RESUMO
Fla-CN is a flavonoid derivative with anti-diabetic and anti-obesity effects; however, its biological targets are still unknown. In this study, we developed bifunctional affinity-based probes to identify the direct targets of Fla-CN. When using probe 3, we observed the co-location of probe 3 and mitochondria in both HepG2 and 3T3-L1 cells. The putative target proteomes were obtained using activity-based protein profiling (ABPP) and photo-affinity labelling. Pyruvate carboxylase, mitochondrial malate dehydrogenase, mitochondrial complex I, and F1FO-ATPase were validated as the direct targets of Fla-CN by surface plasmon resonance (SPR) and biochemical assays. It was elucidated that the Tyr651, Gln870 and Lys912 were the key amino acid residues near the binding site of pyruvate carboxylase with Fla-CN. The direct interaction of Fla-CN and the above four targets allowed elucidation of its complicated molecular mechanism, including the activation of adenosine 5-monophosphate (AMP)-activated protein kinase (AMPK), and the inhibition of gluconeogenesis. Further investigation for activation of AMPK in normal and insulin resistance (IR) HepG2 cells, indicated that Fla-CN could target insulin resistance tissues.
Assuntos
Diabetes Mellitus , Resistência à Insulina , Proteínas Quinases Ativadas por AMP/metabolismo , Flavonoides/química , Flavonoides/farmacologia , Humanos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Piruvato CarboxilaseRESUMO
3-O-[(E)-4-(4-cyanophenyl)-2-oxobut-3-en-1-yl] kaempferol is a novel lead compound to discover anti-diabetic and anti-obesity drugs. The present study reported the scaffold hopping of the lead compound to obtain a new isoxazole derivative, C45, which has improved glucose consumption at the nanomolar level (EC50 = 0.8 nM) in insulin resistant (IR) HepG2 cells. Western blotting showed that C45 markedly enhanced the phosphorylation of AMPK (AMP-activated protein kinase) and reduced the levels of the gluconeogenesis key enzymes PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (glucose 6-phosphatase) in HepG2 cells. The potential molecular mechanism of C45 may be activation of the AMPK/PEPCK/G6Pase pathways. We concluded that C45 might be a valuable candidate to discover anti-diabetic drugs.
Assuntos
Flavonoides/farmacologia , Hipoglicemiantes/farmacologia , Flavonoides/química , Glucose/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hipoglicemiantes/química , Estrutura MolecularRESUMO
A novel chiral derivatization reagent, the N-[1-oxo-5-(triphenylphosphonium)pentyl]- (R)-1,3-thiazolidinyl-4-N-hydroxysuccinimide ester bromide salt (OTPTHE), was developed for the separation and selective detection of chiral DL-amino acids by RP-HPLC analysis. The OTPTHE reacted with DL-amino acids at 60°C maintained for 30 minutes in the presence of 100 mM borate buffer (pH 9.5). The separability of the diastereomeric derivatives was evaluated in terms of the resolution value (Rs) using 13 kinds of DL-amino acids, which were completely separated by reversed-phase chromatography using C18 column at 254 nm. The Rs of the DL-amino acids varied from 1.62 to 2.51. As for the application of the DL-amino acids, the determination of DL-Ser in the human plasma of healthy volunteers was performed based on our developed method. It was shown that linear calibrations were available with high coefficients of correlation (r2 > 0.9997). The limit of detection (S/N = 3) of the DL-Ser enantiomers was 5.0 pmol; the relative standard deviations of the intraday and interday variations were below 4.56%; the accuracy ranged between 95.40%-110.06% and 95.45%-109.80%, respectively; the mean recoveries (%) of the DL-Ser spiked in the human plasma were 99.49%-103.74%. The amounts of DL-Ser in the human plasma of healthy volunteers were determined.
