Scientia Pharmaceutica

The maleimide derivative 1-(4-chlorobenzyl)-3-chloro-4-(3-trifluoromethylphenylamino)-1H-pyrrole-2,5-dione (MI-1) was synthesized as inhibitor of several protein kinases, however, its application is hindered by its poor water solubility. In this study, the mechanisms of the antineoplastic action of MI-1 and its MI-1/M5 complex with M5 carrier (poly (PEGMA-co-DMM)) towards human colon carcinoma HCT116 cells were investigated by using the MTT and clonogenic assays, DNA intercalation with methyl green replacement, alkaline DNA comet assay, and Western-blot analysis. MI-1 compound and its MI-1/M5 complex possessed high toxicity towards colon (HCT116), cervical (HeLa) carcinoma cells and melanoma (SK-MEL-28) cells with GI₅₀ value in a range of 0.75–7.22 µg/mL, and demonstrated high selectivity index (SI ˃ 6.9). The p53 status of colon cancer cells did not affect the sensitivity of these cells to the treatment with MI-1 and its MI-1/M5 complex. M5 polymer possessed low toxicity towards studied cells. The MI-1, MI-1/M5, and M5 only slightly inhibited growth of the pseudo-normal HaCaT and Balb/c 3T3 cell lines (GI₅₀ ˃ 50 μg/mL). The MI-1 and its MI-1/M5 complex induced mitochondria-dependent pathway of apoptosis, damage of the DNA, and morphological changes in HCT116 cells, and affected the G2/M transition checkpoint. The MI-1 intercalated into the DNA molecule, while such capability of MI-1/M5 complex and M5 polymer was much lower. Thus, poly (PEGMA-co-DMM) might be a promising carrier for delivery of the maleimide derivative, MI-1, a novel kinase inhibitor, through improving its solubility in aqueous media and enhancing its antiproliferative action towards human tumor cells. Studies are in progress on the treatment of Nemeth-Kellner lymphoma (NK/Ly)-bearing mice with the MI-1 and MI-1/M5 complex. Full article

Objectives: The current work was designed to study the effect of dehydroepiandrosterone (DHEA) on glucose homeostasis, liver functions and hemostatic disturbances in a rat model of bilateral orchidectomy (ORCH). Methods: 32 male rats (n = 8) were randomly assigned into 4 groups; (i) control (sham operated) group; were normal rats in which all surgical procedures were done without ORCH, (ii) Control + DHEA group: as control group but rats were treated with DHEA for 12 weeks, (iii) orchiectomized (ORCH) group: rats had bilateral orchidectomy and (iv) ORCH + DHEA group: orchiectomized rats treated with DHEA for 12 weeks. Four weeks after ORCH, DHEA treatment began and lasted for twelve weeks. By the end of the experiment, the parameters of glucose homeostasis, lipid profile, liver enzymes, bleeding and clotting times (B.T. and C.T.), prothrombin time (P.T.), activated partial thromboplastin time (aPTT), platelet count and aggregation, von-Willebrand factor (vWF), fibrinogen, plasminogen activator inhibitor (PAI-1), fibrin degradation products (FDP), intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), endothelin-1 were measured. Results: ORCH caused significant deteriorations in the parameters of glucose homeostasis, lipid profile, and liver functions (p < 0.05). In addition, lower androgenicity-induced by ORCH caused a significant rise in PAI-1, fibrinogen, FDPs, ET-1 (p < 0.01) with significant shortening of bleeding and clotting times. DHEA replacement therapy significantly decreased glucose, insulin, PAI-1, fibrinogen, ICAM-1, and VCAM-1 when compared to ORCH rats. Conclusion: DHEA ameliorated the metabolic, hepatic, hypercoagulable, and hypofibrinolysis disturbances induced by ORCH. Full article

The objective of this study was to qualitatively evaluate a Fab-targeting ligand preparation containing free thiol groups in the hinge region by using bevacizumab as a model. The evaluation focused on the purification of fragments through a nonaffinity-based process using a centrifugal ultrafiltration technique and mild reduction conditions for the intact production of F(ab’) fragments with specific inter-heavy-chain disulfide bonds cleavage. Under these conditions, F(ab’) fragments with a defined chemical composition were successfully obtained via proteolytic digestion followed by a controlled reduction reaction process maintaining the integrity of the binding sites. The ultrafiltration purification technique appears to be suitable for the removal of the digestive enzyme but inefficient for the removal of Fc fragments, thus requiring additional processing. A suitable analytical strategy was developed, allowing us to demonstrate the reformation of disulfide bridges between the two reduced cysteines within F(ab’) fragments. Full article

