Nigella sativa oil (NSO) might have beneficial effects in the treatment of diabetic complications. Thirty-two mature male Wistar rats were equally divided into four experimental groups: control, control NSO 2 mL/kg, streptozotocin- (STZ-) induced diabetic, and diabetic (STZ-induced) treated with oral NSO 2 mg/kg for 30 days. Fasting blood glucose (FBG), insulin, and lipid profile levels were determined. Pancreatic and hepatic tissues were used for catalase and GSH. Histopathology, hepatic glycogen contents, insulin immunohistochemistry, and pancreatic islet morphometry were performed. NSO 2 mL/kg was noticed to decrease (P < 0.05) FBG and increase (P < 0.05) insulin levels in diabetic rats than in diabetic nontreated animals. Lipid profile showed significant (P < 0.5) improvement in diabetic rats that received NSO 2 mL/kg than in the diabetic group. Both pancreatic and hepatic catalase and GSH activities revealed a significant (P < 0.05) increment in the diabetic group treated with NSO than in the diabetic animals. NSO improved the histopathological picture and hepatic glycogen contents of the diabetic group as well as increased (P < 0.05) insulin immunoreactive parts % and mean pancreatic islet diameter. NSO exerts ameliorative and therapeutic effects on the STZ-induced diabetic male Wistar rats.Diabetes mellitus is one of the metabolic diseases having several complications.  Diabetes mellitus (DM) is considered as one of the most common chronic metabolic diseases characterized by increased blood glucose due to insulin resistance, insulin deficiency, or both . Mostly,Insulin is the primary hormone which is involved in blood glucose control. Once insulin is released into the blood, it stimulates the entrance of glucose into skeletal muscles and, to a lesser extent, liver and adipose tissue via special transporters, thus controlling glucose homeostasis . InsulinThe efficient management of diabetes requires continuous control to blood sugar level to minimize the risks of diabetic complications . Thus, nNigella sativa (NS), a dicotyledon plant species of the family Ranunculaceae, has been used since long eras in medical and culinary fields [y fields . Its play fields . Thymoquy fields . NS has y fields , 16, immy fields , nephropy fields , 19, neuy fields , antimuty fields , and anty fields  effects.y fields  propertiThe current study aimed to investigate the potential protective actions of NSO in streptozotocin- (STZ-) induced diabetic male Wistar rats. This was done via the manipulation of blood biochemical parameters, oxidative stress, histopathology, and pancreatic insulin immunoreactive parts.Thirty-two adult male Wistar rats, weighing 195–205 g, were kept in metallic cages (4/cage) at Laboratory Animal House, Faculty of Veterinary Medicine, Suez Canal University, Egypt. They were kept under standard natural day-light rhythm with a temperature of 25°C (±1°C) and allowed to ad libitum diet and water supply. The animals were handled and cared according to the ethical guidelines described by Faculty of Veterinary Medicine, Suez Canal University.n = 8): They were normal nondiabetic rats and gavaged daily with distilled water for one month.Control group (Nigella group (n = 8): They were normal nondiabetic rats that were gavaged with 2 mL/kg cold-pressed NSO  containing 0.95% thymoquinone, as determined via HPLC, by gavage tube for one month.Diabetic group: They were STZ-induced diabetic rats and given daily oral dose of distilled water by gavage tube for one month.Nigella-treated group: They were STZ-induced diabetic rats that were given a daily oral dose of 2 mL/kg cold-pressed NSO  containing 0.95% thymoquinone, as determined via HPLC, by gavage tube for one month.Diabetic After 14 days of acclimatization, animals were divided into four equal groups:Experimental rats from diabetic and diabetic treated with NSO groups were induced to diabetes after 16 h fasting by a sole STZ , intraperitoneal (i.p.) injection, in a dose of 45 mg/kg. STZ was freshly prepared in 0.1 M citrate buffer (pH 4.5). The STZ-inoculated rats were allowed ad libitum 20% glucose solution for a duration of 24 h to avoid the occurrence of hypoglycemia. Rats of the control and NSO groups were injected with citrate buffer only .After 30 days of treatments, overnight fasted rats were decapitated under effect of anesthesia, and blood samples were obtained in sterile plain tubes. The sera were stored at −20°C. Pancreas and liver of each experimental animal were excised, rinsed in cold phosphate buffered saline, and dried with filter paper. Pancreas and liver of each rat was divided into 2 parts: one part was kept at −80°C until preparation of pancreatic homogenate for reduced glutathione (GSH) and catalase assay. The remaining part of pancreas and liver was immersed in 10% neutral buffered formalin for histopathological and immunohistochemical examination.Experimental rats were weighed weekly during the experimental period.Fasting blood glucose level was estimated using reagent strips . The levels of insulin in serum were estimated by commercial rat enzyme-linked immunosorbent assay (ELISA) kit  according to enclosed manufacturer's protocol.The high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), total cholesterol (TC), and triglycerides (TG) were measured in sera using enzymatic colorimetric kits  according to Tietz .Catalase and GSH activities in pancreatic and hepatic homogenates were determined using colorimetric kits from Biodiagnostic (Egypt) and BioVision (USA), respectively, according to methods of Aebi  and Tietμm sections were sliced and then subjected to hematoxylin and eosin (H&E) stain according to Bancroft and Gamble [μm sections were subjected to periodic acid Schiff (PAS) stain for glycogen demonstration [Formalin-fixed pancreases and livers were put in paraffin wax, and several 5 d Gamble . Livers stration .