Assuntos
Serina/sangue , Serina/química , Succinimidas/química , Tiazolidinas/química , Calibragem , Fracionamento Químico , Cromatografia Líquida , Humanos , Indicadores e Reagentes/química , Serina/isolamento & purificação , EstereoisomerismoRESUMO
Fla-CN (3-O-[(E)-4-(4-cyanophenyl)-2-oxobut-3-en-1-yl] kaempferol) is a semi-synthesized flavonoid derivative of tiliroside which exhibited anti-diabetic effect in vivo. Our previous study revealed the role of Fla-CN in anti-obesity and anti-diabetes in vivo, but the underlying mechanism remained to be addressed. The present study aimed to investigate the mechanism of anti-adipogenesis in vitro. Fla-CN markedly inhibited intracellular lipid accumulation in a dose-dependent manner, and the inhibitory effect was mainly limited to the early stage of adipocyte differentiation in vitro. Further investigations revealed that Fla-CN up-regulated the expression level of miR-27a/b and suppressed its target genes expression including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα). Furthermore, the phosphorylation of AMP-activated protein kinase (AMPK) was also enhanced by Fla-CN in pre-adipocyte differentiation. These effects were abolished when cells were treated with miR-27a/b inhibitor and AMPK inhibitor Compound C. Additionally, Fla-CN reduced the expressions of adipocyte-specific genes such as sterol regulatory element-binding transcription factor 1c (SREBP-1c), fatty acid synthase (FAS) and adipocyte fatty acid binding protein (aP2). In conclusion, these results suggested a mechanism of Fla-CN for adipocyte differentiation inhibition of 3T3-L1 cells through miR-27a/b induction and AMPK activation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Quempferóis/farmacologia , MicroRNAs/genética , Regulação para Cima/efeitos dos fármacos , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Animais , Ativação Enzimática/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
Novel pregn-17(20)-en-3-amine derivatives were synthesized and their anti-metastatic effects were evaluated in human breast cancer cells using chemotaxis assay. Compared with positive control LY294002, a PI3K inhibitor, derivatives 5a, 19a, 20a, 19g, 20f, 5c, 12e and 12f exhibited significant inhibitory effects against cancer cell migration induced by chemokine epidermal growth factor (EGF). Especially, the IC50 for compound 20f was as low as 0.03 µM. Preliminary structure-activity relationship studies suggested that most 3ß-substituted derivatives were more effective than those 3α-substituted derivatives, provided there was no substituted group at position C-16. Moreover, the α,ß-unsaturated fragment in ring D might be critical for their anti-metastatic activities. Further investigations on compound 20f revealed inhibitory effects on cell adhesion, migration and invasion of MDA-MB-231 cells. The mechanisms for the anti-metastatic effect of 20f might be through the inhibition of the phosphorylations of PI3K, Akt, PKCζ, and integrin ß1 in a dose-dependent manner. Taken together, the novel steroidal alkaloid derivative 20f could be further explored as an effective anti-metastatic agent for the treatment of human metastatic breast cancer.
Assuntos
Aminas/síntese química , Aminas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Aminas/química , Antineoplásicos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Química Sintética , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Integrina beta1/metabolismo , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Relação Estrutura-AtividadeRESUMO
Nine new pregnane alkaloids (1-9), together with eight known alkaloids (10-17), were isolated from the whole herb of Pachysandra terminalis. Their structures were elucidated on the basis of spectroscopic analyses. In addition, the isolates were examined for their ability to inhibit the migration of MDA-MB-231 cells induced by the chemokine epidermal growth factor (EGF). Alkaloids 1, 5, 7, 9, 12, and 17 presented significant anti-metastasis activities compared with the positive reagent, LY294002.
