroinflammation, neuronal loss, and cognitive dysfunctions. However, like other polyphenols, to a certain extent, M. koenigii activities are limited by its bioavailability and in such conditions, enhancement of the efficiency should be conducted. Therefore, future studies need to include more experimental studies on bioavailability and efficiency enhancement in clinical investigations.The current review summarizes the medicinal uses, phytochemistry, and pharmacological properties of


N-glycosylated proteins, regulates multiple immunological processes. However, the contribution of receptor sialylation to inflammatory signaling has been under-investigated. In the current study, we uncovered a role for ST6Gal-I in promoting sustained signaling through two prominent inflammatory pathways, NFκB and JAK/STAT. Using the U937 monocytic cell model, we determined that knockdown (KD) of ST6Gal-I expression had no effect on the rapid activation of NFκB by TNF (≤ 30 min), whereas long-term TNF-induced NFκB activation (2–6 hr) was diminished in ST6Gal-I-KD cells. These data align with prior work in epithelial cells showing that α2–6 sialylation of TNFR1 prolongs TNF-dependent NFκB activation. Similar to TNF, long-term, but not short-term, LPS-induced activation of NFκB was suppressed by ST6Gal-I KD. ST6Gal-I KD cells also exhibited reduced long-term IRF3 and STAT3 activation by LPS. Given that ST6Gal-I activity modulated LPS-dependent signaling, we conducted pull-down assays using SNA  to show that the LPS receptor, TLR4, is a substrate for sialylation by ST6Gal-I. We next assessed signaling by IFNγ, IL-6 and GM-CSF, and found that ST6Gal-I-KD had a limited effect on STAT activation induced by these cytokines. To corroborate these findings, signaling was monitored in bone marrow derived macrophages (BMDMs) from mice with myeloid-specific deletion of ST6Gal-I . In agreement with data from U937 cells, BMDMs with ST6Gal-I knockout displayed reduced long-term activation of NFκB by both TNF and LPS, and diminished long-term LPS-dependent STAT3 activation. However, STAT activation induced by IFNγ, IL-6 and GM-CSF was comparable in wild-type and ST6Gal-I knockout BMDMs. These results implicate ST6Gal-I-mediated receptor sialylation in prolonging the activity of select signaling cascades including TNF/NFκB, LPS/NFκB, and LPS/STAT3, providing new insights into ST6Gal-I’s role in modulating the inflammatory phenotype of monocytic cells.The ST6Gal-I sialyltransferase, an enzyme that adds α2-6-linked sialic acids to  N-glycosylated proteins, has been shown to be vital for granulopoiesis [Sialyltransferases play a seminal role in a variety of immunological processes through their modulation of surface receptors. For example, ST6Gal-I, a Golgi sialyltransferase that adds α2–6 linked sialic acids to opoiesis , 2, thymopoiesis , B cell opoiesis  and antiopoiesis , 5. Moreopoiesis , type 2 opoiesis , 9 and Iopoiesis . While mOne of the most highly studied inflammatory signaling pathways is the NFκB axis. Pivotal to controlling multiple facets of innate and adaptive immunity, the NFκB transcription factor induces the expression of numerous pro-inflammatory genes . DysreguPrior studies from our group have shown that signaling by TNFR1 is modulated by ST6Gal-I-mediated α2–6 sialylation. In both epithelial cancer cells  and U937As with TNFR1, the sialylation status of TLR4 is reported to affect its activation of NFκB . HoweverCCGGCGTGTGCTACTACTACCAGAACTCGAGTTCTGGTAGTAGTAGCACACGTTTTTG), followed by selection with 1 μg/ml of puromycin (Sigma). As a control, cells were stably transduced with an empty lentiviral vector. Puromycin was removed from the medium at least 2 days prior to all experiments.U1 cells were purchased from ATCC and maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic supplements . A stable, polyclonal ST6Gal-I KD cell line was created by transducing U937 cells with lentivirus encoding a shRNA against ST6Gal-I  were purchased from The Jackson Laboratory. These mice were crossed to LysM-Cre mice Ifo/J) to generate myeloid-specific deletion of St6gal1. Control animals consisted of floxed St6gal1 mice with no Cre-recombinase expression. All animal experiments were conducted with prior approval from the University of Alabama at Birmingham (UAB) Institutional Animal Care and Use Committee.C57BL/6 mice homozygous for the floxed Bone marrow cells were flushed from the femurs of 8-12-week-old mice. Following red blood cell lysis , cells were seeded in tissue culture treated dishes and incubated for 3–4 hr. Suspension cells were then pelleted and cultured in RPMI 1640 medium containing 10% FBS, 1% A/A and 20 ng/ml of murine M-CSF (R&D Systems) for 4–6 days to obtain BMDMs.All cell treatments were performed in medium containing 1% serum . To examine signaling, cells were cultured for the indicated times with 10 ng/ml of each molecule–TNF (R&D Systems), LPS (Sigma), IFNγ, IL-6 and GM-CSF .Cells were treated for indicated times and immediately lysed in radioimmune precipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors (Pierce). Total protein concentration was measured by BCA (Pierce). Samples were resolved by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat dry milk in TBS buffer containing 