μm sections on the positively charged slides. Sections were subjected to xylene deparaffinization and then rehydrated with descending series of ethanol, followed by water. The sections were incubated with primary monoclonal anti-insulin antibody  at a rate of 0.5 μg/mL for 2 h at 25°C in a humidified chamber. Then the steps according to the method of Adewole and Ojewole [Paraffin-embedded pancreases were sliced into 4  Ojewole  were folMeasurements of pancreatic islet size were performed via ImageJ program. Insulin-positive islet immunostaining intensity and area % were determined from each slide of each experimental group using ImageJ program after subtracting light background. Seven fields of pancreatic islets were randomly chosen. The intensity of immunohistochemistry (IHC) staining and the percentages of IHC stained regions were obtained by ImageJ program and calculated according to Elgawish et al. . The diaP < 0.05.The data were presented as the mean ± standard error of mean (SEM). Statistically significant differences between groups were calculated using one-way analysis of variance (ANOVA) followed by Duncan's post hoc multiple comparison test . The criterion for significance was set at P < 0.05) than did those of the control nondiabetic rats at the 3rd and 4th weeks of the experiment. However, NSO 2 mL/kg treatment of diabetic rats increased their body weights (P < 0.05) compared to those of diabetic nontreated rats at the 4th week of the experimental period (The body weights of the STZ-induced diabetic group rats reduced significantly (l period .P < 0.05) elevation in FBG levels compared to those of the control rats. Administration of NSO 2 mL/kg to the diabetic rats, for one month, induced a significant (P < 0.05) reduction in the levels of FBG compared to those of the diabetic group. Insulin level revealed a significant (P < 0.05) decrement in the STZ diabetic group than in the control one. Meanwhile treatment of the diabetic rats with NSO 2 mL/kg, for 1 month, significantly (P < 0.05) improved insulin to a level comparable to that of the control group  increment in HDL-c level compared to that of the control group. However, HDL-c level revealed a significant (P < 0.05) decrement in the diabetic group than in the control. Meanwhile, administration of 2 mL/kg NSO to the diabetic rats increased HDL-c significantly (P < 0.05) compared to that of diabetic nontreated rats. Treatment of the control rats with NSO 2 mL/kg for one month induced a significant (P < 0.05) decrement in TC and LDL-c levels compared to those in the control. The diabetic rats revealed elevation in TC, TG, and LDL-c (P < 0.05) than did the control group. However, 30 days' treatment with NSO 2 mL/kg of the diabetic rats induced a significant (P < 0.05) decrement in TC, TG, and LDL-c values compared to those of the diabetic group  in the STZ-induced diabetic rats than in the control. Administration of NSO 2 mL/kg, for one month, to the diabetic rats significantly (P < 0.05) elevated hepatic and pancreatic catalase and GSH activities compared to those in the diabetic group  reduction in the mean diameter in the diabetic rats. Oral treatment with NSO (2 mL/kg), for one month, significantly (P < 0.05) increased the mean diameter of Langerhans islets compared to that in the diabetic nontreated group  variations among the study groups. Regarding the percentage of insulin-positive islet area, the analysis showed a significant (P < 0.05) decrement in the percentage of insulin-positive area in the diabetic group than in the control group. However, NSO treatment of the diabetic rats significantly (P < 0.05) improved positive islet area % compared to that of the diabetic ones  insulin staining intensity.The immunostaining intensity of pancreatic islets' insulin was assessed in a quantitative fashion according to the microdensitometric method. The staining intensities of insulin in the pancreatic islets among different study groups are shown in Diabetes mellitus is considered as one of the systemic, endocrine, and metabolic diseases, which is diagnosed by hyperglycemia. This could be considered with alterations in carbohydrate, lipid, and protein metabolism. This is considered to be due to reduction and/or impairment in antioxidant mechanisms inside the body leading to weight loss , 34.Nigella sativa thymoquinone is considered as an antioxidant and hypoglycemic compound that could counteract the high cost and the adverse effects of pharmacological drugs [Therefore the usage of antioxidants is considered as one of the strategies used for resetting the normal homeostasis in diabetic patients , 36. Numal drugs , which cP < 0.05) decrement in body weights at the 3rd and 4th weeks of experiment. These results are considered to be consistent with Wong and Tzeng [The current study demonstrated that STZ produced a significant  in the diabetic group than in the control one. These results are considered to be consistent with those of Houcher et al. [β cells of pancreatic islets, which blocks insulin secretion leading to hyperglycemia [P < 0.05 in both FBG and insulin levels, respectively, than in those in the diabetic group. These results were consistent with those of Fararh et al. [β cells of pancreatic islets, thus leading to improvement in their insulin production [The serum level of insulin was reduced in the diabetic rats than in the control, while FBG level was increased . These results were parallel to those obtained by Doğan and Çelik . DiabetiIn the current study, the influence of NSO on pancreatic and hepatic oxidative stress was examined in the STZ-induced diabetic male rats. The underlying mechanisms behind the oxidative stress and the free radicals generated by diabetes have been extensively investigated , 57. TheParallel to the results of hepatic and pancreatic antioxidant enzymes, the hepatic and pancreatic histopathology showed deleterious effects on the STZ-induced diabetic rats. These changes include hepatic congestion, fibrosis, centrilobular hepatocyte swelling, and incidence of inflammatory cell infiltration, which were ameliorated by NSO. Pancreatic pathology revealed severe atrophy, necrosis of acinar cells, and severe reduction in islet area %, which were greatly improved by NSO administration. The present results were in accordance with those of Kanter et al. , Widodo The depletion of hepatic glycogen contents in the diabetic rats was due to depletion of insulin that led to increased gluconeogenesis rather than glycogenesis . AdminisThe increment in pancreatic islet immunostained area % by NSO treatment in the diabetic rats, as a result of its antioxidant effect, was in agreement with the findings of Omar and Atia  and WidoBased on the current results, NSO was found to be an effective protection against the adverse consequences of STZ-induced diabetes in Wistar rats. NSO exerts its effect through thymoquinone antioxidant potential, which improved pancreatic and hepatic integrity, thus increasing pancreatic islet immunoreactivity, therefore increasing the serum insulin level, increasing hepatic glycogen content, reducing the elevated blood glucose level, and counteracting diabetic dyslipidemia.