RESUMO
In our previous study, a derivative of tiliroside, 3-O-[(E)-4-(4-ethoxyphenyl)-2-oxobut-3-en-1-yl]kaempferol (Fla-OEt) significantly enhanced glucose consumption in insulin resistant HepG2 cells. This article deals with the antihyperglycemic and antihyperlipidemic effects of Fla-OEt in diet-induced obesity (DIO) mice. Daily administration of Fla-OEt significantly decreased oral glucose tolerance test, intraperitoneal insulin tolerance test and serum lipids. Hyperinsulinemic-euglycemic clamp and the ratio of high-density-lipoprotein/low-density-lipoprotein with Fla-OEt treatment were increased comparing with high-fat diet (HFD) group, so lipid metabolism was improved. Histopathology examination showed that the Fla-OEt restored the damage of adipose tissues and liver in DIO mice. Moreover, compared with HFD group, Fla-OEt treatment significantly increased the phosphorylation of AMPK and ACC in adiposity tissues, liver, and muscles. The mechanism of its action might be the activation of AMPK pathway. It appears that Fla-OEt is worth further study for development as a lead compound for a potential antidiabetic agent.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Flavonoides/farmacologia , Hipoglicemiantes/farmacologia , Obesidade/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Dieta Hiperlipídica , Flavonoides/administração & dosagem , Hipoglicemiantes/administração & dosagem , Resistência à Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Obesos , Obesidade/tratamento farmacológico , Fosforilação/efeitos dos fármacosRESUMO
3-O-[(E)-4-(4-cyanophenyl)-2-oxobut-3-en-1-yl]kaempferol (Fla-CN), a semi-synthesized flavonoid derivative of tiliroside, reduces whole-body adiposity, ameliorates metabolic lipid disorder, improves insulin sensitivity and benefits other disorders characterized by insulin resistance in high fat diet induced obesity mice. The improvement of insulin sensitivity and the reduction of weight gain are correlated with the changes of leptin and adiponectin levels. As a result, Fla-CN treatment could increase the expressions of pAMPK and miR-27 in the liver and adipose tissues. Meanwhile, we discovered that the expressions of various adipogenesis genes were downregulated, which were target genes of miR-27. This is the first report for the action of miR-27 by flavonoid derivative in rodents. The action of Fla-CN might be through multiple approaches including AMPK activation and enhancement in miR-27 expression.
Assuntos
Fármacos Antiobesidade/uso terapêutico , Hipoglicemiantes/uso terapêutico , Quempferóis/uso terapêutico , Obesidade/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Fármacos Antiobesidade/farmacologia , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Dieta Hiperlipídica , Flavonoides , Hipoglicemiantes/farmacologia , Resistência à Insulina , Quempferóis/farmacologia , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Obesidade/patologiaRESUMO
Three new pregnane alkaloids, named terminamines H-J (1-3), together with two known alkaloids (4 and 5), were isolated from the ethanol extract of Pachysandra terminalis. The structures of isolated compounds were elucidated by spectroscopic methods, including (1)H and (13)C NMR, 2D NMR, and HR-ESI-MS. Compounds 1, 4, and 5 revealed significant anti-metastasis activities. In addition, compound 1 inhibited the expression of p-PKCζ in MDA-MB-231 cells, and compound 4 inhibited the expressions of p-PKCζ in MDA-MB-231 and A549 cells.
Assuntos
Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Pachysandra/química , Pregnanos/isolamento & purificação , Alcaloides/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Pregnanos/química , Pregnanos/farmacologiaRESUMO
The preparation of novel E-salignone derivatives and their biological evaluation as potential antimetastatic agents is described. The E-salignone amide derivatives were prepared from epiandrosterone and androsterone, and characterized by analytical (1) H NMR, (13) C NMR, and mass spectrometry. The derivatives were evaluated for antimetastatic activity in MDA-MB-231 cells by using a transwell assay. Comparing with the positive control, LY294002, compounds 19b, 19d, and 19e exhibited significant inhibitory effects on the EGF-induced invasion of MB-MDA-231 cells. Moreover, compound 19b also had antimigration effects in wound-healing assay. Compound 19b may represent a novel antimetastatic agent for treating breast cancer.
Assuntos
Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/prevenção & controle , Pregnenos/síntese química , Pregnenos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Cromonas/farmacologia , Feminino , Humanos , Estrutura Molecular , Morfolinas/farmacologia , Invasividade Neoplásica , Metástase Neoplásica , Pachysandra/química , Pregnenos/química , Pregnenos/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
The purpose of this study was to develop novel dissolving microneedle arrays fabricated from hyaluronic acid (HA) as a material and to improve the transdermal permeability of relatively high molecular weight drugs. In this study, fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4kDa (FD4) was used as a model drug with a relatively high molecular weight. The microneedle arrays significantly increased transepidermal water loss (TEWL) and reduced transcutaneous electrical resistance (TER), indicating that they could puncture the skin and create drug permeation pathways successfully. Both TEWL and TER almost recovered to baseline levels in the microneedle array group, and relatively small pathways created by the microneedles rapidly recovered as compared with those created by a tape stripping treatment. These findings confirmed that the microneedle arrays were quite safe. Furthermore, we found that the transdermal permeability of FD4 using the microneedle arrays was much higher than that of the FD4 solution. Furthermore, we found that the microneedle arrays were much more effective for increasing the amount of FD4 accumulated in the skin. These findings indicated that using novel microneedle arrays fabricated from HA is a very useful and effective strategy to improve the transdermal delivery of drugs, especially relatively high molecular weight drugs without seriously damaging the skin.
