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1.
Braz. j. biol ; 82: e244735, 2022. tab, graf
Article in English | LILACS | ID: biblio-1249280

ABSTRACT

Abstract L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G - 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.


Resumo A L-asparaginase, que catalisa a degradação da L-asparagina em L-aspartato e amônia, é uma enzima de importância terapêutica no tratamento do câncer, especialmente dos linfomas e da leucemia. O presente estudo descreve a produção recombinante, propriedades e potencial anticancerígeno da enzima de Pyrococcus abyssi, um archaeon hipertermofílico. Existem dois genes que codificam para a asparaginase no genoma desse organismo. Um gene de 918 bp, que codifica 305 aminoácidos, foi amplificado por PCR e clonado na cepa BL21 (DE3) de E. coli usando o plasmídeo pET28a (+). A produção da enzima recombinante foi induzida sob 0,5mM de IPTG, purificada por desnaturação seletiva por calor e cromatografia de troca iônica. A enzima purificada foi analisada quanto à cinética, estrutura in silico e propriedades anticancerígenas. A enzima recombinante apresentou peso molecular de 33 kDa, atividade específica de 1.175 U / mg, valor de KM 2,05 mM, temperatura ótima de 80º C e pH 8. Nenhuma atividade enzimática detectável foi encontrada quando a L-glutamina foi usada como substrato. Estudos in silico mostraram que a enzima existe como um homodímero, com Arg11, Ala87, Thr110, His112, Gln142, Leu172 e Lys232 sendo os resíduos do local ativo putativo. A mudança de energia livre calculada por estudos de docking molecular da enzima e do substrato foi encontrada como ∆G - 4,5 kJ / mol, indicando a afinidade da enzima com o substrato. Valores de IC50 de 5U / mL a 7,5U / mL foram determinados para células FB, células caco2 e células HepG2. Uma quantidade de enzima (5U / mL) apresentou inibição de crescimento de 78% a 55% das células caco2 e HepG2, respectivamente. Em conclusão, a enzima recombinante produzida e caracterizada no presente estudo é uma boa possibilidade para o tratamento do câncer. Os procedimentos adotados na presente pesquisa podem ser aplicados para estudos in vivo.


Subject(s)
Humans , Asparaginase/biosynthesis , Asparaginase/pharmacology , Pyrococcus abyssi/enzymology , Antineoplastic Agents/pharmacology , Substrate Specificity , Enzyme Stability , Recombinant Proteins/biosynthesis , Recombinant Proteins/pharmacology , Caco-2 Cells , Escherichia coli/genetics , Molecular Docking Simulation , Hydrogen-Ion Concentration
2.
Electron. j. biotechnol ; 52: 76-84, July. 2021. graf, ilus
Article in English | LILACS | ID: biblio-1283597

ABSTRACT

BACKGROUND: Butyrate is a histone deacetylase inhibitor that induces apoptosis and inhibits cell proliferation of colorectal cancer cells. To improve its anticancer activity, butyrate has been evaluated mixed with drugs and different molecules. Plant antimicrobial peptides are attractive anticancer alternative molecules because they show selective cytotoxic activity against different cancer cell lines. In this work, we explore if the plant defensin c-thionin (Capsicum chinense) can improve butyrate activity on Caco-2 cell line and we also determined the mechanism of death activated. RESULTS: The combined treatment of c-thionin (3.5 mM) and butyrate (50 mM) showed higher cytotoxicity on Caco-2 cells with respect to single treatments. Also, the combined treatment reduced cell proliferation and exhibited a higher rate of apoptosis than single treatments. Combined treatment induced caspases 8 and 9 activation to an extent comparable with that of butyrate while c-thionin did not activate caspases. Additionally, reactive oxygen species generation preceded the onset of apoptosis, and superoxide anion production was higher in cells treated with the combined treatment. CONCLUSIONS: The c-thionin from Habanero chili pepper improved the butyrate cytotoxicity on Caco-2 cells. This effect occurred through apoptosis induction associated with reactive oxygen species production. Therefore, the combination of butyrate with cytotoxic antimicrobial peptides could be an attractive strategy for cancer therapy.