Strongest in vitro antioxidant and AChE inhibitory activity exhibiting fraction, BuPC, was examined for inhibition kinetic study by Lineweaver–Burk and Dixon plots. BuPC was further used for fabrication ZnOPC and characterized by UV-visible spectroscopy, Fourier Transform Infrared (FTIR), Field Emission Scanning Electron Microscopy (FESEM), Energy Dispersive X ray (EDX), and Dynamic Light Scattering (DLS) analysis. Old male swiss albino mice were randomly divided into seven groups and treated for 21 days. Subsequently spatial memory was determined by two behavioral models [Elevated plus maze (EPM) and Hebbs William maze (HWM)] and supernatant of brain homogenate was analyzed for enzymatic antioxidant level and AChE inhibitory activity. Zinc content of blood plasma and brain was estimated. Results showed prolonged transfer latency (TL) and time taken to reach reward chamber (TRC) by scopolamine was not ameliorated by the ZnOPC group, whereas BuPC group showed significant reduction in scopolamine induced increase in TL and TRC compared to control and scopolamine treated groups. ZnOPC alleviated enzymatic antioxidant activity and AChE as compared to donepezil and BuPC treated groups. Study concludes that ZnOPC attenuated spatial learning and memory by increase in oxidative stress and decrease in AChE activity at both dose levels. Our results suggest that BuPC exhibited a strong neuroprotective effect on cognitive deficit mice and it may be employed as a strong substance for the treatment of dementia whereas the green synthesized ZnOPC was not proficient to reverse the memory impairment induced by scopolamine.Over recent years, utilization of green synthesized nanomaterials has been widely growing on human body because of its special properties. With the increasing acceptance of nanoparticle approach for various clinical treatments, the biosafety and toxicological effects on the vital organs such as central nervous system, have received more concern. Main focus of this study was to evaluate acute exposure of  Dementia is a mental syndrome associated with different types of neurodegenerative disease, such as Alzheimer’s and Parkinson’s disease, vascular dementia and frontotemporal dementia, etc. . ClinicoProsopis cineraria L. Druce (PC) also known as king of dessert belongs to Fabaceae family and a plant of Indian traditional medicine, used for management of various ailments. It is evergreen, small to moderate size tree, distributed in dry and arid region of India, mainly Rajasthan, Haryana, Gujarat and Punjab, as well as Oman, Afghanistan, and Pakistan  is the second most abundant essential micronutrient in the human body which is necessary for DNA replication, transcription, protein synthesis, cell growth and division. Dietary Zn deficiency may cause learning impairment as a result of alteration in Zn homeostasis in the brain . Zn playin vivo oxidative stress, which ultimately results in cognitive impairment and memory loss via cell injury and neuronal death, as well as on other brain functions , 5,5′-dithio-bis-(2-nitro) benzoic acid (DTNB), acetylthiocholine iodide (ATCI), AChE, rutin and gallic acid were purchased from Sigma-Aldrich . Berberine chloride, ascorbic acid, ethylene di amine tetra acetic acid (EDTA) and nitro blue tetrazolium (NBT) were procured from HiMedia Laboratories Pvt. Ltd., Mumbai. Folin-Ciocalteu’s phenol reagent was obtained from Merck KGaA . All solvents and reagents used in our study were of analytical grade.Fresh leaves of PC were procured from the rural area of Rewari, Haryana (India) in June 2015. The plant was identified and authenticated by botanist Dr. R. M. Kadam, Department of Botany, Mahatma Gandhi Mahavidyalaya, Latur, Maharashtra, India. The voucher specimen (DI 15) of plant has been deposited in the herbarium of same institution.Fresh leaves of plant (5 kg) were collected, washed with water, drained, shade dried and coarsely powdered. Dried powder was extracted with petroleum ether (60–80°C) to remove fatty material. Defatted plant material was macerated to prepare hydroethanolic extract . Maceration procedure repeated thrice, filtered and all three filtrates were combined. Filtrate was dried under vacuum to obtain the hydroethanolic extract (551 g).n-butanol, respectively. All three resultant fractions, i.e., chloroform , ethyl acetate  and n-butanol , separately collected, dried under reduced pressure and preserved at -20°C.Dried extract was subjected to fractionation by liquid-liquid partition in increasing order of polarity. The hydroalcoholic extract was dissolved in water, transferred into a separating funnel and then partitioned with chloroform, ethyl acetate and Total phenolic compounds content of crude hydroethanolic extract of PC was estimated by Folin-Ciocalteu method . The resTotal flavonoid content was determined by aluminum chloride colorimetric technique and expressed as mg rutin equivalent (RE)/g of extract from standard rutin solution calibration curve . The expAbility of PC fractions  to donate hydrogen or electron was estimated by colorimetric method using stable free radical DPPH . BrieflyAll three fractions of PC were subjected to determination of their ability to inhibit AChE by using Ellman’s assay with slight modifications . ReactioKi) was calculated from intercept in Dixon plot {concentration of BuPC vs. 1/(Absorbance/min)}.BuPC fraction was used to analyze the kinetics of inhibition by using previously described method . Assay wPreparation of nanoparticles using plant extract is known as green synthesis. Co-precipitation method was used . 