In South Africa, plants belonging to the Restionaceae family possess an ecological dominance. As a result, they have been the subject of numerous morphological, anatomical, and evolutionary studies. However, few studies have focused on their phytochemical profile and their potential pharmacological activities. The genus Elegia L. is the second largest of this family comprising 52 species, which are mainly used as materials for thatching. Limited studies on the chemical constituents of Elegia species and their importance as medicinal plants have been undertaken. This review provides constructive and extensive information about the botanical characterization, distribution, traditional uses, phytochemistry and pharmacology of the genus Elegia. A comprehensive search of previously published literature was performed for studies on this genus, using databases with different key search words. This survey documented 52 Elegia species summarizing their previous taxonomic classification. In addition, 14 species were found to be studied for their phytochemical profile, revealing 14 chemical compounds. Concerning their biological activities, only one species (E. tectorum (L.f.) Moline and H.P.Linder) is reported for its anti-wrinkle activity. Moreover, two species are locally used for thatching and as materials for brooms. The present review highlights the Elegia genus as an important source of bioactive phytochemicals with flavonol glycosides being the main metabolites and reveals the uncharted territory of this genus for new research studies. Full article

The aim of this study was to develop and validate a dissolution test for favipiravir release in a tablet dosage form using ultra-high performance liquid chromatography (UHPLC). The dissolution method was developed by testing the solubility of favipiravir in media with different pH values. The results demonstrated that the best dissolution was achieved in phosphate buffer with a pH of 6.8. The amount of favipiravir that was released was about 100% after 30 min. The UHPLC method presented linearity (R = 1.000) in the concentration range of 0.044–0.44 mg/mL. The recovery parameter that was achieved ranged from 102.5% to 104.2%. The system suitability, repeatability, and intermediate precision RSD% results were found to be 0.36%, 1.99%, and 2.49%, respectively. In addition to these parameters and results, an F-test was performed using the Minitab 18 Statistical Software program for the intermediate precision and repeatability results. The standard and sample solutions were found to be stable for 2 days in their respective dissolution medium. This analytical method was also found to be selective for favipiravir. In conclusion, a simple and feasible dissolution method with a short run time of 2.5 min was developed and validated successfully. The obtained results demonstrated that the dissolution test developed here is adequate for its purpose and can be applied as the dissolution method for favipiravir in film-coated tablets for release analyses. Full article

Among all modern antibiotics, fluoroquinolones are well known for their broad spectrums of activity and efficiency toward microorganisms and viruses. However, antibiotic resistance is still a problem, which has encouraged medicinal chemists to modify the initial structures in order to combat resistant strains. Our current work is aimed at synthesizing novel hybrid derivatives of ciprofloxacin and norfloxacin and applying docking studies and biological activity evaluations in order to find active promising molecules. We succeeded in the development of a synthetic method towards 1,2,3-triazole-substituted ciprofloxacin and norfloxacin derivatives. The structure and purity of the obtained compounds were confirmed by ¹H NMR, ¹³C NMR, ¹⁹F NMR, LC/MS, UV-, IR- spectroscopy. Docking studies, together with in vitro research against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 27853, Candida albicans NCTC 885-653 revealed compounds in which activity exceeded the initial molecules. Full article

Many specified impurities in vildagliptin’s finished product have been disclosed in the literature that are above their qualification threshold. We used the impurity B (amide impurity) as a case example to explore whether existing literature can be leveraged to determine the safe level of impurity and thereby develop a patient-centric specification (PCS) for impurities. No-observed-adverse-effect level (NOAEL) was derived from rate metabolism information and converted to human equivalent dose (HED). The HED was estimated as 6.5 mg/day. The high qualification levels are supported by repeat dose toxicity studies performed in rats, mice and dogs. Maximum theoretical amount (MTA) was correlated with the maximum observed amount (MOA) to verify whether the exposure was due to impurity and/or metabolite. MOA/MTA was found ≥1 suggesting that metabolism contributed to the amount excreted in feces and therefore could be used to further justify a higher specification limit than the usual one of ≤0.5%. Quite often the drug metabolism and degradation pathways overlap, resulting in the formation of identical constituents. Therefore, metabolism data can be leveraged for deriving safe levels of degradation impurities and develop PCS for impurities. Full article

