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ThesisItem Open Access GREEN SYNTHESIS OF SILVER NANOPARTICLES USING HERBAL PLANTS EXTRACT WITH EVALUATION OF ANTICANCER ACTIVITY(Department of Pharmaceutical Sciences SIHAS, Faculty of Health Sciences SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY & SCIENCES ALLAHABAD-211007 INDIA 2019-20, 2020) PATHAK, MANISH KUMAR; Verma, Prof. (Dr) AmitaAim: In present research, Green synthesis of AgNPs using extracts of Scindapsus officinalis, Polygonatum verticillatum and Prunus cerasoides with evaluation of their cytotoxicity activity against Hepatic and breast cancer cell lines. Material & methods: AgNPs were used to characterized by ultraviolet –visible spectrophotometry, Fourier-transform infrared spectrophotometry (FTIR), Scanning electron microscopy (SEM) with elemental mapping, Transmission Electron Microscope (TEM) , X-ray diffraction (XRD), Energy-dispersive X-ray spectroscopy (EDAX). In-vitro cytotoxic action evaluated by MTT assay method. Result & discussion: Silver nanoparticles were identified by change of color & their absobtion found at between 300-400 nm measured by UV-visible spectroscopy, FTIR spectral analysis confirmed phenolic compounds presence, morphology & size visualized in SEM, TEM used for determination of size, shape & light scattering analysis. Synthesized silver nanoparticles were spherical in shape with size less than 50 nm. XRD analysis was affirmed the crystalline nature of metal particles. In-vitro cytotoxic results of synthesized AgNPs of different plants showed an excellent IC50 value against HepG-2 & MCF-7 cell lines. Conclusion: The current study reveals green synthesized AgNPs possess high cytotoxic action against HepG-2 & MCF-7 cell lines which suggested the potential therapeutic use of these silver nanoparticles as alternative medicine for the treatment of hepatic & breast cancer cases.ThesisItem Open Access A STUDY ON GLYCOCONJUGATED NANOPARTICULATE SYSTEMS FOR THE DELIVERY OF ANTICANCER BIOACTIVE(Department of Pharmaceutical sciences Faculty of Health Sciences Sam Higginbottom university of Agriculture, Technology and Sciences (Formerly Allahabad Agricultural Institute) Allahabad- 211007, 2018) Soni, Namrata; pandey, Dr. HimanshuThe lung is a common location of a secondary tumor that has metastasized from the primary source of tumor. The relapse of lung metastases usually occurs during conventional chemotherapy, hence it is no longer effective against the metastasis. Such a life-threatening condition urgently needs a new systemic treatment that can target the anticancer bioactive specifically to tumor cells. Thus the present study aim to construct Glycosylated SLNs loaded with gamcitabine (an anticancer bioactive) for efficiently targeting the lung cancer cells. GmcH-SLNs were prepared by solvent injection method and surface modification done with different carbohydrate molecules like mannose, galactose, fucose, dextran and lactose using ring opening technique. The prepared glycosylated SLNs were characterised for different parameter like particle size, shape, surface charge and FTIR for different peaks shows conjugation, drug entrapment, drug release, heamolytic toxicity, stability study, cellular uptake, cell cytotoxicity, and in-vivo study. The SLNs were nanometre in size and spherical in shape. The surface charge was positive but less. The all glycosylated formulation have higher entrapment efficiency, sustained drug release profile at physiological pH 7.4,lower heamolytic toxicity. All developed formulation were stable at cold and in dark condition. The cell uptake and cell cytotoxic study were perform on A-549 cell line, and all glycosylated formulations were highly cytotoxic and higher cellular uptake then plain SLNs due to lactin receptor mediated uptake. The In-vivo study also revealed that the glycosylated SLNs have higher targeting potential on lung tumor cells due to lactin receptor mediated uptake.ThesisItem Open Access EXTRACTION, FORMULATION AND EVALUATION OF SELECTED HERBAL DRUGS FOR ITS ANTIULCER ACTIVITY(DEPARTMENT OF PHARMACEUTICAL SCIENCES Shalom Institute of Health and Allied Sciences Allahabad, 2018) VERMA, BHUPESH KUMAR; Pandey, Dr. HimanshuGastric ulcer is an illness that affects a