1 g of Optical characteristics of BuPC and ZnOPC were observed with the help of UV-Visible spectrophotometer . The synthesized sample was made ready for scanning by re-suspending in sterile de-ionized water.-1.Spectrum was recorded using FT-IR spectrophotometer to identity the presence of various bioactive phytoconstituents which are known to be responsible for reduction and stabilization of ZnONP (Perkin-Elmer Spectrum 1000). FT-IR spectra were obtained at room temperature by using KBr pallets of BuPC and ZnOPC in the wavelength range of 4500–400 cmThe FESEM  associated with energy dispersive X-ray spectroscopy  was used to identify the surface morphology and metal analysis of synthesized ZnOPC. Dry sample powder was sprinkled over the carbon tape and then coated with gold for FESEM analysis.Sample was suspended in sterile de-ionized water and then analysis was performed by using Zetasizer Nano  just after sonication for 15 min. Zetasizer was equipped with a He/Ne red laser at 633 nm wavelength along with a detector set at 173° which results in size measurement with high sensitivity. Hydrodynamic diameter, zeta potential and polydispersity index (PdI) were determined as a function of time.ad libitum. Before starting the behavioral experiment, mice were acclimatized to laboratory conditions for 7 days. The Institutional Animal Ethics Committee (IAEC) approved the experimental protocol .Male swiss albino mice (8 weeks old) of 25–30 g weight were used for evaluation of antiamnesic activity. Throughout the experimental duration, mice were kept in polypropylene cages in well maintained room with standard laboratory conditions, i.e., temperature (25 ± 2°C), relative humidity (55–60%), 12:12 light and dark cycle. Animals were freely accessible to water and balanced rodent pellet food All mice were randomly divided into seven groups comprising nine animals in each group.Group I (Control): Normal saline at dose level of 10 ml/kg/day p.o.Group II (SCO): Vehicle and scopolamine at dose level of 0.6 mg/kg/day i.p.Group III (DON): Donepezil at dose level of 1 mg/kg/day p.o.Group IV (BuPC400): BuPC at dose level of 400 mg/kg/day p.o.Group V (BuPC600): BuPC at dose level of 600 mg/kg/day p.o.Group VI (ZnOPC15): Green synthesized ZnOPC at dose level of 15 mg/kg/day p.o.Group VII (ZnOPC30): Green synthesized ZnOPC at a dose level of 30 mg/kg/day p.o.All animals were treated with above mentioned drug regime, whereas vehicle was administered to Group II for 21 days. On last day (day 21), all groups (2–7), except group I, were administered with scopolamine (0.6 mg/kg i.p.) after 60 min of respective treatment for induction of memory deficit. After 30 min of scopolamine injection, acquisition trials were performed on all groups and retention of memory was examined after 24 h, on day 22. After being tested by one behavioral model, animals were provided a washout period of 15 days for testing by another model.Elevated plus maze is an exteroceptive behavioral model widely used for evaluation of potency of various drugs on learning and memory in rodents. It consists of two open arms (16 cm × 5 cm) and two covered arms (16 cm × 5 cm × 12 cm) extended from a central platform (5 cm × 5 cm). Height of the maze was 25 cm from the floor . On traiHebbs William maze (HWM) is an incentive-based exteroceptive behavioral model useful for measurement spatial working memory of rodents. Structure of HWM consists of three components, (i) animal chamber, extended into (ii) the middle chamber (exploratory area) and (iii) a reward chamber at the other end of maze, where food was placed as a reward. Guillotine removable doors were used in all three compartments. On 1st day (day 21), after drug treatment protocol, mouse was placed individually in animal chamber, keeping the door opened to facilitate the entry into the next chamber. As animal moved into next chamber, door of the start box was immediately closed to prevent back entry. Time taken by the animal to reach reward chamber (TRC) from start box on 1st day, reflects the learning index, was recorded. Before shifting to home cage, each animal was allowed to explore the maze for another 3 min with all the doors open. Retrieval of memory of this training task was examined after 24 h of the 1st-day trial. Significant reduction in TRC value indicates improvement of memory .After behavioral test, all mice from every group were sacrificed by cervical dislocation. Trunk blood was collected and preserved into tubes containing EDTA as an anticoagulant. With great care, whole brain were quickly removed and weighed. Brain tissues of each group were processed for biochemical estimation, Zn content determination and for histological examination. Isolated brain was homogenized for biochemical estimation in ice cold sodium phosphate buffer (pH 7.4). The brain homogenate was then centrifuged at a speed of 14500 rpm for 15 min at 4°C. Resultant clear supernatant was taken for lipid peroxidation index, antioxidant enzyme assay and AChE activity in brain.AChE activity in the mice brain was measured by using slightly modified Ellman’s method, as described above, using ATCI as a substrate. Reaction was initiated by pouring 0.1 M sodium phosphate buffer and 2.5 mM DTNB followed by addition 2 mM ATCI in a volumetric flask. Then, brain homogenate supernatant was added into the prepared reaction mixture and incubated at 30°C for 10 min. This resulted into the formation of yellow color owing to DTNB reduction by certain substrates in brain homogenate and non-enzymatic hydrolysis of other substrates. The sample was observed spectrophotometrically at 412 nm for change in absorbance and activity was represented as nM/min/mg protein .Thiobarbituric acid reactive substance (TBARS) is considered as a marker of lipid peroxidation. The extent of lipid peroxidation was estimated by determining the concentration of thiobarbituric acid in brain . Assay mBrain SOD activity was measured according to method described by 2O2 (0.16 M). Resultant reaction mixture was then incubated for 1 min at 37°C and the assay was completed by pouring of 1 ml of reagent (dichromate: acetic acid). Green color was formed after boiling the reaction mixture for 15 min and then absorbance was examined spectrophotometrically at 570 nm (2O2 consumed/min/mg protein.Assay was started with the addition of 100 μL supernatant into 150 μL phosphate buffer (0.01 M) having pH 7.0 followed by addition of 250 μL Ht 570 nm . CAT act3 (5 mM) and 1 ml of glutathione (4 mM). 