Sublingual Administration of Teucrium Polium-Loaded Nanofibers with Ultra-Fast Release in the Treatment of Diabetes Mellitus: In Vitro and In Vivo Evaluation.

In this study, Teucrium polium (TP) methanolic extract, which has antidiabetic activity and protects the ß-cells of the pancreas, was loaded in polyethylene oxide/sodium alginate nanofibers by electrospinning and administered sublingually to evaluate their effectiveness in type-2 diabetes mellitus (T2DM) by cell culture and in vivo studies. The gene expressions of insulin, glucokinase, GLUT-1, and GLUT-2 improved in TP-loaded nanofibers (TPF) on human beta cells 1.1B4 and rat beta cells BRIN-BD11. Fast-dissolving (<120 s) sublingual TPF exhibited better sustainable anti-diabetic activity than the suspension form, even in the twenty times lower dosage in streptozotocin/nicotinamide-induced T2DM rats. The levels of GLP-1, GLUT-2, SGLT-2, PPAR-γ, insulin, and tumor necrosis factor-alpha were improved. TP and TPF treatments ameliorated morphological changes in the liver, pancreas, and kidney. The fiber diameter increased, tensile strength decreased, and the working temperature range enlarged by loading TP in fibers. Thus, TPF has proven to be a novel supportive treatment approach for T2DM with the features of being non-toxic, easy to use, and effective.

Casein fibres for wound healing.

The name casein is given to a family of phosphoproteins which is commonly found in milk. Until recently, this was a constituent of milk that was commonly discarded; however today, it is widely used in health supplements all over the world. In this work, a high loading (50 wt%) of casein is mixed with a solution of polycaprolactone (PCL) to produce bandage-like fibres with an average fibre diameter of 1.4 ± 0.5 µm, which would be used to cover wounds in a series of tests with diabetic rats. Mouse fibroblast cell viability tests show that the casein-loaded fibres had little cytotoxicity with over 90% observed viability. A 14-day in vivo trial involving three groups of rats, used as control (no treatment), pure PCL fibres and casein-loaded fibres, showed that the casein within the fibres contributed to a significantly more extensive healing process. Histological analysis showed increased development of granulation tissue and follicle regrowth for the casein-loaded fibres. Further analysis showed that casein-loaded fibres have significantly lower levels of TNF-α, TGF-ß IL-1ß, NF-κB and IL-6, contributing to superior healing. The results presented here show an economical and simple approach to advanced wound healing.

Rosa canina L. improves learning and memory-associated cognitive impairment by regulating glucose levels and reducing hippocampal insulin resistance in high-fat diet/streptozotocin-induced diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Recent studies claim that Type-2 diabetes mellitus (T2DM) and Alzheimer's disease (AD) overlap in several common pathological pathways which from neuronal damage to impaired memory performance. It is known that the use of Rosa canina L. (R. canina) as medicine in folk medicine dates back to ancient times and is used in the treatment of nervous diseases in Persian medicine. However, the effect of R. canina on diabetes-related cognitive decline and memory impairment has not yet been studied. AIM OF THE STUDY: We evaluated the impact of T2DM on AD-like alterations and examined the molecular mechanism of a possible effect of R. canina on cognitive alterations in diabetic rats. MATERIALS&METHODS: R. canina ethanol extract was obtained by maceration method. This study was performed with male Sprague-Dawley rats fed with a high-fat diet (HFD) for 8 weeks, low-dose streptozotocin (STZ; 35 mg/kg IP) injection for 4 weeks, and R. canina (250 mg/kg; per oral) and metformin (400 mg/kg; per oral) administration for 4 weeks. The weight and blood glucose of rats were measured weekly. To evaluate glucose tolerance area under the curve (AUC) was calculated by performing an oral glucose tolerance test. Then the rats were subjected to behavioural tests, and their hippocampus and cortex tissues were obtained for biochemical and morphological analyses. RESULTS: R. canina could manage glucose responsiveness by reducing post-prandial blood glucose levels, preventing weight loss, and raising serum insulin levels in T2DM-induced rats. Behavioural tests showed that R. canina significantly improves diabetes-related cognitive decline in recall and long-term memory. Treatment with R. canina significantly reversed HFD/STZ-induced increases in insulin, amyloid-ß, amyloid precursor protein levels, and acetylcholinesterase activity in the prefrontal cortex and hippocampus. Furthermore, histological analyzes revealed the protection of R. canina against neuronal disruption in the cortical and hippocampal CA3 region caused by chronic hyperglycemia. CONCLUSION: Analyzed collectively, these results suggest that R. canina can correct T2DM-related cognitive decline may be attributed to insulin pathway modulation, prevention of amyloid deposition, and increased cholinergic transmission.