considerable number of people worldwide. The development and progression of gastric ulcer depends to some extent on the type of the food consumed by the patient. It has been shown that spicy food, fatty food or foods containing caffeine stimulates acid secretion in stomach and increase the risk of ulcer formation. It is among the most serious diseases in the world. A localized loss of gastric as well as duodenal mucosa leads to the formation of peptic ulcer. It arises when the normal mucosal defensive factors such as mucus, mucosal blood flow, formation of bicarbonate ions and prostaglandin E2 are impaired or over powered. Also by the aggressive factor includes acid, pepsin, NSAIDs and helicobacter pylori. Several indigenous plants and mineral preparations for the treatment of gastric ulcer were mentioned in the Indian system of medicines. The drug obtained from the medicinal plants are safe, cheaper, easily available and with no fear of any side effects. Moreover, these are more compatible the human body constitution and suits to the local and cultural need of the people. The indigenous method of preparation maintains the purity of the drug. The present study deals with extracts of plants named Capparis zeylanica (root), Asparagus racemosus (entire plant) and Jatropha multifida (leaves) known for their antiulcer activity). These plant extracts was then subsequently studied phytochemically and detailed acute toxicological effects based on the OECD guidelines. The effective dose was found to be 400 mg/kg/day, 500 mg/kg/day, 500 mg/kg/day of C. zeylanica, A. racemosus and J. multifida respectively. In this present study, The antiulcer effect observed in pylorus ligated and phenylbutazone induced ulcerative rats justifies the use of plants for the gastroprotective action. C. zeylanica caused a significant gastroprotection where as A. racemosus showed a moderate antiulcer activity. J. multifida showed moderate to trivial action against pylorus ligation and phenylbutazone induced ulcer. The commercialization of herbal drug market with increasing dependence of manufacturers of herbal formulations on suppliers of readymade extracts has necessitated the standardization of plant drugs and their extracts. The combination of three plants is the most effective. This combination was named as CAJ-500. CAJ-500 showed significant effects as antiulcer formulation. The beneficial multiple properties present in medicinal plants offer exciting opportunity to develop them into novel therapeutics for ulcer.ThesisItem Open Access Biosynthesis, Structural Characterization and Biological Evaluation of Silver Nanoparticles Using Some Medicinal Plants(FACULTY OF HEALTH SCIENCES SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY & SCIENCES ALLAHBAD-211007 (U.P.), INDIA 2018, 2018) SINGH, DEEPIKA; Verma, Prof. (Dr.) AmitaOxidative stress and inflammation plays a pivotal role in the expansion and progression of hepatic cancer and renal cancer. Nanoparticle based drug delivery can quickly enhance the restorative capability of hepatic cancer and renal cancer. Silver nanoparticles synthesize from plant source of great importance due to their small size, economic, non- hazardous and different biomedical application. In the current study, the impacts of oxidative stress and proinflammatory markers of biosynthesized silver nanoparticles of Phyllanthus Emblica, Madhuca longifolia, Carissa carandas leaves against diethylnitrosamine (DEN) induced hepatocellular carcinoma (HCC) and renal cancer in wistar rats till 16 weeks with its underlying mechanism. The physico-chemical properties of biosynthesized silver nanoparticles were determined by Ultra-Visible spectroscopy, Fourier transform infrared spectroscopy, field emission scanning electron microscope, energy dispersive x-ray Analysis, x-ray diffraction studies and transmission electron microscopy. Silver nanoparticles significantly enhanced the process of recovery from hepatic cancer and renal cancer at both the dose level in animal models, which was ascertained by increased body weight, reduced hepatic knobs on the outer surface of liver and renal, reduced level of serum biochemical parameters, decreased lipid peroxidation, increased membrane bound enzymes, increased enzymatic and