1 ml of H2O2 (4 mM) was added after incubating the mixture at 37°C for 5 min. Glutathione concentration was determined by recording the absorbance of solution with the help of spectrophotometer at 412 nm.Assay was performed by DTNB method described by 3 for 3 h at 160°C. Atomic absorption spectrometer  was used to analyze Zn concentration in brain and plasma with the help of acetylene gas as fuel and air as an oxidizer. Concentration of Zn was determined from the calibration curves (prepared separately with suitable concentrations of the standard solution of Zn). Detected Zn concentration was up to the limit of 0.05 ppm and expressed as ng/g of the dry mass of brain and μg/ml of plasma.Amount of Zn was evaluated in brain and blood plasma of treated animals. Plasma was collected after centrifugation of collected blood samples. Carefully isolated brains were immediately weighed, stored in dry ice and then digested in concentrated HNOIsolated brain tissues from all groups were immediately stored in 10% formalin solution. After 24 h, tissue was subjected to dehydration with gradual concentration of ethanol. Dehydrated brain tissue was cleaned by xylene and entrenched in paraffin wax. Brain sections of 0.5 μm thickness were prepared and then stained with hematoxylin and eosin dye for photomicroscopic observation .p < 0.05 was considered as statistically significant.All results were represented as mean ± standard deviation. Data were statistically analyzed by one-way ANOVA followed by Newman–Keuls multiple comparison test. Value of Total phenolic contents of PC were found to be 123.12 ± 1.43 mg of GAE/g of dry extract. The amount of flavonoid content was estimated to be 145.17 ± 0.32 mg of RE/g of dry extract.Figure 1A. Data summarizes highest antioxidant activity by ascorbic acid  followed by BuPC and CHFPC with IC50 value 0.74 ± 0.07, 19.85 ± 1.34, and 42.80 ± 2.23 μg/ml, respectively (Table 1). Least DPPH inhibition was obtained in EAPC . Antioxidant activity of all fractions was observed to be dose dependant.Free radical scavenging ability of all fractions was evident of their antioxidant activity assessed by DPPH inhibition at 517 nm as shown in 50 values of different fractions of hydroethanolic extract of PC and berberine chloride on AChE inhibition is shown in Table 2. Figure 2A indicates the concentration dependant effect of all fractions of hydroethanolic extract of PC on inhibition of AChE. Berberine chloride  and BuPC showed the highest inhibitory activities than other fractions with IC50 value of 0.30 ± 0.03 μg/ml and 11.06 ± 3.61 μg/ml, respectively, followed by CHFPC (22.35 ± 2.73 μg/ml). Lowest AChE inhibitory activity  was observed in EAPC.ICin vitro AChE inhibition therefore, it was further evaluated for kinetic study. Figure 3A shows the kinetics of AChE inhibition by BuPC via Lineweaver–Burk plots [slope = Km/Vmax and intercept with X-axis = (-1/Km)]. It represents the mixed kinetics and competitive inhibition. Ki constant signified the binding affinity between enzyme and inhibitor. It was taken into account from the point of intersection of three lines toward the negative values of the X-axis in Dixon plots. From Figure 3B, Ki value was found to be 39.82 ± 1.17 μg/ml.The BuPC showed strongest in vitro antioxidant activity and AChE inhibition, it was further utilized to fabricate ZnOPC. UV–visible absorption spectra of BuPC and green synthesized ZnOPC given in Figure 4. ZnOPC showed a characteristic surface plasmon resonance absorption band at 374 nm, subjected to the absorption of ZnONP into the intrinsic band-gap due to transition of electron from the valence band to the conduction band. The absorbance at 374 nm signified the formation of ZnONP, which is well concordant with reported work . Spectrum of ZnOPC showed bands at 3450.79, 2918.42, 1763.98, 1640.53, 1385.91, 1355.05, 1015.57, 827.50, and 346.24 cm-1  which were assigned to –OH stretching of alcohols and phenols, -CH stretching of alkanes, C = O stretching, C = C stretching of aromatic amine, C–N stretching vibration of amine, C-H bending of alkane group (Fourier transform infrared (FT-IR) technique identifies the specific bond vibration peak owing to the reduction and capping of ZnONPs via probable secondary metabolites of plant extract. FT-IR spectra of BuPC exhibited characteristic peaks, i.e., 3330.55, 2918.53, 1650.58, 1446.66, 1376.66, and 1232.69 cmne group . Hence, ne group .Figure 6) on higher magnification showed cauliflower like structure, made up of cluster of small and almost spherical structure having size range of 40–80 nm with rough surface.ZnOPC surface morphology was observed with the help of FESEM micrographs. Photomicrographs  which is relatively larger as compared to the sizes reported by FESEM. The nanoparticles size variation could be due to the characteristic of polydispersity which is considered in terms of polydispersity index value of 0.565. Polydispersity is an indicator of a distribution defines the degree of non-uniformity could be as a result of aggregation of nanoparticles which is attributed with the variation in calculated particle size than the actual size of the particles. Zeta potential was found to be -12 mV.Dynamic light scattering technique is basically utilized for determination of the hydrodynamic diameter of nanoparticles in suspension on the basis of their