Subject(s)
Humans , Butyrates , Capsicum/chemistry , Adenocarcinoma , Colonic Neoplasms , Cell Cycle , Reactive Oxygen Species , Apoptosis , Caco-2 Cells , Defensins , Thionins
3.
São Paulo; s.n; 2021. 128 p.
Thesis in Portuguese | LILACS | ID: biblio-1352769

ABSTRACT

Introdução - O risco de doenças cardiovasculares, dentre elas a aterosclerose, está associado com a hipercolesterolemia, resultado de fatores de risco comportamentais, metabólicos e genéticos. Intervenções dietéticas como o uso do guaraná (Paullinia cupana), podem ser úteis, uma vez que é um alimento rico em compostos bioativos como as catequinas, que possuem elevada atividade antioxidante, inibem a oxidação da lipoproteína de densidade baixa (LDL) e a peroxidação lipídica, sendo capazes de reduzir a concentração plasmática de colesterol, por mecanismos ainda não totalmente elucidados. Objetivo - Investigar os efeitos do extrato aquoso de guaraná (Paullinia cupana) em pó sobre mecanismos envolvidos com a absorção de colesterol em modelos in vitro e em células Caco-2. Métodos - O extrato aquoso do guaraná em pó foi submetido à digestão in vitro, os fenólicos totais foram quantificados pelo método de Folin-Ciocalteu e a determinação do perfil de fenólicos foi realizada por cromatografia líquida de alta eficiência (HPLC), com detector UV/VIS a 210 nm. A atividade antioxidante foi determinada pela capacidade de absorbância de radical oxigênio (Oxygen Radical Absorbance Capacity - ORAC) e foram avaliadas a capacidade de inibição da lipase pancreática, de ligação com ácidos biliares, de interação com a fosfatidilcolina micelar e de inibição da solubilização micelar do colesterol de forma in vitro. Foi realizado ensaio de permeação do colesterol por um período de 2 h em células Caco-2 semeadas e diferenciadas em placas transwell de 24 poços com uma solução de micelas de colesterol e extrato de guaraná em diferentes concentrações. O teor de colesterol permeado foi determinado por HPLC/UV a 206 nm. Foi realizado o ensaio de Western blotting para determinar o conteúdo proteico dos transportadores de colesterol (NPC1L1, ABCG5, ABCG8) e LXR-alfa em células intestinais Caco-2 após a incubação com diferentes concentrações do extrato em 2 e 16 horas. Resultados: O conteúdo total de fenólicos do extrato de guaraná foi 104,36 (± 3,62) mg EAG/g de guaraná em pó. Após o processo de digestão, este valor foi de 48,62 (± 3,97) mg EAG/g de guaraná em pó, demonstrando a bioacessibilidade dos polifenóis. Foram identificados os compostos procianidina B1 (1,56 ± 0,04 mg/g) e B2 (2,34 ± 0,06 mg/g), catequina (24,26 ± 0,63 mg/g), epicatequina (20,39 ± 1,20 mg/g) e cafeína (34,93 ± 0,75 mg/g) no extrato não digerido. Após digestão in vitro, somente a catequina, epicatequina e cafeína foram bioacessíveis com 181,16 %, 136,78 % e 213,51 % de bioacessibilidade, respectivamente. A atividade antioxidante do extrato aquoso não digerido foi menor que do digerido, sendo os valores finais de ORAC de 2512 (± 399) e 4321 (± 778) µmol Eq Trolox/g de guaraná em pó, respectivamente. O extrato exibiu uma atividade inibitória da enzima lipase pancreática de forma dose-dependente, com IC50 de 1033 µg/mL. O extrato também demonstrou uma capacidade de ligação ao taurocolato de sódio de 45,63 (± 8,50) % e de 44,30 (± 8,88) % com concentrações de 1,0 mg/mL e 0,5 mg/mL, respectivamente, correspondendo a 71,78 (± 13,36) % e 93,11 (± 18,65) % da capacidade de ligação da colestiramina quando utilizada nas mesmas concentrações. Observou-se uma redução de 20,47 (± 10,24) % e 17,06 (± 12,88) % na concentração da fosfatidilcolina micelar quando o guaraná foi utilizado nas concentrações de 1,0 e 0,50 mg/mL, respectivamente, em comparação com a micela padrão sem amostra e uma inibição da solubilização micelar do colesterol de 10,14 % pelo guaraná a 1,0 mg/mL. Após o experimento de permeação, as concentrações de colesterol no compartimento basolateral foram de 197,89 (± 3,95), 151,76 (± 1,39) e 94,51 (± 9,49) µg/mL, quando 0,025, 0,050 ou 0,075 mg/mL de guaraná foram utilizados, respectivamente, demonstrando redução de 23,31 % e 52,24 % à medida que a concentração de guaraná utilizada aumentou. Observou-se uma redução do conteúdo proteico do NPC1L1 após 16 horas de incubação com guaraná nas concentrações de 0,050 e 0,075 mg/mL. Em relação ao ABCG5, observou-se um aumento do conteúdo nas concentrações de 0,025 e 0,075 mg/mL de guaraná após 02 horas. Não foram observadas diferenças importantes nos conteúdos de ABCG8 e do LXR-alfa. Conclusões: O extrato aquoso de guaraná em pó demonstrou uma redução da absorção de colesterol em células Caco-2. O extrato também demonstrou uma atividade inibitória da lipase pancreática dose-dependente, uma boa capacidade de ligação aos ácidos biliares e de interação com a fosfatidilcolina micelar, além de uma capacidade de inibição da solubilização micelar do colesterol de forma in vitro. O extrato também foi capaz de modular negativamente o conteúdo proteico do NPC1L1 e positivamente o conteúdo do ABCG5. A sua ingestão pode ser considerada uma importante fonte alimentar com potencial efeito hipocolesterolêmico, capaz de reduzir a absorção de lipídios no intestino, podendo ser utilizado como adjuvante na regulação da hipercolesterolemia, provavelmente devido à presença de polifenóis.