non-enzymatic antioxidants parameters viz. catalase, glutathione peroxidase, superoxide dismutase and alteration in the level of proinflammatory cytokines and mediators viz. tumor necrosis factor (TNF-α) and nuclear factor (NF-κB). Histopathological studies also affirmed the recovered hepatocellular and renal architecture with increased intercellular space and regained the shape of nuclei in cytoplasm in silver nanoparticles treated group. Our outcomes implicate successfully biofabrication of plants (Phyllanthus Emblica, Madhuca longifolia, and Carissa carandas) silver nano-particles and exhibited a chemoprotective potential in the prevention and intervention of hepatic cancer and renal cancer.ThesisItem Open Access Biofabrication, Characterization & Pharmacological Evaluation of ZnO Nanoparticles of Selected Medicinal Plants(Department of Pharmaceutical Sciences Shalom Institute of Health & Allied Sciences SHUATS, Allahabad, 2018) Yadav, Ekta; Verma, Prof. AmitaOver recent years, utilization of green synthesized nanomaterials has been widely growing on human body because of its special properties. With increasing acceptance of nanoparticle approach for various clinical treatments, biosafety and toxicological effects on vital organs such as central nervous system, have received more concern. Main focus of this study was to evaluate acute exposure of n-butanol fraction of Prosopis cineraria (L.) Druce hydroethanolic extract (BuPC) and Trianthema portulacastrum Linn. (BuTP) and their green synthesized zinc oxide nanoparticles (ZnOPC and ZnOTP, respectively) on spatial cognition behavior, and to assess underlying mechanism by estimation of enzymatic antioxidative status along with acetylcholinesterase (AChE) activity in mice brain. Strongest in vitro antioxidant and AChE inhibitory activity exhibiting fraction, BuPC and BuTP were examined for inhibition kinetic study by Lineweaver–Burk and Dixon plots. BuPC and BuTP were further used for fabrication of ZnOPC as well as ZnOTP and characterized by UV-visible spectroscopy, Fourier Transform Infrared (FTIR), Field Emission Scanning Electron Microscopy (FESEM) and Energy Dispersive X ray (EDX). Male swiss albino mice were used of in vivo antiamnesic activity evaluation 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 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 and ZnOTP group, whereas BuPC as well as BuTP groups showed significant reduction in scopolamine induced increase in TL and TRC compared to control and scopolamine treated groups. ZnOPC and ZnOTP alleviated enzymatic antioxidant activity and AChE as compared to donepezil, BuPC and BuTP treated groups. Study concludes that ZnOPC and ZnOTP attenuated spatial learning and memory by increase in oxidative stress and decrease in AChE activity at both dose levels. Our results suggest that BuPC and BuTP exhibited a strong neuroprotective effect on cognitive deficit mice and it may be employed as a substance for treatment of dementia whereas the green synthesized ZnOPC and ZnOTP were not proficient to reverse memory impairment induced by scopolamine. Impediment process of wound healing is attributed to reactive oxygen species and inflammation. PC and TP leaves reported to possess antioxidant, antifungal, anti inflammatory and antibacterial properties, which could make them promising wound healing agent. Current study was aimed to estimate wound healing activity of BuPC, BuTP, ZnOPC and ZnOTP by excision and incision wound models along with assessment of possible underlying mechanism. Strongest antioxidant activity bearing extract fractions, BuPC and BuTP, were further analyzed quantitatively by total phenolic, total flavonoid content and High Performance Liquid Chromatography coupled with Diode Array Detector (HPLC-DAD). Synthesized ZnOPC and ZnOTP were characterized by UV-Visible spectroscopy, Fourier transform infrared (FT-IR) studies, Field Emission Scanning Electron Microscopy (FESEM) and Energy Dispersive X-ray (EDX) studies. Wound healing potential of BuPC, BuTP, ZnOPC and ZnOTP was evaluated by excision and incision wound models. Analyses confirmed the formation of spherical nanoparticles. Significant results (p<0.05) of wound contraction rate, epithelialization and histopathology of