A series of 5-methyl-7-phenyl-3H-thiazolo[4,5-b]pyridin-2-ones has been designed, synthesized, and characterized by spectral data. Target compounds were screened for their antimicrobial activity against some pathogenic bacteria and fungi, and most of them showed moderate activity, especially compound 3g, which displayed the potent inhibitory effect against Pseudomonas aeruginosa and Escherichia coli with MIC value of 0.21 μM. The active thiazolopyridine derivatives 3c, 3f, and 3g were screened for their cytotoxicity effects on HaCat, Balb/c 3T3 cells using MTT assay, which revealed promising results. In silico assessment for compounds 3c, 3f, and 3g also revealed suitable drug-like parameters and ADME properties. The binding interactions of the most active compound 3g were performed through molecular docking against MurD and DNA gyrase, with binding energies and an inhibitory constant compared to the reference drug ciprofloxacin. The tested thiazolo[4,5-b]pyridines constitute an exciting background for the further development of new synthetic antimicrobial agents. Full article

The potential therapeutic benefit of transdermal delivery systems for some active pharmaceutical ingredients (APIs) has been well-established for decades within the scientific community. However, together with the clinical efficacy, there is the need for an evaluation of the stability of such APIs in bases with known transdermal capabilities, which is necessary to provide the compounding pharmacist with confidence when providing transdermal products. In this study, the stability of danazol, metformin HCl, and resveratrol as individual ingredients, as well as metformin HCl, resveratrol, and Vitamin D3 in combinations at bracketed high and low concentrations, were evaluated over a period of 6 months, using a ready-to-use transdermal vehicle for compounding pharmacies (Pentravan^® or Pentravan^® Plus). The five formulations tested (F1: Danazol 50 mg/g + Miodesin^TM 85 mg/g in Pentravan^®, F2: Metformin HCl 200 mg/g in Pentravan^®, F3: Resveratrol 200 mg/g in Pentravan^®, F4: Metformin HCl 100 mg/g + Resveratrol 100 mg/g + Vitamin D3 5000 IU in Pentravan^®, and F5: Metformin HCl 200 mg/g + Resveratrol 200 mg/g + Vitamin D3 5000 IU in Pentravan^® Plus) presented a beyond-use date of at least 6 months, presenting high convenience for the compounding pharmacies. Full article

Knowledge within the field of phytochemistry research has accelerated at a tremendous speed. The excess of literature reports featuring plants of high ethnopharmacological importance, in combination with our interest in the Asteraceae family and traditional medicine, led us to acknowledge the value of the Achillea L. genus. In a broad context, the various Achillea species are used around the globe for the prevention and treatment of different diseases, including gastrointestinal problems, haemorrhages, pneumonia, rheumatic pains, diuresis, inflammation, infections, and wounds, as well as menstrual and gynaecologic abnormalities. The present review aims to provide and summarize the recent literature (2011–2021) on the phytochemistry of the Achillea genus. In parallel, this study attempts to bridge the reports on the traditional uses with modern pharmacological data. Research articles that focused on secondary metabolites, traditional uses and pharmacological activities were collected from various scientific databases such as Pubmed, ScienceDirect, Reaxys and Google Scholar. This study revealed the presence of 141 phytochemicals, while 24 traditionally used Achillea spp. were discussed in comparison to current data with an experimental basis. Full article