Oral empagliflozin-loaded tri-layer core-sheath fibers fabricated using tri-axial electrospinning: Enhanced in vitro and in vivo antidiabetic performance.

Empagliflozin (EM) was successfully loaded in polycaprolactone/poly (L-lactic acid)/polymethyl methacrylate (PCL/PLA/PMMA) fibers. In the rat ß-cell line (BRIN-BD11), the insulin expression ratio of pancreatic ß-cells was stimulated at high and low glucose by culturing with tri-layer EM-loaded fiber (EMF) for 48 h. The expression ratios of glucokinase and GLUT-2 proteins increased after EMF treatment. According to the in vitro drug release test, 97% of all drug contained in fibers was released in a controlled manner for 24 h. The pharmacokinetic test revealed that the bioavailability was improved ∼4.8-fold with EMF treatment compared to EM-powder and blood glucose level was effectively controlled for 24 h with EMF. Oral administration of EMF exhibited a better sustainable anti-diabetic activity even in the half-dosage than EM-powder in streptozotocin/nicotinamide-induced T2DM rats. The levels of GLP-1, PPAR-γ, and insulin were increased while the levels of SGLT-2 and TNF-α were decreased with EMF treatment. Also, EMF recovered the histopathological changes in the liver, pancreas, and kidney in T2DM rats and protected pancreatic ß-cells. Consequently, EMF is suggested as an unprecedented and promotive treatment approach for T2DM with a higher bioavailability and better antidiabetic effect compared to conventional dosage forms.

A novel multi-target strategy for Alzheimer's disease treatment via sublingual route: Donepezil/memantine/curcumin-loaded nanofibers.

Drug delivery systems that not only show efficacy through multiple therapeutic pathways but also facilitate patient drug use and exhibit a high bioavailability profile represent a promising strategy in the treatment of Alzheimer's disease (AD). Here, donepezil (DO)/memantine (MM)/curcumin (CUR)-loaded electrospun nanofibers (NFs) were produced for the treatment of AD. DSC, XRD, and FT-IR studies demonstrated the complete incorporation of the drug into PVA/PVP NFs. The disintegration profile was improved by loading the drugs in PVA/PVP with fast wetting (less than 1 s), the start of disintegration (21 s), and dispersion in 110 s. The desired properties for sublingual application were achieved with the dissolution of NFs in 240 s. The cell viability in DO/MM/CUR-loaded NFs was similar to the control group after 48 h in the cell culture. DO/MM/CUR-loaded NFs enhanced the expressions of BDNF (13.5-fold), TUBB3 (8.9-fold), Neurog2 (5.6-fold), NeuroD1 (5.8-fold), Nestin (166-fold), and GFAP (115-fold). DO/MM/CUR-loaded NFs and powder of these drugs contained in these fibers were daily administered sublingually to intracerebroventricular-streptozotocin (icv-STZ) treated rats. DO/MM/CUR-loaded NFs treatment improved the short-term memory damage and enhanced memory, learning ability, and spatial exploration talent. Results indicated that the levels of Aß, Tau protein, APP, GSK-3ß, AChE, and TNF-α were significantly decreased, and BDNF was increased by DO/MM/CUR-loaded NFs treatment compared to the AD group. In the histopathological analysis of the hippocampus and cortex, neuritic plaques and neurofibrillary nodes were not observed in the rats treated with DO/MM/CUR-loaded NFs. Taken together, the sublingual route delivery of DO/MM/CUR-loaded NFs supports potential clinical applications for AD.

Effects of Colchicum baytopiorum leaf extract on cytotoxicity and cell death pathways in C-4 I and Vero cell lines.

PURPOSE: The present study aimed to research the cytotoxic effects of Colchicum baytopiorum extract on normal and cancerous cells and reveal the cell death mechanisms in cancerous cells triggered by this effect. METHODS: Within this framework, the cells' index values obtained with an xCELLigence Real Time Cell Analysis DP device, selectivity index (SI), apoptotic index (AI) based on a DAPI application and time-related activities of caspase 3,7 and 9 with a spectrofluorometer were inspected. The expressions of apoptosis/autophagy/entosis/necroptosis/anoikis-related genes were researched with qRT-PCR. RESULTS: It was determined that C. baytopiorum extract had displayed a high selectivity [(SI)=4], increased AI (p<0.01) and activation of caspases 3,7 and 9 (p<0.05). It was observed that the mRNA expressions of Atg12, Atg16, Atg5, Atg7, bad, bak, bax, bcl-xL, Beclin1, caspase3, FLIP, Puma, LC3, mcl-1, Bit1, Rho, RIP1, ROCK and TRAF2 genes in C-4 I cells to which the plant extract was applied had increased significantly in comparison with the control group (FC≥1.5). A lowering was detected in the mRNA levels of IAP, SRC kinase and TNF. CONCLUSION: Consequently, it was revealed that the plant extract used had increased the gene expressions in the autophagic cell death pathway in C-4 I cells along with apoptosis and thus, it could be a promising candidate for cervix carcinoma treatment.