Introduction - The risk of cardiovascular diseases, such as atherosclerosis, is associated with hypercholesterolemia, a result of behavioral and metabolic risk factors. Dietary interventions with the use of guarana (Paullinia cupana) may be useful, since it is a food rich in bioactive compounds such as catechins, which have high antioxidant activity, inhibit oxidation of LDLc and lipid peroxidation, being able to lower blood cholesterol levels, by mechanisms not yet fully elucidated. Objective - To investigate the effects of guarana (Paullinia cupana) powder aqueous extract on mechanisms involved with the absorption of cholesterol in vitro and in Caco-2 cells. Methods - The guarana powder aqueous extract was subjected to in vitro digestion, the total phenolics content were quantified by the Folin-Ciocalteu method and the determination of the phenolic profile was performed by high performance liquid chromatography (HPLC) UV/VIS at 210 nm. The antioxidant activity was determined by Oxygen Radical Absorbance Capacity (ORAC) and the ability to inhibit pancreatic lipase, to binding with bile acids, the interaction with micellar phosphatidylcholine and the inhibition of micellar solubilization of cholesterol in vitro were evaluated. Cholesterol permeation assay was performed for a period of 2 hours in Caco-2 cells seeded and differentiated in 24- transwell plates with a solution of cholesterol micelles and guarana extract at different concentrations. The permeated cholesterol content was determined by HPLC/UV at 206 nm. The Western blotting assay was performed to determine the protein content of cholesterol transporters (NPC1L1, ABCG5, ABCG8) and LXR-alpha in Caco-2 cells after incubation with different concentrations of the extract in 2 and 16 hours. Results: The total phenolic content of the guarana extract was 104.36 (± 3.62) mg EAG/g of guarana powder. After the digestion process, this value was 48.62 (± 3.97) mg EAG/g of guarana powder, demonstrating the bioaccessibility of the polyphenols. Procyanidin B1 (1.56 ± 0.04 mg/g) and B2 (2.34 ± 0.06 mg/g), catechin (24.26 ± 0.63 mg/g), epicatechin (20.39 ± 1.20 mg/g) and caffeine (34.93 ± 0.75 mg/g) were identified in the undigested extract. After in vitro digestion, only catechin, epicatechin and caffeine were bioaccessible with 181.16 %, 136.78 % and 213.51 % of bioaccessibility, respectively. The antioxidant activity of the undigested aqueous extract was lower than that digested, with the final ORAC values of 2512 (± 399) and 4321 (± 778) µmol Eq Trolox/g of guarana powder. The extract exhibited an inhibitory activity of the pancreatic lipase enzyme in a dose-dependent manner, with an IC50 of 1033 µg/mL. The extract also demonstrated a sodium taurocholate binding capacity of 45.63 (± 8.50) % and 44.30 (± 8.88) % with concentrations of 1.0 mg/mL and 0.5 mg/mL, respectively, corresponding to 71.78 (± 13.36)% and 93.11 (± 18.65)% of the cholestyramine binding capacity when used at the same concentrations. A reduction of 20.47 (± 10.24) % and 17.06 (± 12.88) % was observed in the concentration of micellar phosphatidylcholine when guarana was used at concentrations of 1.0 and 0.50 mg/mL, respectively, compared to the standard micelle without sample and an inhibition of 10.14 % micellar cholesterol solubilization by guarana at 1.0 mg/mL. After the permeation experiment, the cholesterol concentrations in the basolateral compartment were 197.89 (± 3.95), 151.76 (± 1.39) and 94.51 (± 9.49) µg/mL, when 0.025, 0.050 or 0.075 mg/mL of guarana were used, respectively, showing a reduction of 23.31 % and 52.24 % as the concentration of guarana used increased. A reduction in the protein content of NPC1L1 was observed after 16 hours of incubation with guarana at concentrations of 0.050 and 0.075 mg/mL. After 02 hours, an increase in the content of ABCG5 at concentrations of 0.025 and 0.075 mg/mL of guarana was observed. No significant differences were observed in the contents of ABCG8 and LXR-α. Conclusions: The aqueous extract of guarana powder showed a reduction in the absorption of cholesterol in Caco-2 cells. The extract also demonstrated a dose-dependent pancreatic lipase inhibitory activity, a good ability to bind bile acids and to interact with micellar phosphatidylcholine, in addition to an ability to inhibit micellar solubilization of cholesterol in vitro. The extract was also able to negatively modulate the protein content of NPC1L1 and positively the content of ABCG5. Its intake can be considered an important dietary source with a potential hypocholesterolemic effect, capable of reducing the absorption of lipids in the intestine, and can be used as an adjunct in the control of hypercholesterolemia, probably due to the presence of polyphenols.