Brownian movements. The average hydrodynamic diameter was observed with DLS is 117.5 nm  = 23.46, p < 0.0001] on the reference memory were shown in Figure 9. Scopolamine (p < 0.001) administered group showed significantly higher TL than control group during retention trial on day 22. Newman–Keuls multiple comparison test revealed that BuPC600 group (p < 0.001) significantly reduced TL, similar to DON group (p < 0.001), followed BuPC400 group (p < 0.01), shows reversal of amnesia induced by scopolamine. No significant change in TL (p > 0.05) by ZnOPC at both dose levels (15 and 30 mg/kg) indicates that it was not able to reverse the scopolamine induced amnesia.In the EPM model, significant overall differences between groups  on the reference memory. As shown in Figure 10, significant decrease in TRC was observed in groups treated by donepezil (p < 0.001) and BuPC  as compared to scopolamine treated group. Conversely, no significant change (p > 0.05) in TRC of ZnOPC (15 and 30 mg/kg) treated group was detected when compared to SCO group. Scopolamine injected group showed significant increase in TRC (p > 0.001) as compared to control group.To determine retrieval of memory, all groups analyzed for effect on TRC on 22nd day, showed significant differences among all treatment groups  were observed . Significant (p < 0.001) increase in AChE activity in brain homogenate of scopolamine treated animals was observed in comparison to control group indicates marked destruction in memory. Scopolamine induced increase in AChE activity was significantly ameliorated by BuPC  via restoration of cholinergic deficit. Results indicate inhibition of AChE activity with the administration of BuPC at both dose levels (400 and 600 mg/kg), whereas ZnOPC (15 and 30 mg/kg) was not able to significantly reverse the scopolamine-induced increased enzyme activity. Donepezil significantly (p < 0.001) reversed scopolamine induced increase in AChE activity.Since BuPC antagonized the scopolamine-induced amnesia, its effect on AChE enzyme activity in brain homogenate was examined by using Ellman’s method. For the AChE specific activity estimated in the brain homogenates, significant overall differences between all groups  were noticed . As compared to control group, scopolamine treated mice significantly elevated (p < 0.001) TBARS levels in brain homogenate. Donepezil (p < 0.001) and BuPC  substantially inhibited the scopolamine-induced elevated lipid peroxidation compared to SCO group. ZnOPC (p > 0.05), at both dose levels, was not found to be significant in decreasing the TBARS level elevated by scopolamine.For the lipid peroxidation level analyzed in the brain homogenates, significant overall differences between groups [Figure 13A) at 1.34 ± 0.08 ng/g (44%) and 1.73 ± 0.04 ng/g (86%) on exposure of ZnOPC 15 and 30 mg/kg, respectively, and similarly, plasma Zn concentration  was observed to be 479 ± 33.12 μg/ml (37%) and 502 ± 45.70 μg/ml (50%). While no significant change was detected in BuPC600 group as compared to control group.Atomic absorption spectrometer was used to determine the total Zn concentration in brain and plasma of mice treated with BuPC, ZnOPC and normal saline. Results revealed concentration dependant significant increase in total Zn concentration in brain .Histopathological examination of brain section illustrated the scopolamine mediated deleterious effects evidenced by elevation in vacuolated cytoplasm and focal gliosis, while pyramidal cells were diminished as compared to control group. Distinct reversal of scopolamine induced alterations in brain tissue was observed in BuPC pre administered groups. On the contrary, ZnOPC treated groups showed more number of dilated vacuolation and less number of pyramidal cells than scopolamine group, exhibits neurotoxic effect of ZnOPC . Memory impairment induced by scopolamine in mice was significantly attenuated by pretreatment with BuPC. But it was observed that ZnOPC treatment was not able to significantly reverse the scopolamine induced memory deficit. This is in well agreement with past study, which revealed chemically synthesized ZnONP damages the spatial learning and memory capability in rats through disruptive effect at the homeostasis of synaptic Zn concentration leading to direct hyperactive long-term potentiation and insufficient depotentiation in hippocampus  with the neurodegenerative disease, i.e., Alzheimer’s disease  which results in cellular toxicity. Along with this, ZnONP exposure also grounds brain damage and neurotoxicity via disturbance in the normal metabolism of neurotransmitters, ultimately affecting long-term synaptic plasticity and causing imbalance between spatial cognition stability and flexibility. Even though green synthesized nature of ZnONP, it is potentially harmful and hazardous toward central nervous system. Hence critical evaluation is essential before wide utilization to guarantee safe use.The current study depicts that preadministration with BuPC alleviate TL and TRC in scopolamine induced cognition deficit albino mice. It is potential This study was carried out in accordance with the recommendations of Committee for the Purpose of Control And Supervision of Experiments on Animals (CPCSEA) guidelines. The protocol was approved by the Institutional Animal Ethics Committee (IAEC), SHIATS.in vitro and in vivo studies. PY and EY analyzed and interpreted the data as well as drafted the manuscript. AV designed, supervised the study, and edited the manuscript. All authors contributed in the finalization of the manuscript.EY performed the majority of experimental studies. DS and PY assisted in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A strategy was reported to prepare boron nitride nanosheets (BNNSs) by a molten hydroxide assisted liquid exfoliation from hexagonal boron nitride (h-BN) powder. BNNSs with an average thickness of 3 nm were obtained by a facile, low-cost, and scalable exfoliation method. Highly thermally conductive polyimide (PI) composite films with BNNSs filler were prepared by solution-casting process. The in-plane thermal