Accelerated diabetic wound healing by topical application of combination oral antidiabetic agents-loaded nanofibrous scaffolds: An in vitro and in vivo evaluation study.

The combination of oral antidiabetic drugs, pioglitazone, metformin, and glibenclamide, which also exhibit the strongest anti-inflammatory action among oral antidiabetic drugs, were loaded into chitosan/gelatin/polycaprolactone (PCL) by electrospinning and polyvinyl pyrrolidone (PVP)/PCL composite nanofibrous scaffolds by pressurized gyration to compare the diabetic wound healing effect. The combination therapies significantly accelerated diabetic wound healing in type-1 diabetic rats and organized densely packed collagen fibers in the dermis, it also showed better regeneration of the dermis and epidermis than single drug-loaded scaffolds with less inflammatory cell infiltration and edema. The formation of the hair follicles started in 14 days only in the combination therapy and lower proinflammatory cytokine levels were observed compared to single drug-loaded treatment groups. The combination therapy increased the wettability and hydrophilicity of scaffolds, demonstrated sustained drug release over 14 days, has high tensile strength and suitable cytocompatibility on L929 (mouse fibroblast) cell and created a suitable area for the proliferation of fibroblast cells. Consequently, the application of metformin and pioglitazone-loaded chitosan/gelatin/PCL nanofibrous scaffolds to a diabetic wound area offer high bioavailability, fewer systemic side effects, and reduced frequency of dosage and amount of drug.

Evaluation of burst release and sustained release of pioglitazone-loaded fibrous mats on diabetic wound healing: an in vitro and in vivo comparison study.

In order to provide more effective treatment strategies for the rapid healing of diabetic wounds, novel therapeutic approaches need to be developed. The therapeutic potential of peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist pioglitazone hydrochloride (PHR) in two different release kinetic scenarios, burst release and sustained release, was investigated and compared with in vitro and in vivo tests as potential wound healing dressings. PHR-loaded fibrous mats were successfully fabricated using polyvinyl-pyrrolidone and polycaprolactone by scalable pressurized gyration. The results indicated that PHR-loaded fibrous mats expedited diabetic wound healing in type-1 diabetic rats and did not show any cytotoxic effect on NIH/3T3 (mouse embryo fibroblast) cells, albeit with different release kinetics and efficacies. The wound healing effects of fibrous mats are presented with histological and biochemical evaluations. PHR-loaded fibrous mats improved neutrophil infiltration, oedema, and inflammation and increased epidermal regeneration and fibroblast proliferation, but the formation of hair follicles and completely improved oedema were observed only in the sustained release form. Thus, topical administration of PPAR-γ agonist in sustained release form has high potential for the treatment of diabetic wounds in inflammatory and proliferative phases of healing with high bioavailability and fewer systemic side effects.

Evaluation of Paclitaxel Effects in The Pattern of Expression of Survival and Apoptotic Genes Regulators in HeLa Cells.