Subject(s)
Cholesterol , Caco-2 Cells , Paullinia , Polyphenols , Cardiovascular Diseases , Hypercholesterolemia
4.
Article in Chinese | WPRIM | ID: wpr-888177

ABSTRACT

In this study, we studied the solubility and permeability of matrine, oxymatrine, sophoridine, and oxysophocarpine, four alkaloids in the Mongolian herbal medicine Sophorae Flavescentis Radix, and evaluated the absorption mechanism with the Caco-2 cell model, so as to provide a basis for the new drug development and efficacy evaluation of Sophorae Flavescentis Radix. The results showed that all the four alkaloids had high solubility and high permeability and can be well absorbed, belonging to the class-I drugs of Biopharmaceutical Classification System(BCS). The absorption(AP→BL) and excretion(BL→AP) of matrine and oxymatrine were not affected by the concentration while the absorption depended on P-gp protein. The absorption(AP→BL) and excretion(BL→AP) of sophoridine and oxysophocarpine were positively related to the concentration and time, and the absorption process was independent from P-gp protein. The results provide scientific reference and an experimental basis for the development of Mongolian medical prescriptions containing Sophorae Flavescentis Radix.


Subject(s)
Alkaloids , Biological Products , Caco-2 Cells , Drugs, Chinese Herbal , Herbal Medicine , Humans , Sophora
5.
Article in English | WPRIM | ID: wpr-922263

ABSTRACT

To investigate the effect and mechanism of metformin on intestinal epithelial barrier injury in ulcerative colitis. A cell model of colitis was established by co-culture of human colon cancer cell line Caco-2 and human monocyte cell line THP-1. The colitis model cells were treated with metformin at concentration of for Flow cytometry was used to detect Caco-2 cell apoptosis, and Western blotting was used to detect the protein expression of tight junction proteins and endoplasmic reticulum stress-related proteins. After metformin treatment, the apoptosis rate of Caco-2 cells was decreased from (14.22±2.34)% to 0.61)% (=3.119, <0.05), and the expression levels of tight junction protein-1 and claudin-1 increased (=5.172 and 3.546, both <0.05). In addition, the expression levels of endoplasmic reticulum-related proteins glucose regulated protein (GRP) 78, C/EBP homologous protein (CHOP) and caspase-12, as well as the phosphorylation level of PRKR-like endoplasmic reticulum kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α) decreased (all <0.05). Metformin may alleviate the intestinal epithelial barrier damage in colitis by reducing intestinal epithelial cell apoptosis and increasing the expression of tight junction proteins, which may be associated with the inhibition of endoplasmic reticulum stress-induced apoptotic pathway.