0.1% Tween 20 (TBS-T), and then incubated with antibodies against ST6Gal-I , pNFκB–p65 , total NFκB–p65 , p-STAT1 , total STAT1 , p-STAT3 , total STAT3 , p-STAT5 , total STAT5 , p-IRF3 , total IRF3  and TLR4 . Protein loading was verified using horseradish peroxidase (HRP)–conjugated anti-actin  or HRP-conjugated anti-tubulin . Membranes were incubated with appropriate HRP-coupled secondary antibodies , and protein was detected by ECL (Pierce), Clarity (Bio-Rad), or SuperSignal West Femto substrate (Pierce). At least two independent experiments for each cell treatment and associated immunoblots were performed.To confirm that ST6Gal-I knockdown corresponded with a decrease in α2–6 sialic acid on the cell surface, staining was performed with SNA lectin, which binds to α2–6 sialic acid. Briefly, cells were incubated with FITC-conjugated SNA  at a 1:400 dilution for 20 minutes at 4°C. SNA staining was then quantified via flow cytometry using an LSR-II (BD Bioscience).To verify that TLR4 is a substrate for ST6Gal-I and that α2–6 sialylation of TLR4 corresponds with ST6Gal-I manipulation, 400 μg of U937 cell lysates (harvested and quantified as previously described) were incubated with 50 μl of SNA-conjugated agarose beads  overnight at 4°C. α2–6–sialylated proteins bound to the beads were then precipitated by centrifugation, washed with PBS, resuspended in 1x SDS-PAGE sample buffer (Invitrogen) plus 10% 2-mercaptoethanol (Sigma) and incubated at 95°C for 5 min. Proteins were resolved by SDS-PAGE and immunoblotted for TLR4 .RNA was extracted from U937 cells treated for 6 hr with TNF or LPS using the protocol for the PureLink RNA Mini Kit (Invitrogen). Total RNA concentration was measured, and cDNA was synthesized using the M-MLV Reverse Transcriptase protocol (Promega). qPCR samples were prepared using TaqMan Fast Advanced Master Mix (Thermo). Primers for IL-6 (Hs00985639_m1), IL-8 (Hs01555410_m1), and TNF (Hs00174128_m1) were obtained from Applied Biosystems. The StepOne Plus Real-Time PCR System (Applied Biosystems) was used to determine mRNA levels. The data were analyzed using the comparative CT method to obtain relative quantitation values, which were normalized to GAPDH . Values for cells treated with TNF or LPS were normalized to values for untreated cells to yield fold increases in cytokine induction. At least three independent experiments were conducted, with each independent experiment performed in triplicate.U937 cells were treated with TNF or LPS for 6 hr and cell culture supernatants were collected. As a control, supernatants from untreated cells were also collected 6 hr after seeding. ELISA kits for IL-6 , IL-8  and TNF (430204 Biolegend) were used to measure cytokine abundance in conditioned media. At least three independent experiments were conducted, with each independent experiment performed in triplicate.In prior studies using epithelial cancer cell models, we determined that α2–6 sialylation of TNFR1 had no effect on the initial activation of NFκB by TNF, but instead prolonged TNF-dependent NFκB activation . To studConsidering the prominent role that NFκB plays in mediating inflammation, we evaluated a potential function for ST6Gal-I in regulating another pathway known to initiate NFκB activation, the LPS/TLR4 signaling axis. As with TNF stimulation of NFκB, ST6Gal-I activity had little effect on early (≤ 30 min) LPS-induced NFκB signaling . HoweverWe evaluated the effects of α2–6 sialylation on known inflammatory targets of NFκB transcriptional activity, such as IL-6, IL-8 and TNF. qRT-PCR experiments revealed that ST6-KD cells had reduced mRNA expression of IL-6, IL-8 and TNF compared to EV cells following 6 hr of TNF treatment . LikewisWhile the canonical LPS pathway centers on NFκB, it has been suggested that LPS can indirectly activate JAK/STAT signaling, further amplifying the inflammatory response to LPS , 19. AccGiven that ST6Gal-I activity modulated LPS-induced NFκB and STAT3 activation, we evaluated whether the receptor responsible for propagating the LPS signal, TLR4, was a direct substrate for ST6Gal-I-mediated sialylation. Using agarose beads conjugated to SNA, α2–6 sialylated proteins were precipitated, and the precipitates were then immunoblotted for TLR4. As shown in Because knockdown of ST6Gal-I led to a decrease in the long-term activation of NFκB by LPS, we evaluated activation of IRF3, a downstream target of the adaptor protein, TRIF. IRF3 is thought to be responsible for later-stages of TLR4-induced NFkB signaling . While nSince ST6Gal-I activity influenced TNF and LPS-induced signaling, we interrogated a role for ST6Gal-I in other cytokine-mediated inflammatory pathways. U937 cells were treated with IFNγ and examined for activation of STAT1 and STAT3, both of which are well-known downstream targets of IFNγ. As shown in St6gal1 gene were crossed to LysM-Cre mice (“LysMCre/ST6fl/fl”). Mice with floxed St6gal1, but lacking Cre-recombinase expression (“ST6fl/fl”), were used as the control. Monocytes were isolated from the bone marrow of the mice and cultured under established conditions to generate BMDMs. Cre-mediated deletion of St6gal1 in LysMCre/ST6fl/fl mice was confirmed by immunoblotting for ST6Gal-I in BMDM lysates Click here for additional data file.