healed tissues of rats confirmed promising wound healing property of BuPC, BuTP, ZnOPC and ZnOTP. In addition, inflammatory markers, biochemical estimation such as hydroxyproline content of granulation tissue, and profile of antioxidant enzymes also supported wound healing potential of BuPC, BuTP, ZnOPC and ZnOTP. Present study advocated faster attenuation of wound via antioxidant and anti-inflammatory activities of ZnOPC and ZnOTP green synthesized nano-ointment approach as compared to crude fractionated plant extracts (BuPC and BuTP).ThesisItem Open Access DESIGN, SYNTHESIS AND PHARMACOLOGICAL ACTIVITY OF SOME NITROGEN CONTAINING HETEROCYCLES(Department of Pharmaceutical Sciences Faculty of Health Sciences SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY AND SCIENCES ALLAHABAD-211007, 2018) SHUKLA, PARJANYA KUMAR; Verma, Prof. (Dr.) AmitaOn the basis of background of literature available on nitrogen containing heterocyclic compounds, our research work designed to synthesize different new compounds of biologically active nitrogen containing heterocyclic moieties like thiadiazole, 1,2,4-triazole, isatin and thiazolidinone and to evaluate their pharmacological activities. Efficient one pot synthesis schemes of these compounds well deign by uses of different synthetic environments well accomplished by different catalysts, solvents and reagents. All the synthesized compounds were tested for their in vitro antibacterial, antifungal as well as antitubercular activity against agar disc diffusion method and minimum inhibitory concentration technique in comparison with different standard reference drugs and the results were presented in the given the form of tabular and graphical representation. The in-vivo pharmacological screening of the synthesized compounds done for analgesic, anti-inflammatory and antidiabetic activities.ThesisItem Open Access Fabrication and Characterization of Novel Nanofibrous Scaffolds for the Controlled Delivery of Tolnaftate to Treat Dermatophytic Infections(FACULTY OF HEALTH SCIENCES SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY AND SCIENCES (FORMERLY ALLAHABAD AGRICULTURAL INSTITUTE) ALLAHABAD 2018, 2018) MISRA, SHASHI KIRAN; Pandey, Dr. HimanshuFilamentous dermatophytes are immensely mortifying and keratinophilic eukaryotes, infected ¼th of world’s community till date. In an opportunistic role they are extremely devastating, as dermatophytes take advantage of a host with weakened immune system. Patients suffering from AIDS, cancer, old age senescence, diabetes, cystic fibrosis become more vulnerable to dermatophytosis. These pathogenic dermatophytes are keratinophilic fungi include species of Microsporum, Trichophyton and Epidermophyton which secrete proteolytic enzyme and affect keratin by making environment alkaline around at the site of action. The conventional remedial in form of cream, powder, lotion and gels prove effective as prophylactic at a prelim phase. At advanced and severe state of infection, these measures prove rather helpless due to some shortcomings associated with their use as the drug releases immediately and before reaching the pathogen site it interacts with healthy tissues also. The interaction may trigger on host’s immune system, and create allergic symptoms like itching or burning sensations in the host. Herein, we report fabrication of PEGylated nanofibrous scaffold, possessing enhanced localized antifungal activity against dermatophytes through development of nanoconjugate (TOL-GN) from amalgam of thiocarbamate derivative tolnaftate (TOL) and bioactive carbon allotrope graphene (GN), loaded on Eudragit polymers (ERL100/ ERS100). The rationale behind fabrication of nanofibrous scaffold was to synergize topical antifungal activity of TOL against pathogenic dermatophytes. In this regard, balanced combination of biocompatible ERL100 and ERS100 were selected to provide better adhesion on site of dermatophytosis, ample absorption of exudates during treatment and also customizing controlled drug release. Drug and polymers were found to be compatible and stable to each other through FTIR, XRD and DSC/ TGA studies. Surface topography analyzed that the scaffolds were regular, defect free, comprising distinct pockets with nanoscaled diameter. Excellent