conductivity of PI composite films with 7 wt% BNNSs is up to 2.95 W/mK, which increased by 1,080% compared to the neat PI. In contrast, the out-of plane thermal conductivity of the composites is 0.44 W/mK, with an increase by only 76%. The high anisotropy of thermal conductivity was verified to be due to the high alignment of the BNNSs. The PI/BNNSs composite films are attractive for the thermal management applications in the field of next-generation electronic devices. Furthermore, low cost and light weight polymer-based materials for next-generation electronic device, power systems, and communication equipment are needed. Polymers such as polyimide (PI) has been widely used as an electronic packaging material due to good thermal and mechanical properties7. In particular, it possesses a low dielectric constant, low loss tangent, high thermal stability and high storage modulus8. However, PI exhibit a poor thermal conductivity in the order of 0.1 W/mK11, which cannot meet the requirement of fast heat conduction for the advanced electronic devices. The general strategy to improve the thermal transport performance is using thermally conductive fillers such as carbon materials15, metal or ceramic materials19 are added to the polymer matrix20.With the rapid development of electronics industry, there is an increasing demand for electrically insulating polymer-based materials with enhanced capability of heat dissipation21 has attracted much attention due to its excellent electrical insulation and high thermal conductivity22. As the low aspect ratio of h-BN filler, conventional PI composite achieve the thermal conductivity of 1–5 W/mK by utilizing large loading volume fraction h-BN filler (of up to ~50%). Large loading volume fraction in polymer composites leads to many problems such as mechanical property deterioration and processing difficulty as well as cost enhancement. In the contrast, large aspect ratio boron nitride nanosheets (BNNSs) exhibit many potential applications including ultraviolet light emitter, field emitters, and a superior substrate for graphene-based electrical devices28 and a superior thermal conductivity ranges from 1,700–2,000 W/mK29. Furthermore, BNNSs are an electrical insulation material with dielectric constant of 2–430. Therefore, BNNSs can be an ideal thermal conductive filler for polymer composites.However, the carbon and metal materials are highly electrically conductive and small additions of these fillers into polymers result in high electrical conductivity of the composites, which restricts the application. Meanwhile, hexagonal boron nitride (h-BN) is a typical ceramic filler31, ultrasonication32, and high energy electron beam irradiation33, chemical vapor deposition34, and liquid exfoliation35. However, BNNSs still suffer from a low cost, high yield, and facile exfoliation method. Micromechanical cleavage and electron beam irradiation technique are inefficient and unscalable. Furthermore, chemical vapor deposition method usually involved expensive templates and complicated fabrication processes, which appears to be tedious and expensive for large-scale production. Though the liquid-exfoliation method is popular method, still suffers from low product yield and sometimes the toxic reagents have to be employed37. The high yielding and high-quality of BNNSs by exfoliation of h-BN powder remains a tough challenge.Recently, many efforts have been used to prepare BNNSs including micromechanical cleavage38. This method has several advantages, such as cheap precursors, high yields, and without the use of organic solvents, catalysts and vacuum systems. More importantly, we developed a PI composite film incorporated with BNNSs. As a result, the incorporation of low loading BNNSs into the PI matrix shows a significant enhancement of thermal conductivity, especially along the in-plane direction. The composites are promising for using as a heat dissipation material in next-generation electronic device.Herein, BNNSs were prepared by a molten hydroxide assisted liquid exfoliation from h-BN powder. BNNSs with an average thickness of 3 nm were obtained in a high product yield of 19%The h-BN powders were purchased from ESK Ceramics GmbH & Co. (Germany) with lateral size of 7 µm. Sodium hydroxide and potassium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd (China). Poly(amic acid) synthesized by pyromellitic dianhydride and 4,4-oxydianiline was obtained from Ningbo Cen Electrical Material Co., Ltd (China). All chemicals were of analytical reagent grade and used without further purification.The molten alkali-assisted exfoliation of h-BN was following by two steps. Firstly, NaOH (2.84 g) and KOH (2.16 g) were finely ground, and then h-BN micropowder (1.0 g) was added. The mixture was further ground into a homogeneous form and transferred to a 100 mL Teflon-lined stainless steel autoclave. The sealed autoclave was heated and kept at 180 °C for 2 h. After cooling down to room temperature, the solid product was collected from the autoclave and dispersed into 300 mL deionized water. The dispersion was sonicated for 1 h using a tip sonicator . Subsequently the sample was filtered, re-dispersed in deionized water, and centrifuged to remove hydroxides and other unreacted materials. After centrifugation, the supernatant containing the product was collected. It is noted that the precipitate can be used for the next cycle of liquid exfoliation in the same alkaline solution as above mentioned.