Subject(s)
Apoptosis , Caco-2 Cells , Colitis, Ulcerative , Endoplasmic Reticulum Stress , Humans , Metformin/pharmacology
6.
Article in Chinese | WPRIM | ID: wpr-879135

ABSTRACT

The absorption is the key to the resulted efficacy of orally administered drugs and the small intestine is the main site to absorb the orally administered drug. In this paper, internationally recognized human colon adenocarcinoma cell line(Caco-2) monola-yer model which can simulate small intestinal epithelial cell was used to comparatively study the absorption and transportation diffe-rences of total coumarins and main individual coumarin in Angelica dahurica 'Yubaizhi' by separately using 6-and 12-well plates. It was found that apparent permeability coefficient(P_(app)) values of oxypeucedanin hydrate, byakangelicin and phellopterin were at the quantitative degree of 1 × 10~(-5) cm·s~(-1) when the individual administration was conducted independently, indicating that they were well-absorbed compounds. P_(app) ratio of their bi-directional transportation was close to 1, indicating that they can be absorbed across Caco-2 monolayer by passive diffusion mechanism without carrier mediation during the transportation. The similar trend of transportation was also observed for imperatorin, isoimperatorin and bergapten. The P_(app) values of oxypeucedanin hydrate, byakangelicin and bergapten were at quantitative degree of 1 × 10~(-5) cm·s~(-1) when the administration of total coumarins in Angelica dahurica 'Yubaizhi' was conducted, indicating that they were well-absorbed compounds. The results were consistent with those of independent administration of individual coumarins. Whereas, the P_(app) values of imperatorin, phellopterin and isoimperatorin in the total coumarins decreased, indicating that the interaction between compounds may exist although the P_(app) value ratio of bi-directional transportation was between 0.5 and 1.5. The results laid the foundation for intestinal absorption study of Angelica dahurica 'Yubaizhi' coumarins in compound Chinese medicine.


Subject(s)
Angelica , Caco-2 Cells , Coumarins , Drugs, Chinese Herbal , Humans , Intestinal Absorption , Plant Roots
7.
Article in Chinese | WPRIM | ID: wpr-879129

ABSTRACT

Nanocrystals self-stabilized Pickering emulsion(NSSPE) is a new kind of emulsion where only nanocrystals of poorly soluble drugs are used as stabilizers. Our previous study showed that NSSPE with Ligusticum chuanxiong oil as the main oil phase can significantly promote oral absorption of puerarin. The present study aimed to explore its absorption mechanism in oral administration. The in vitro dissolution test was carried out to study the effect of NSSPE on release of puerarin. The effects and mechanism of NSSPE on uptake and transport of puerarin across Caco-2 cell were investigated. The results showed that the drug release rate of NSSPE was similar to that of nanocrystals, with their cumulative dissolution of puerarin not affected by pH of releasing mediums, both significantly higher than that of crude material. The uptake of puerarin in NSSPE was concentration-dependent and significantly higher than that of solution or surfactant stabilized emulsion. Genistein and indomethacin, inhibitors of lipid rafts/caveolin, could significantly reduce the uptake of puerarin in NSSPE. Compared with solution, NSSPE and surfactants stabilized emulsion obviously increased transport rate K_a and apparent permeability coefficient P_(app) of puerarin in AP → BL direction, but there was no significant difference in BL → AP direction. It could be inferred that there were both passive and active transport mechanisms, as well as lipid raft/caveolin mediated endocytosis for absorption of NSSPE. The promoted oral absorption of puerarin in NSSPE was mainly related to the existing nanocrystal form which could promote dissolution, puerarin as well as Ligusticum chuanxiong oil which could promote drug transmembrane transport and inhibit drug efflux. It is the unique structure and composition of the compound NSSPE that promoted the oral absorption of puerarin.


Subject(s)
Caco-2 Cells , Drugs, Chinese Herbal , Emulsions , Humans , Isoflavones , Nanoparticles
8.
Article in Chinese | WPRIM | ID: wpr-879012