Assuntos
Ácido Hialurônico/administração & dosagem , Ácido Hialurônico/química , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/química , Pele/metabolismo , Administração Cutânea , Animais , Dextranos/administração & dosagem , Dextranos/química , Sistemas de Liberação de Medicamentos/métodos , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Humanos , Microinjeções/métodos , Peso Molecular , Agulhas , Permeabilidade , Ratos , Segurança , Absorção CutâneaRESUMO
Four new flavonoids including three C-glycosidic flavonoids, named tetrastigma A-D (1-4) and five known flavones (5-9) were isolated from the herbs of Tetrastigma obtectum (Wall.) Planch. The structures of 1-4 were elucidated by 1D- and 2D-NMR and HR mass spectrometric methods. Compound 1 caused significant enhancement in glucose consumption by insulin-resistant HepG2 cells compared with control cells. In addition, compound 1 stimulated phosphorylation of AMPK, which plays an important role in glycometabolism.
Assuntos
Flavonoides/farmacologia , Glucose/metabolismo , Resistência à Insulina , Vitaceae/química , Proteínas Quinases Ativadas por AMP/metabolismo , Flavonoides/química , Flavonoides/isolamento & purificação , Células Hep G2 , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/isolamento & purificação , Hipoglicemiantes/farmacologia , Estrutura Molecular , Fosforilação , Extratos Vegetais/química , Extratos Vegetais/farmacologiaRESUMO
By repeated column chromatography, including silica gel, macroporous resin, and preparative HPLC, a new compound (1) was isolated and purified. On the basis of spectroscopic methods, the structure of 1 was elucidated as ( - ) -epiafzelechin-3, 5-di-O-beta-D-apiofuranoside (1). In the bioassay screening experiments, glucose consumption assays in IR HepG2 cells and colorimetric assay of surface GLUT4myc translocation were used to assess the effects on glucose metabolism of compound 1. Both compound 1 and its derivatives--naringin could improve glucose consumption in IR HepG2 cells and enhance GLUT4 translocation in skeletal muscle cell L6myc in a dose-dependent manner, indicating that these two compouds showed potential anti-diabetic activities in vitro.
Assuntos
Catequina/análogos & derivados , Glucose/metabolismo , Glicosídeos/farmacologia , Hipoglicemiantes/farmacologia , Polypodiaceae/química , Catequina/farmacologia , Relação Dose-Resposta a Droga , Transportador de Glucose Tipo 4/metabolismo , Células Hep G2 , Humanos , Transporte Proteico/efeitos dos fármacosRESUMO
The aim of the present study was to identify potentially useful natural compounds for the development of novel therapeutic agents to inhibit metastasis. A phytochemical investigation of Pachysandra terminalis resulted in the isolation of seven new pregnane alkaloids, terminamines A-G (1-7), and seven known alkaloids (8-14). The structures of 1-7 were elucidated by 1D- and 2D-NMR spectroscopic and mass spectrometric methods. Compounds 1-5 and 8-14 inhibited the migration of MB-MDA-231 breast cancer cells induced by the chemokine epithelial growth factor. In addition, compound 1 inhibited phosphorylation of integrin ß(1), which plays an important role in MB-MDA-231 cell adhesion and metastasis.