+, Na+, and OH− into the interlayer space of h-BN, driven by the high chemical potential. In particular, Na+ and K+ firstly absorbed on the surface of h-BN and then diffused into the space between adjacent BN lattices, leading to curling or crinkling of top BN layer39. After more ions entered the interlayer space, the curling up sheet peeled off from the parent counterpart owing to functionalized BN with hydroxides. This behavior enlarges the interlayer spacing near the edge. Then use molten hydroxide treated BN powder as precursor for liquid exfoliation and ultrasonication. The results indicate that the molten hydroxide treatment of BN powder could significantly improve the yield of BNNSs in the liquid exfoliation stage. The yield enhancement can be ascribed to the surface pretreatment, which effectively enlarges the BN sheets interlayer spacing and weaken the interaction between neighboring BN layers. Furthermore, the liquid exfoliation with dissolved hydroxides also induces the ion insertion, which further accelerates the exfoliation yield.To interpret the formation mechanism of BNNSs prepared by molten alkali-assisted exfoliation method, a schematic diagram is presented in Fig. A solution method was adopted to prepare the PI/BNNSs composites. Firstly, BNNSs powders were dispersed in dimethylacetamide (1 mg/mL) by applying the ultrasonic wave for 10 min at room temperature. At the same time, poly(amic acid) was dissolved in dimethylacetamide at room temperature. Then, the BNNSs solution was added to a solution of poly(amic acid)/dimethylacetamide, and the resulting mixture was put into speedmixer to stirred for 4 min at 3200 rpm to obtain homogeneous mixture. Finally, the prepared mixture was casted on a clean glass substrate followed by thermally imidization in a vacuum oven at 80 °C for 2 h and 120, 150, 200, 250, 300 and 350 °C for 1 h, respectively.−1. Raman spectra were obtained by Raman spectrometer with laser wavelength of 532 nm . Thermal conductivities of the composites were determined with laser flash apparatus . The sample size for in-plane and out-of plane measurement was round with a diameter of 25.4 and 12.7 mm, respectively. The infrared (IR) photos were captured by IR camera .The samples were examined with a Quanta FEG250 field emission scanning electron microscopy  at an acceleration voltage of 20 kV. Samples were broken and the fractured surface were coated with a thin layer of gold powder to avoid the accumulation of charge and improve the conductivity, and a transmission electron microscopy JEM-2100 , respectively, with an acceleration voltage of 200 kV. The samples were dispersed in ethanol using ultrasonic mixing for 15 min and some pieces were collected on 200 mesh carbon coated copper grids. Atomic force microscope (AFM) measurement was conducted on a multimode scanning probe microscope from Digital Instruments with NanoscopeIa controller. X-ray photoelectron spectroscopy (XPS) was carried out with Kratos AXIS ULTR DLD spectrometer. Fourier transform infrared (FTIR) spectra were obtained using a Nicolet 6700 FTIR  between 400 and 4000 cm40. The spacing between adjacent fringes and the number of layers was measured 8 layers, respectively.The morphologies of the pristine BN powder and exfoliated BNNSs were studied using SEM. As shown in Fig. The thickness of the BNNS was approximately 3.07 nm, as shown in Fig. −1, corresponding to B−N stretching  and at 815 cm−1, which is attributed to B−N−B bending 42. However, the exfoliated BNNSs also exhibits a broad peak at 3205 cm−1, which is attributed to −OH vibration. This peak shows a significant decreasing after annealing process, indicating our proposed exfoliation method leads to hydroxyl functionalization of BNNSs. In order to investigate the surface composition and the functional groups, the XPS spectra of the BNNSs are shown in Fig. 44. In addition, the B 1s core level spectrum in Fig. 46.As shown in Fig. 47.The appearance of neat PI and PI composites are shown in Fig. Figure Figure In summary, BNNSs were prepared by a molten hydroxide assisted liquid exfoliation from h-BN powder. Highly thermally conductive PI/BNNSs composite films were produced by a simple solution-casting process. The in-plane thermal conductivity of PI composite films with 7 wt% BNNSs loading is up to 2.95 W/mK, which increased by 1,080% as compared to that of neat PI. In contrast, the out-of plane thermal conductivity of the composites is 0.44 W/mK, with an increase by only 76%. The thermal conductivity of PI/BNNSs composite shows highly anisotropy behavior according to the heat transfer direction. The PI/BNNSs composite films are very attractive for the thermal management (heat spreading) in electronics.Supplementary Information


Ganglionated plexus (GP) plays an important role in the initiation and maintenance of atrial fibrillation (AF). The GP ablation has been found to be effective for AF treatment. In this case, we reported an AF case in which the pulmonary vein (PV) potentials of the anterior region of the left superior PV were eliminated by an inferior right GP ablation. Ganglionated plexus (GP) clusters were identified as sites where vagal reflexes were evoked by transcatheter high‐frequency stimulation. Regarding the GP ablation, radiofrequency (RF) applications for a circumferential pulmonary vein (PV) isolation were performed to cover the superior left and anterior right GP sites. Inferior GP sites were observed far from the circumferential PV isolation line. Therefore, RF applications to the inferior GP were performed separately from the PV isolation line.A 71‐year‐old man with hypertension underwent a cavotricuspid isthmus (CTI) ablation for a CTI‐dependent atrial flutter (AFL) at the age of 62 years . Drug resistance paroxysmal atrial fibrillation (AF) occurred after the AFL ablation, and therefore, an AF ablation was performed under the use of three‐dimensional electroanatomical mapping . Ultrasound echocardiography revealed no structural heart disease and a left atrial (LA) dimension of 34 mm. The LA volume calculated by computed tomography was 72 cm™, St. Jude Medical Inc., St. Paul, MN, USA) were performed at the anterior part of the left superior and inferior PVs, roof, anterior part of the right superior and inferior PVs, and site of the inferior left GP in that order. A 20‐pole ring catheter with a 17.5 mm size  placed at the left superior PV (LSPV) and intracardiac electrocardiogram (ECG) showing the pre‐ablation LSPV potentials are shown in Figure Radiofrequency applications with a 7.5Fr, 3.5‐mm‐tip‐irrigated ablation catheter  and increase the AERP heterogeneity YF, YM, YU, KY, YM, MF, SM, YW, HT, TO, YM, YH: are the members at out division of cardiology.None.