ABSTRACT

To evaluate the effects of Hydroxypropyl methylcellulose acetate succinate(HPMCAS MF) on absorption of silybin(SLB) from supersaturable self-nanoemulsifying drug delivery system which was pre-prepared at the early stage experiment. The cell toxicity of self-emulsifying preparation was evaluated by the MTT method, and the in vitro membrane permeability and absorption promoting effect of the self-emulsifying preparation were evaluated by establishing a Caco-2 cell monolayer model. The in vivo and in vitro supersaturation correlation was evaluated via the blood concentration of SLB. The results of MTT showed that the concentration of the preparation below 2 mg·mL~(-1)(C_(SLB) 100 μg·mL~(-1)) was not toxic to Caco-2 cells, and the addition of polymer had no significant effect on Caco-2 cells viability. As compared with the solution group, the transport results showed that the P_(app)(AP→BL) of the self-emulsifying preparation had a very significant increase; the transport rate of silybin can be reduced by polymer in 0-30 min; however, there was no difference in supersaturated transport between supersaturated SLB self-nanoemulsion drug delivery system(SLB-SSNEDDS) and SLB self-nanoemulsion drug delivery system(SLB-SNEDDS) within 2 hours. As compared with SLB suspension, pharmacokinetic parameters showed that the blood concentration of both SLB-SNEDDS and SLB-SSNEDDS groups were significantly increased, and C_(max) was 5.25 times and 9.69 times respectively of that in SLB suspension group, with a relative bioavailability of 578.45% and 1 139.44% respectively. C_(max) and relative bioavailability of SLB-SSNEDDS were 1.85 times and 197% of those of SLB-SNEDDS, respectively. Therefore, on the one hand, SSNEDDS can increase the solubility of SLB in gastrointestinal tract by maintaining stability of SLB supersaturation state; on the other hand, the osmotic transport process of SLB was regulated through the composition of its preparations, and both of them could jointly promote the transport and absorption of SLB to improve the oral bioavailability of SLB.


Subject(s)
Administration, Oral , Biological Availability , Caco-2 Cells , Drug Delivery Systems , Emulsions , Humans , Methylcellulose/analogs & derivatives , Nanoparticles , Particle Size , Silymarin , Solubility
9.
Article in Chinese | WPRIM | ID: wpr-878948

ABSTRACT

Network pharmacology and liver fibrosis(LF) model in vitro were used to analyze the underly mechanism of anti-liver fibrosis effect that induced by Piperis Longi Fructus and its major active compounds. TCMSP and TCMIP were used to search for the chemical constituents of Piperis Longi Fructus, as well as the oral bioavailability(OB), drug-likeness(DL), intercellular permeability of intestinal epithelial cells(Caco-2) and Drug-likeness grading were set as limiting conditions. The related target genes of Piperis Longi Fructus were queried by TCMSP database, while related targets of LF were screened by GeneCards databases. Interaction network was constructed using Cytoscape 3.7.1. These above data were imported into STRING database for PPI network analysis. Enrichment of gene ontology(GO) and pathway analysis(KEGG) within Bioconductor database were utilized to note functions of related targets of Piperis Longi Fructus. Finally, the core targets and pathways were preliminarily verified by in vitro experiments. The effects of piperlongumine(PL), the major active component of Piperis Longi Fructus, on proliferation of rat liver stellate cells(HSC-T6) and expression of α smooth muscle actin(α-SMA) and collagen Ⅰ were investigated. The major factors TNF-α of tumor necrosis factor(TNF) pathway and NF-κB p65, IL-6 protein expressions of LF process were examined. A total of 12 active compounds such as PL were obtained by analyzing the bioavailability and drug-like properties, which inferred to 48 targets. The functional enrichment analysis of GO obtained 1 240 GO items, mainly involving in process of biology and molecular function. A total of 99 signaling pathways were enriched in the KEGG pathway enrichment analysis, including TNF signaling pathway, cGMP-PKG signaling pathway, calcium signaling pathways. CCK-8 assay showed that PL inhibited proliferation of HSC-T6 induced by transforming growth factor-β1(TGF-β1). Western blot analysis found that treated with PL suppressed the protein expressions of α-SMA, collagen Ⅰ, TNF-α and p65 in HSC-T6. Enzyme linked immunosorbent assay(ELISA) showed that PL inhibited the expressions of TNF-α and IL-6 in the cluture supertant of HSC-T6 cells. In conclusion, PL could play an anti-liver fibrosis role by regulating TNF/NF-κB signaling pathway. This study provided the mechanism basis of anti-LF effects induced by Piperis Longi Fructus and its major active compounds, which might help for the further study of the mechanism and key targets of Piperis Longi Fructus.