Assuntos
Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Pachysandra/química , Pregnanos/isolamento & purificação , Pregnanos/farmacologia , Alcaloides/química , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Medicamentos de Ervas Chinesas/química , Feminino , Humanos , Estrutura Molecular , Metástase Neoplásica/tratamento farmacológico , Ressonância Magnética Nuclear Biomolecular , Pregnanos/químicaRESUMO
The aim of the present study was to develop novel insulin-loaded microneedle arrays (MNs) fabricated from hyaluronic acid (HA), and characterize their applicability in the transdermal delivery of insulin. The shape of MNs was observed via scanning electron microscopy. The characteristics of these novel insulin-loaded MNs, including hygroscopy, stability, drug release profiles, and dissolution properties, were evaluated from a clinical application point-of-view. Transepidermal water loss (TEWL) was measured to investigate the piercing properties of MNs, and the recovery of the skin barrier after the removal of MNs to confirm their safety. Additionally, the transdermal absorption of insulin from MNs was examined via an in vivo absorption study in diabetic rats. The length of MNs was 800 µm with a base diameter of 160 µm and a tip diameter of 40 µm. MNs were found to maintain their skin piercing abilities for at least 1h, even at a relative humidity of 75%. After storing insulin-loaded MNs for a month at -40, 4, 20, and 40 °C, more than 90% of insulin remained in MNs at all temperatures, indicating that insulin is highly stable in MNs at these storage conditions. It was also found that insulin is rapidly released from MNs via an in vitro release study. These findings were consistent with the complete dissolution of MNs within 1h of application to rat skin in vivo. Therefore, the novel HA MNs possess self-dissolving properties after their dermal application, and insulin appears to be rapidly released from these MNs. A significant increase in TEWL was observed after the application of MNs. However, this parameter recovered back to baseline within 24h after the removal of MNs. These findings indicate that the transdermal transport pathway of insulin, which was created by the MNs, disappeared within 24h, and that the skin damage induced by the MNs was reversible. Furthermore, a dose-dependent hypoglycemic effect and transdermal delivery of insulin were observed after a dermal treatment with insulin-loaded MNs in vivo. A continuous hypoglycemic effect was observed after 0.25 IU of insulin was administered to skin via MNs. Additionally, lower peak plasma insulin levels, but higher plasma insulin concentrations after 2 h, were achieved with 0.25 IU of insulin administered via MNs as compared to the subcutaneous administration of insulin of the same dose. Pharmacodynamic and pharmacokinetic parameters indicated that insulin administered via MNs was almost completely absorbed from the skin into the systemic circulation, and that the hypoglycemic effect of insulin-loaded MNs was almost similar to that of the subcutaneous injection of insulin. These findings indicate that the novel insulin-loaded MNs fabricated from HA are a very useful alternative method of delivering insulin via the skin into the systemic circulation without inducing serious skin damage. Therefore, HA MNs may be an effective and safe method of transdermal insulin delivery in the clinic.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Agulhas , Administração Cutânea , Animais , Glicemia/análise , Diabetes Mellitus Experimental/sangue , Ácido Hialurônico/química , Hipoglicemiantes/sangue , Hipoglicemiantes/farmacocinética , Insulina/sangue , Insulina/farmacocinética , Masculino , Ratos , Ratos WistarRESUMO
Seven new dibenzocyclooctadiene lignans, named propinquain E-K, together with 11 known lignans were isolated from the ethanol extract of the roots of Schisandra propinqua subsp. sinensis. The structures of the new compounds were elucidated by spectroscopic methods, including ¹H- and ¹³C-NMR, 2D-NMR, HR ESI-MS, and CD spectra. Two dibenzocyclooctadienes revealed inhibitory effects on lymphocyte proliferation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Lignanas/isolamento & purificação , Linfócitos/efeitos dos fármacos , Schisandra/química , Animais , Lignanas/química , Lignanas/farmacologia , Estrutura MolecularRESUMO
In a study to evaluate the structural elements essential for the antidiabetic activity of flavonoids, we synthesized two series of flavonoids, 5,7-dihydroxyflavanones and 5,7-dihydroxyflavones. In a screening for potential antidiabetic activity, most of the flavonoids showed a remarkable in vitro activity, and compounds 1f, 2d, and 3c were significantly more effective than the positive control, metformin. The biological activity was mainly affected by structural modification at the ring B moiety of the flavonoid skeleton. The results suggest that 5,7-dihydroxyflavonoids can be considered as promising candidates in the development of new antidiabetic lead compounds.
Assuntos
Flavanonas/síntese química , Flavonoides/síntese química , Flavonoides/farmacologia , Hipoglicemiantes/síntese química , Hipoglicemiantes/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Flavanonas/química , Flavanonas/farmacologia , Flavonoides/química , Células Hep G2 , Humanos , Hipoglicemiantes/química , Transdução de Sinais/efeitos dos fármacosRESUMO
A series of new tiliroside derivatives were synthesized and characterized by analytical (1)H NMR, (13)C NMR and mass spectrometry. All of the compounds were evaluated for anti-diabetic properties in vitro using HepG2 cells. Compounds 3c, 3d, and 3i-l caused significant enhancements in glucose consumption by insulin-resistant HepG2 cells compared with control cells and cells that were exposed to metformin (an anti-diabetic drug). Moreover, compound 3l significantly activated adenosine 5'-monophosphate-activated protein kinase activity and reduced acetyl-CoA carboxylase activity. Thus, the tiliroside derivative 3l offers potential to be developed as a new approach for treating type II diabetes.