We aim to determine the regulation of apoptosis by paclitaxel-induced and understand cancer dynamics to treatment targets for HeLa cells by identifying decrease/increase genes expression on HeLa cells. In this study, the anti-tumor effects of Paclitaxel (PAC) on HeLa cells have been studied in order to determine the cellular and molecular mechanisms of these effects. PAC has been applied to HeLa cells in 6 different doses (3, 7.5, 15, 30, 60, 120 nM) for 48 hours and the IC50 dose MTT method, has been determined with apoptic index (AI) DAPI. Morphological aspects have been demonstrated using light, phase contrast and fluorescent microscopes, additionally activation of Caspase 3,7 and 10 have been shown using florescent spectroscopy. RT-PCR and qRT-PCR have been used to evaluate pro/anti-apoptotic gene expression. According to the parameters being evaluated; PAC has reduced cell multiplication based on dosage and time (p<0.01). 15 nM has been determined as the IC50 value. AI value has been determined as 42%. In the molecular level analyses in addition to the increase in Caspase3,7,10 activation, RT-PCR results show that bax, bak, bcl-x, bik, mcl-1 genes are expressed in the control group as well as the experimental 15 nM group; whereas bak, bcl-x ve bik genes have a decrease in expression compared to the control group. qRT-PCR results show that Apaf1, Bad, Bax, Bcl2L11, Caspase1, Caspase10, Caspase4, Caspase7, Dffa, Fas, Htra2, Lrdd, NFKB1, NFKB2, PMAIP1, RELA, RELB, TNFRSF10A, TNFRSF10C, TNFRSF10D, TNFRSF1A, TNFRSF21, TNFRSF25 gene expressions have increased significantly. On the other hand, BAG1, BBC3, Bcl2L1, Bcl2L10, Bid, Caspase2, Caspase6, Caspase8, Caspase9, FADD, FAM96A, FasLG, HRK, SOCS3, TNF, TNFSF10, TRAF5, TRAF6 mRNA levels are significantly decreased.

In vitro anticancer and cytotoxic activities of some plant extracts on HeLa and Vero cell lines.

PURPOSE: The aim of our study was to evaluate the effect of in vitro anticancer and cytotoxic activity of the methanolic extracts of 14 medicinal plants, 8 of which are endemic species in Anatolia, against the human HeLa cervical cancer cell line and to compare to the normal African green monkey kidney epithelial cell line (Vero) using the MTT colorimetric assay. METHODS: Values for cytotoxicity measured by MTT assay were expressed as the concentration that causes 50% decrease in cell viability (IC50, µg/mL). The degree of selectivity of the compounds can be expressed by its selectivity index (SI) value. High SI value (>2) of a compound gives the selective toxicity against cancer cells (SI = IC50 normal cell/IC50 cancer cell). RESULTS: Dose-dependent studies revealed IC50 of 293 mg/mL and >1000 mg/mL for Cotinus coggygria Scop., IC50 of 265 µg/mL and >1000 mg/mL for Rosa damascena Miller, IC50 of 2 µg/mL and 454 mg/mL for Colchicum sanguicolle K.M. Perss, IC50 of 427 µg/mL and >1000 µg/mL for Centaurea antiochia Boiss. var. praealta (Boiss & Bal) Wagenitz on the HeLa cells and the Vero cells, respectively. Four plants showed significant SI values which were 227 for Colchicum sanguicolle K.M. Perss (endemic species), >3.8 for Rosa damascena Miller, >3.4 for Cotinus coggygria Scop. and >2.3 for Centaurea antiochia Boiss. var. praealta (Boiss & Bal)Wagenitz (endemic species). CONCLUSION: According to our study, 4 methanolic extracts of 14 tested plants exhibit greater activity on the HeLa cell line and little activity on the Vero cell line, meaning that these plants can be evaluated for potential promising anticancer activity.

Screening for antitumor activity of various plant extracts on HeLa and C 4-I cell lines.

PURPOSE: Cancer is a long process that leads the organism to death and is associated with the normal cells acquiring the ability to divide permanently. Nowadays, the use of natural products in cancer therapy has a great importance. In addition, working with plants that are endemic to Turkey and determining the biological activities of these plant extracts, is extremely important due to the potential for new drug development. There is no comparative study available in the literature on the antitumor effects of Colchicum sanguicolle, a new found species of the genus Colchicum in Turkey, Crateagus microphylla, of the genus Crateagus and Centaurea antiochia of the genus Centaurea. In this study, we tried to demonstrate the antitumor effect of these plant extracts on HeLa and C 4-1 cells. METHODS: Five different doses (0.001, 0.01, 0.05, 0.25 and 0.5 mg/ml) of the three plant types were prepared and applied for 24, 48 and 72 hrs on the cervical cancer derived cell lines. Subsequently, the growth rate was evaluated with the mitochondrial dehydrogenase enzyme method. RESULTS: Colchicum sanguicolle extracts showed the most effective antitumor activity. For the Colchicum sanguicolle extract, the IC50 dose for HeLa cells was 0.01 mg/ml at 48 hrs, while for the C-4 I cells it was 0.001 mg/ml at 48 hrs. These results showed that C-4 I cells were more sensitive to the Colchicum sanguicolle extracts. Conclus?on: The results of from this study regarding the antitumor effect of plant extracts of endemic varieties of Turkey may have an important place in design and development of anticancer drugs and would make contributions to other studies to be conducted in this area.