The rapid development in bacterial resistance to many groups of known antibiotics forces the researchers to discover antibacterial drug candidates with previously unknown mechanisms of action, one of the most relevant being the inhibition of tRNA (Guanine37-N1)-methyltransferase (TrmD). The discovery of selective TrmD inhibitors in the series of carboxamide derivatives of thienopyrimidines became a background for further modification of the similar structures aimed at the development of promising antibacterial agents. As part of this research, we carried out the construction of heterocyclic hybrids bearing the moieties of thieno[2,3-d]pyrimidine and benzimidazole starting from 3,5-dimethyl-4-oxo-2-thioxo-1H-thieno[2,3-d]pyrimidine-6-carboxylic acid, which was used as the pivotal intermediate. The hybrid molecule of 6-(1H-benzimidazol-2-yl)-3,5-dimethyl-2-thioxo-1H-thieno[2,3-d]pyrimidin-4-one prepared via condensation of the carboxylic acid with ortho-phenylenediamine was further alkylated with aryl/hetaryl chloroacetamides and benzyl chloride to produce the series of S-alkyl derivatives. The results of molecular docking studies for the obtained series of S-alkyl benzimidazole-thienopyrimidines showed their high affinity to the TrmD isolated from the P. aeruginosa. The results of antimicrobial activity screening revealed the antimicrobial properties for all of the studied molecules against both Gram-positive and Gram-negative bacteria and the Candida albicans fungal strain. The highest antimicrobial activity was determined for 2-{[6-(1H-benzimidazol-2-yl)-3,5-dimethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-2-yl]thio}-N-(4-isopropylphenyl)acetamide, which also had the highest affinity to the TrmD inhibitor’s binding site according to the docking studies results. Full article

Taxadiene synthase (TDS) is the rate-limiting enzyme of Taxol biosynthesis that cyclizes the geranylgeranyl pyrophosphate into taxadiene. Attenuating Taxol productivity by fungi is the main challenge impeding its industrial application; it is possible that silencing the expression of TDS is the most noticeable genomic feature associated with Taxol-biosynthetic abolishing in fungi. As such, the characterization of TDS with unique biochemical properties and autonomous expression that is independent of transcriptional factors from the host is the main challenge. Thus, the objective of this study was to kinetically characterize TDS from endophytic bacteria isolated from different plants harboring Taxol-producing endophytic fungi. Among the recovered 23 isolates, Bacillus koreensis and Stenotrophomonas maltophilia achieved the highest TDS activity. Upon using the Plackett–Burman design, the TDS productivity achieved by B. koreensis (18.1 µmol/mg/min) and S. maltophilia (14.6 µmol/mg/min) increased by ~2.2-fold over the control. The enzyme was purified by gel-filtration and ion-exchange chromatography with ~15 overall folds and with molecular subunit structure 65 and 80 kDa from B. koreensis and S. maltophilia, respectively. The chemical identity of taxadiene was authenticated from the GC-MS analyses, which provided the same mass fragmentation pattern of authentic taxadiene. The tds gene was screened by PCR with nested primers of the conservative active site domains, and the amplicons were sequenced, displaying a higher similarity with tds from T. baccata and T. brevifolia. The highest TDS activity by both bacterial isolates was recorded at 37–40 °C. The Apo-TDSs retained ~50% of its initial holoenzyme activities, ensuring their metalloproteinic identity. The activity of purified TDS was completely restored upon the addition of Mg²⁺, confirming the identity of Mg²⁺ as a cofactor. The TDS activity was dramatically reduced upon the addition of DTNB and MBTH, ensuring the implementation of cysteine-reactive thiols and ammonia groups on their active site domains. This is the first report exploring the autonomous robust expression TDS from B. koreensis and S. maltophilia with a higher affinity to cyclize GGPP into taxadiene, which could be a novel platform for taxadiene production as intermediary metabolites of Taxol biosynthesis. Full article

The algae Kappaphycus alvarezii is considered an important raw material for industrial practices, producing high economic value of various derived products. However, the quality of this commodity, which can be indicated by the level of phenolic compounds, may vary due to growth factors, including cultivation sites. An analytical UV-Vis spectrophotometry method coupled with chemometrics was proposed to standardize the red alga based on the content of phenolic compounds. The correlation between the UV-Vis spectra and UPLC–PDA results, combined with a multivariate calibration of the K. alvarezii extracts, was analyzed. The extracts were prepared using an ultrasound-based technique and subsequently subjected to UV-Vis spectral measurements at 200–800 nm and UPLC–PDA at 260 and 330 nm. Chemometric techniques and partial least squares (PLS) were applied to the acquired data to build a reliable analysis of the phenolics in the K. alvarezii extracts. The result showed that the wavelength combination of 200–450 and 600–690 nm provided a valid method for quantitative analysis of the studied phenolics that belong to hydroxybenzoic acid, hydroxycinnamic acid, and flavonoid with a coefficient of regression (R²) > 0.96 in the calibration and validation models, along with an RMSEC and RMSEP value < 8%. The method was then employed to characterize the K. alvarezii samples from 13 different cultivation areas. Principal component analysis (PCA) generated principal components that produced a clear distribution among the samples of K. alvarezii based on phenolic compounds corresponding to the geographical origin. Full article