Subject(s)
Animals , Caco-2 Cells , Hepatic Stellate Cells/metabolism , Humans , Liver Cirrhosis/genetics , NF-kappa B/metabolism , Rats , Signal Transduction
10.
Acta Physiologica Sinica ; (6): 42-50, 2021.
Article in Chinese | WPRIM | ID: wpr-878234

ABSTRACT

This study was designed to evaluate the role of short-chain fatty acid butyrate acid on intestinal morphology and function, and atherosclerotic plaque formation in apolipoprotein E-knockout (ApoE


Subject(s)
Animals , Apolipoproteins E/genetics , Atherosclerosis/prevention & control , Butyrates/pharmacology , Caco-2 Cells , Diet, High-Fat/adverse effects , Fatty Acids, Volatile , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Plaque, Atherosclerotic
11.
Biol. Res ; 53: 12, 2020. tab, graf
Article in English | LILACS | ID: biblio-1100918

ABSTRACT

BACKGROUND: Mast cells (MCs) have been found to play a critical role during development of inflammatory bowel disease (IBD) that characterized by dysregulation of inflammation and impaired intestinal barrier function. However, the function of MCs in IBD remains to be fully elucidated. RESULTS: In our study, we used exosomes isolated from human mast cells-1 (HMCs-1) to culture with NCM460, HT-29 or CaCO2 of intestinal epithelial cells (lECs) to investigate the communication between MCs and lECs. We found that MCs-derived exosomes significantly increased intestinal epithelial permeability and destroyed intestinal barrier function, which is attributed to exosome-mediated functional miRNAs were transferred from HMCs-1 into lECs, leading to inhibit tight junction-related proteins expression, including tight junction proteins 1 (TJP1, ZO-1), Occludin (OCLN), Claudin 8 (CLDN8). Microarray and bioinformatic analysis have further revealed that a panel of miRNAs target different tight junction-related proteins. Interestingly, miR-223 is enriched in mast cell-derived exosome, which inhibit CLDN8 expression in IECs, while treatment with miR-223 inhibitor in HT-29 cells significantly reversed the inhibitory effect of HMCs-1-derived exosomes on CLDN 8 expression. Most importantly, enrichment of MCs accumulation in intestinal mucosa of patients with IBD compared with those healthy control. CONCLUSIONS: These results indicated that enrichment of exosomal miR-223 from HMCs-1 inhibited CLDN8 expression, leading to destroy intestinal barrier function. These finding provided a novel insight of MCs as a new target for therapeutic treatment of IBD.


Subject(s)
Humans , Animals , Cattle , MicroRNAs/metabolism , Epithelial Cells/metabolism , Intestinal Mucosa/metabolism , Mast Cells/metabolism , Permeability , Inflammatory Bowel Diseases/metabolism , Cells, Cultured , Caco-2 Cells/cytology , Computational Biology , Tissue Array Analysis , Exosomes/metabolism , Claudins/metabolism , Occludin/metabolism , Zonula Occludens-1 Protein/metabolism
12.
Article in Chinese | WPRIM | ID: wpr-775043

ABSTRACT

OBJECTIVE@#To establish a congenital chloride diarrhea (CCD)-associated SLC26A3 c.392C>G (p.P131R) polymorphism-expressing cell model, and to investigate its biological function.@*METHODS@#The sequence of the SLC26A3 gene in GenBank was used to design the upstream and downstream single-guide RNA (sgRNA) that could specifically recognize the 392 locus of the SLC26A3 gene, and the sgRNA was mixed with the pSpCas9-puro vector after enzyme digestion to construct an eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3). Caco-2 cells were transfected with the recombinant plasmid and synthesized single-stranded DNA oligonucleotides (ssODNs), and Taqman genotyping assay and Sanger sequencing were used to identify the expression of SLC26A3 c.392C>G (p.P131R) in Caco-2 cells. Wild-type Caco-2 cells were selected as normal control group and the Caco-2 cells with successful expression of SLC26A3 c.392C>G (p.P131R) was selected as P131R group. Both groups were treated with 100 ng/mL tumor necrosis factor-α (TNF-α), and then the normal control group was named as TNF-α group, and the P131R group was named as TNF-α+P131R group. Electric cell-substrate impedance sensing (ECIS) assay was used to evaluate the change in the monolayer barrier function of intestinal epithelial cells in the above four groups, and Western blot was used to measure the change in the expression of SLC26A3 protein in the normal control group and the P131R group.@*RESULTS@#The eukaryotic recombinant expression plasmid (pSpCas9-SLC26A3) was successfully constructed. Both Taqman genotyping assay and Sanger sequencing confirmed the successful establishment of the Caco-2 cell model of SLC26A3 c.392C>G (p.P131R) expression. ECIS assay showed that compared with the normal control group, the P131R group had a significant increase in the monolayer permeability of intestinal epithelial cells (PG (p.P131R) can reduce the expression of SLC26A3 protein, increase the monolayer permeability of intestinal epithelial cells, and thus lead to diarrhea.