Assuntos
Flavonoides/química , Flavonoides/farmacologia , Glucose/metabolismo , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Potentilla/química , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Ativação Enzimática/efeitos dos fármacos , Flavonoides/síntese química , Células Hep G2 , Humanos , Hipoglicemiantes/síntese química , Extratos Vegetais/síntese química , Extratos Vegetais/química , Extratos Vegetais/farmacologiaRESUMO
OBJECTIVES: The aim was to assess the effect of trypsin on the transdermal delivery of macromolecules by applying its specific biochemical properties to the stratum corneum of the skin. METHODS: Fluorescein isothiocyanate (FITC)-labelled dextrans (FDs), with molecular weights of 4 to 250 kDa, and FITC-insulin were used as model macromolecules and a model polypeptide, and the in-vitro transdermal permeation experiments, with or without trypsin (0.1-2.5%), were carried out using rat skin and cultured human epidermis. The mechanism for the enhancement of trypsin was also studied using fluorescence and conventional light microscopy. KEY FINDINGS: Trypsin significantly increased the transdermal permeability of all FDs through the rat skin (2.0- to 10.0-fold). It also markedly enhanced the permeation of FD4 through three-dimensional cultured human epidermis (3.1-fold), which was used to evaluate the transport pathways other than the transfollicular route. Furthermore, the permeation flux of FITC-insulin was increased by 10.0-fold with trypsin pretreatment (from 0.02 +/- 0.00 to 0.20 +/- 0.07 microg/cm(2) per h). Mechanistic studies indicated that trypsin affects both the intercellular pathway and the hair follicular route, and may alter stratum corneum protein structures, thereby affecting skin barrier properties. CONCLUSIONS: This study suggests that trypsin could be effective as a biochemical enhancer for the transdermal delivery of macromolecules including peptide and protein drugs.
Assuntos
Excipientes/química , Absorção Cutânea , Tripsina/química , Administração Cutânea , Animais , Dextranos/administração & dosagem , Dextranos/farmacocinética , Fluoresceína-5-Isotiocianato/administração & dosagem , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Humanos , Técnicas In Vitro , Insulina/administração & dosagem , Insulina/farmacocinética , Masculino , Microscopia de Fluorescência , Permeabilidade , Ratos , Ratos Wistar , Pele/efeitos dos fármacos , Pele/metabolismo , SuínosRESUMO
Trypsin has been extensively used in laboratory settings for in vitro epidermal separation and keratinocyte isolation for over 50 years. The aim of this study was to assess the enhancing effect of trypsin on the transdermal delivery of insulin by applying its specific biochemical properties to react with the stratum corneum (SC) of skin. Bovine insulin was used as a model peptide to investigate in vitro permeation through rat skin and in vivo hypoglycemic effects of bovine insulin with or without the trypsin pretreatment. Trypsin significantly increased the transdermal permeability of bovine insulin in pH 3.0 solution, but no effect was observed in pH 6.0 solution. The permeation flux of bovine insulin from pH 3.0 solution was promoted 5.2-fold with 0.25% trypsin pretreatment when compared with the control. The enhancement of trypsin was dependent on the concentration in the range of 0.5-2.5%. Furthermore, with trypsin pretreatment, the plasma glucose level was reduced to less than 60% of the initial value after 8 h of in vivo permeation of bovine insulin with pH 3.0 solution, but did not return to the initial value during an 8-h experiment. Mechanistic studies with Fourier transform-infrared and attenuated total reflectance analysis and electrical resistance measurements suggest that trypsin alters the SC protein structure from the alpha- to the beta-form and decreases the electrical resistance of skin, thereby decreasing the SC barrier and enhancing the permeation of insulin. We conclude that trypsin would be effective as a biochemical enhancer for the transdermal delivery of peptide and protein drugs such as insulin.