Causes of the progression of periodontitis such as an imbalance between the immune response by the host by the release of inflammatory mediators in the response of the oral pathogenic dysbiotic biofilm have been identified. New insights on specific cell signaling pathways that appear during periodontitis have attracted the attention of researchers in the study of new personalised approaches for the treatment of periodontitis. The gold standard of non-surgical therapy of periodontitis involves the removal of supra and subgingival biofilm through professional scaling and root planing (SRP) and oral hygiene instructions. In order to improve periodontal clinical outcomes and overcome the limitations of traditional SRP, additional adjuvants have been developed in recent decades, including local or systemic antibiotics, antiseptics, probiotics, anti-inflammatory and anti-resorptive drugs and host modulation therapies. This review is aimed to update the current and recent evolution of therapies of management of periodontitis based on the adjunctive and target therapies. Moreover, we discuss the advances in host modulation of periodontitis and the impact of targeting epigenetic mechanisms approaches for a personalised therapeutic success in the management of periodontitis. In conclusion, the future goal in periodontology will be to combine and personalise the periodontal treatments to the colonising microbial profile and to the specific response of the individual patient. Full article

The exposure of reactive oxygen species is one of the aging triggers at cellular level. The antioxidants have been used as strategic efforts in overcoming the accumulation of ROS. Previous research using crude extracts of clove bud and leaves showed its potential as an antioxidant agent. However, no data were available regarding the antioxidant and antiaging activities of subsequent fractions of clove extracts. Therefore, this study aimed to analyze the antioxidant and antiaging activities of the n-hexane and ethanol fractions from clove bud and leaves. Antioxidant and antiaging activities were tested at the cellular level using the yeast model Schizosaccharomyces pombe. The highest flavonoid content was shown by clove leaf n-hexane fraction (25.6 mgQE·g⁻¹). However, ethanol fraction of clove bud (FEB) showed the highest antioxidant activity based on TBA and antiglycation assays. FEB (8 μg·mL⁻¹) and leaf ethanol fraction (FEL) (10 μg·mL⁻¹) were able to induce yeast tolerance against oxidative stress. In addition, FEB could induce mitochondrial activity and delay the G1 phase of the cell cycle. FEB was found to be rich in gallic acid and (15Z)-9,12,13-trihydroxy-15-octadecenoic. Based on the data, FEB shows the potential antiaging activity, which is promising for further development as biopharmaceutical product formulations. Full article

In this work, antiparasitic peptidomimetics inhibitors (PEP) of falcipain-3 (FP3) of Plasmodium falciparum (Pf) are proposed using structure-based and computer-aided molecular design. Beginning with the crystal structure of PfFP3-K11017 complex (PDB ID: 3BWK), three-dimensional (3D) models of FP3-PEPx complexes with known activities (${ \mathrm{IC}}_{50}^{\exp }$) were prepared by in situ modification, based on molecular mechanics and implicit solvation to compute Gibbs free energies (GFE) of inhibitor-FP3 complex formation. This resulted in a quantitative structure–activity relationships (QSAR) model based on a linear correlation between computed GFE (ΔΔG_com) and the experimentally measured ${ \mathrm{IC}}_{50}^{\exp }$. Apart from the structure-based relationship, a ligand-based quantitative pharmacophore model (PH4) of novel PEP analogues where substitutions were directed by comparative analysis of the active site interactions was derived using the proposed bound conformations of the PEPx. This provided structural information useful for the design of virtual combinatorial libraries (VL), which was virtually screened based on the 3D-QSAR PH4. The end results were predictive inhibitory activities falling within the low nanomolar concentration range. Full article