swelling index and remarked hydrophilicity were obtained which gratified essential benchmark for fabrication of nanofibrous scaffolds to alleviate dermatophytosis. In vitro drug release followed kinetics of Korsermeyer- Peppas model, suggested diffusion based mechanism from scafolds. Microdilution assay was carried out against extremely devastating dermatophytes i.e. Trichophyton rubrum, Microsporum canis, Microsporum gypseum and Microsporum fulvum. C3 nanofibers exhibited more prolong and preeminent activity against T.rubrum than D3 nanofibers. In vivo activity on dermatophytic swiss albino mice revealed superior antifungal activity of C3 nanofibers on successive 7 days of application and offered biocompatible topical rostrum to tailor localized and controlled drug delivery at the site of infection. The investigation offered futuristic potential usage of nanoconjugate TOL- GN loaded polyacrylate nanofibers as dressing materials/ scaffolds for effective management of dermatophytosis.ThesisItem Open Access QUINAZOLINE CLUBBED s-TRIAZINE DERIVATIVES AS KINASE INHIBITOR: DESIGN, SYNTHESIS AND ANTICANCER ACTIVITY(DEPARTMENT OF PHARMACEUTICAL SCIENCES FACULTY OF HEALTH SCIENCES SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY AND SCIENCES ALLAHABAD (U.P.) INDIA, 2017) PATHAK, PRATEEK; Verma, Prof. (Dr.) Amita; Kumar, Dr. VikasThree different congeneric series of quinazoline derivatives were synthesized via multi steps reaction and evaluated for their in-vitro anticancer activity and further in-ovo antiangiogenic activity using cancer induced chick egg against HeLa (Human cervical cancer), MCF-7 (Human breast cancer cell), HL-60 (Human promyelocytic leukemia cell) and HepG2 (Human Hepatocellular carcinoma cell) and TPC-1 (Thyroid cancer cell) respectively during both assays. Derivatives 12d, 12j, 12k found significantly active against HeLa cell line, derivatives 13f, 14f, 14h, 14k, 14l, 14m showed significant potency against TPC-1 cell line, derivatives 12b, 12l, 13m against HeLa and MCF-7 cell lines both during MTT assay. Whereas, cancer induced CAM assay stated that derivatives 12l, 12m, 12d were potent against HeLa and MCF-7 cell lines, derivatives 12g, 14b, 14k were significant active against MCF-7 cell line. Apart from these derivatives 12j against HeLa, derivative, derivative 13d against TPC-1 and MCF-7, derivatives 14a, 14e, 14i, 14j, and 14l against TPC-1 were significantly potent. The observed activity was further substantiated by docking study on VGFR2 and FAK. On the basis of SAR, it may be concluded that the potency of drugs depends on the nature of aliphatic substitution and the heterocyclic ring system.ThesisItem Open Access Synthesis, Characterization and biological activity of 4 aminoquinoline 1, 2, 4 triazole conjugated Benzothiazole Derivatives(Department of Pharmaceutical Sciences Faculty of Health Sciences SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY & SCIENCES (FORMERLY ALLAHABAD AGRICULTURE INSTITUTE) ALLAHABAD-211007 2018, 2018) ANJALI; Gupta, Dr. Pushpraj S.Increasing bacterial resistance is concomitant with the volume of antibiotic consumed, as well as missing doses when taking antibiotics. Inappropriate prescribing of antibiotics has been credited to a number of cases of drug resistance. To conquer drug resistance the discovery of new derivatives are obligatory as preexisting drugs are now less affective, most drugs existing presently at the market are varied heterocyclic compounds, amongst these compounds the five-membered heterocycles represent a broad and differentiated group with extensive range of biological activity. The current generations drugs necessitate definite variation to improve efficacy and to conquer multiple drug resistance. From literature survey it was proved that aminoquinoline, triazole and benzothiazole rings are present in antimicrobial, analgesic and anti-inflammatory agents. The presented work explores synthesis, characterization and evaluation of antimicrobial activity of new heterocyclic compounds bearing quinoline derivatives having Benzothiazole and 1,2,4 triazole nucleus. Structures were confirmed by FT-IR, 1H NMR, Elemental analysis, Mass spectral analytical studies. Method: A series of derivatives of 4-(Substituted