Nicotine is widely used in pharmaceutical industries, especially for smoking cessation in the form of transdermal patches. Nicotine gel in the patches has limitations from nicotine instability and high volatility. Thus, a nicotine preservation technique is needed. In this study, a nicotine encapsulation process using methyl-β-cyclodextrin (MβCD) is investigated and compared with β-cyclodextrin (βCD) to evaluate the preservation and skin permeation of nicotine. The M06-2X/6-31G(d,p) density functional theory calculations indicate a 1:1 host–guest molar ratio for the inclusion complex of nicotine with βCD and MβCD, which have been validated by experimental studies. The encapsulation efficiencies of βCD and MβCD to encapsulate nicotine are 59.96% and 63.76%, respectively. The preservation study of the inclusion complexes compared to pure nicotine shows a stability improvement of nicotine after being encapsulated. After 21 days, the percentages of the nicotine/βCD and nicotine/MβCD inclusion complexes that remain are 89.32% and 76.22%, while only 65.56% of pure nicotine remains. Besides the one-hour skin permeation tests, the amounts of nicotine permeated through pig skin from the nicotine/βCD and nicotine/MβCD inclusion complex gels are 14 and 10 times as much as the pure nicotine gel, respectively. Therefore, the encapsulation of nicotine with βCD and MβCD can be used to enhance the stability and skin permeation application of nicotine-containing products. Full article

Linalool, a volatile terpene alcohol, is responsible for a characteristic aroma in food, beverages, and cosmetics. However, linalool’s low aqueous solubility and high volatility limit the applications and shelf life of linalool-containing products. Nanoencapsulation using beta-cyclodextrin (BCD), methyl-beta-cyclodextrin (MBCD) and hydroxypropyl-beta-cyclodextrin (HPBCD) was studied to improve the aqueous solubility and stability of linalool. Linalool has two enantiomers with distinct flavors and odors which affect product quality. The enantiomeric selectivity of the cyclodextrins (CDs) toward racemic linalool standard was evaluated. A computational simulation was performed to predict the conformations and interactions of the inclusion complexes. The 1:1 host-guest ratio from the computer simulation was implemented in the experimental study. Phase solubility study shows an improvement in linalool aqueous solubility after being encapsulated by CDs. The encapsulation efficiencies of linalool/BCD, linalool/MBCD, and linalool/HPBCD inclusion complexes are 66.30%, 51.38% and 32.31%, respectively. Nanoencapsulation by CDs can preserve linalool in the form of inclusion complexes compared to its free form. The amount of remaining linalool in linalool/BCD, linalool/MBCD, and linalool/HPBCD inclusion complexes are 89.57%, 87.07%, and 74.86%, respectively which are considerably larger than that of pure linalool (42.30%). CDs also show the enantiomeric selectivity toward (R)-linalool as evident from (R)-linalool percentage of 54.53% in the inclusion complex. Full article

Background: Nicotine is the active alkaloid in cigarettes. It was reported that tobacco smoking has many hazards; one of these hazards is the effect on the cognitive function of the prefrontal cortex. The aim of our study is to investigate the antioxidant effects of ginger, cinnamon oils, and their combination on morphological changes in the prefrontal cortex that were induced by nicotine. Materials and methods: Fifty adult male albino rats were divided into five groups: group I (control group), group II (nicotine), group III (nicotine + cinnamon), group IV (nicotine + ginger), and group V (nicotine + cinnamon + ginger). The coronal sections from the anterior part of the rat brain at the site of prefrontal cortex were examined by light microscope for (H&E and immunohistochemical staining with TNF-α and GFAP), while the ultrastructure morphology was examined by transmission electron microscopy. Levels of the oxidative stress markers (MDA, GSH) in the rats’ brain tissue homogenate were biochemically assessed. Results: Compared to the control group, the rats that were treated with nicotine (group II) showed a significant oxidative stress in the form of marked elevation of MDA and decrease in GSH, apoptotic changes especially in the pyramidal cells in the form of neuronal cell degeneration and pyknosis, and an elevation in the inflammatory marker TNF-α and GFAP expressions. These changes were observed to a lesser degree in rat group (III) and group (IV), while there was a marked improvement achieved by the combined usage of cinnamon and ginger oils, together compared to the nicotine group. Conclusions: Ginger and cinnamon are powerful antioxidants which ameliorate the degenerative and oxidative effects produced by nicotine on a rat’s prefrontal cortex. Full article