Benzothiazol-2-ylamino)-5-[2-(7-chloro-quinolin-4-ylamino)-ethylamino] Substituted methyl-2,4-dihydro-[1,2,4] triazole-3-thione (4a-4k,5a-5k,6a-6k) were synthesized via three major step protocol. In the first major step formation of 4-Amino-5-[2-(7-chloro-quinolin-4-ylamino)-ethylamino]-4H-[1,2,4] triazole-3-thiol take place via five series of reaction where at first step 4,7-dichloroquinoline react with 1,3-diamino propane however second step involve reaction between derivative (1a) with phenyl chloroformate in presence of triethylamine leads to the formation of 1-[2-(7-chloro-quinolin-4-ylamino)-ethyl]-carbamic acid phenyl ester (1b) . Formation of N-{2-[(7-chloro quinolin-4 yl) amino] ethyl} hydrazine carboxamide (1c) achieved in third step where hydrazine monohydrate react with compound 3 using methanol as solvent further forth step involve potassium dithiocarbazinate (1d) synthesis where derivative 1c reacted with KOH and carbon disulfide using ethanol as solvent. Fifth synthetic protocol involve synthesis of 4-amino-5-[2-(7-chloro-quinolin-4-ylamino)-ethyl amino]-4H-[1,2,4]triazole-3-thiol (1e) where derivative (1d) unite with hydrazine hydrate. The second major step formation of 2-Chloro-substituted Benzothiazole (2b) via two step reaction involving the substituted aniline with ammonium thiocyanate followed by bromination in the presence of glacial acetic acid leads to formation of Substituted benzothiazol-2-yl amine (2a), Further it is reacted with sodium nitrite in the solution of phosphoric acid followed by addition of brine solution and copper sulphate solution. The third major step involve the formation of 4-(Substituted Benzothiazol-2-ylamino)5-[2-(7-chloro-quinolin-4-ylamino)-ethylamino]-2substituted methyl-2,4-dihydro-[1,2,4] triazole-3-thione(4) via formation of 4 (Substituted Benzothiazol-2-ylamino)-5-[2-(7-chloro-quinolin-4-ylamino)-ethyl amino]-4H-[1,2,4]triazole-3-thiol (3) where 2b reacts with 1e in the presence of equimolar amount of the 4-chlorobenzoyl chloride in the presence of sodium hydroxide solution. Synthesized derivatives (4a-4k, 5a-5k, 6a-6k) were evaluated for antibacterial, antifungal, anti-inflammatory and analgesic activity. Antimicrobial activity tested by agar disc diffusion method and Minimum inhibitory concentration against gram positive (Bacillus subtilis, Bacillus cereus, Staphylococcus aureus) and gram negative (Escherichia. coli, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa) and antifungal activity against Aspergillus niger, Candila albicans, Aspergillus clavatus. Synthesized derivatives 4i,4j,5a,5b,5c,5d,5e,6f,6j showed higher zone of inhibition and low MIC against given microbes compared with standard drug Ciprofloxacin and Fluconazole at different dilutions. The statistical analysis of data was done by using SPSS Trial version 20 and Microsoft Excel 2013 to correlate the MIC and Zone of Inhibition. The vivo pharmacological activity evaluation includes anti-inflammatory activity by carrageenan-induced rat paw edema method and analgesic activity by Tail flick method and hot plate method. The statistical analysis of data was done by using one-way ANOVA followed by Dunnett’s test using Graph Pad Instant software. The synthesized derivatives 4b having piperazine ring while 5k having 2-Methyl aniline nucleus and compound 5c having thiourea nucleus showed more than 50% paw edema inhibition at 100mg/Kg dose for 3 hours and 4 hours respectively compared to standard drug Meloxicam at 13.5 mg/Kg orally. Analgesic activity evaluation by tail flick model measured at 0 h, 1 h, 2 h, 3 h and 4 h for all test groups (150 mg/kg orally) compared with standard drug Diclofenac sodium by digital analgesiometer. Study showed that among all synthesized derivatives, the compounds 4e, 5e, 6e emerged as most potent analogues indicating the significant role of acetamide group in analgesic activity while synthesized compound having thioacetamide (6d) and 4-Bromo aniline (6g) in fluorine substituted benzothiazole showed improved antinociceptive activity in tail flick response. Compounds 4b, 4g, 5b, 6e, 6k exhibited significant activity by increasing the time of response of paw linking and jumping when compared with standard drug Diclofenac sodium (1mg/Kg b.wt.) in hot plate method.