Subject(s)
Caco-2 Cells , Chloride-Bicarbonate Antiporters , Genetics , Diarrhea , Genetics , Humans , Intestinal Mucosa , Metabolism, Inborn Errors , Genetics , Polymorphism, Single Nucleotide , Sulfate Transporters , Genetics , Tight Junctions , Tumor Necrosis Factor-alpha
13.
Article in Chinese | WPRIM | ID: wpr-773694

ABSTRACT

Tripterygium wilfordii multiglycoside( GTW),an extract derived from T. wilfordii,has been used for rheumatoid arthritis and other immune diseases in China. However its potential hepatotoxicity has not been investigated completely. Firstly,the content of triptolid( TP) in GTW was 0. 008% confirmed by a LC method. Then after oral administration of GTW( 100,150 mg·kg-1) and TP( 12 μg·kg-1) in female Wistar rats for 24 h,it was found that 150 mg·kg-1 GTW showed more serious acute liver injury than 12 μg·kg-1 TP,with the significantly increased lever of serum ALT,AST,TBA,TBi L,TG and bile duct hyperplasia even hepatocyte apoptosis. The expression of mRNA and proteins of liver bile acid transporters such as BSEP,MRP2,NTCP and OATP were down-regulated significantly by GTW to inhibit bile acid excretion and absorption,resulting in cholestatic liver injury. Moreover,GTW was considered to be involved in hepatic oxidative stress injury,although it down-regulated SOD1 and GPX-1 mRNA expression without significant difference in MDA and GSH levels. In vitro,we found that TP was the main toxic component in GTW,which could inhibit cell viability up to 80% in Hep G2 and LO2 cells at the dose of 0. 1 μmol·L-1. Next a LC-MS/MS method was used to detect the concentration of triptolid in plasma from rats,interestingly,we found that the content of TP in GTW was always higher than in the same amount of TP,suggesting the other components in GTW may affect the TP metabolism. Finally,we screened the substrate of p-glycoprotein( p-gp) in Caco-2 cells treated with components except TP extrated from GTW,finding that wilforgine,wilforine and wilfordine was the substrate of p-gp. Thus,we speculated that wilforgine,wilforine and wilfordine may competitively inhibit the excretion of TP to bile through p-gp,leading to the enhanced hepatotoxity caused by GTW than the same amount of TP.


Subject(s)
Animals , Caco-2 Cells , Chemical and Drug Induced Liver Injury , Pathology , Chromatography, Liquid , Diterpenes , Toxicity , Drugs, Chinese Herbal , Toxicity , Epoxy Compounds , Toxicity , Female , Glycosides , Toxicity , Humans , Liver , Phenanthrenes , Toxicity , Plant Extracts , Toxicity , Rats , Rats, Wistar , Tandem Mass Spectrometry , Tripterygium , Toxicity
14.
Chinese Medical Journal ; (24): 1071-1078, 2019.
Article in English | WPRIM | ID: wpr-772221

ABSTRACT

BACKGROUND@#Colorectal cancer is the third most common cancer worldwide and still lack of effective therapy so far. Petasin, a natural product found in plants of the genus Petasites, has been reported to possess anticancer activity. The present study aimed to investigate the anticolon cancer activity of petasin both in vitro and in vivo. The molecular mechanism of petasin was also further explored.@*METHODS@#Caco-2, LoVo, SW-620, and HT-29 cell lines were used to detect the inhibitory effect of petasin on colon cancer proliferation. Cell viability was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was analyzed by flow cytometry. Hoechst 33258 staining was used to visualize morphological changes. Cell migration was assessed using a wound-healing migration assay, and cell invasion was investigated using Transwell chambers. Western blotting assays were employed to evaluate the expression levels of proteins in the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway. Finally, in vivo activity of petasin was evaluated using the SW-620 subcutaneous tumor model established in Balb/c nude mice. Twelve rats were randomly divided into control group and 10 mg/kg petasin group. The tumor volume was calculated every 7 days for 28 days. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to assess the apoptotic effect of petasin. Differences between two groups were assessed by analysis of independent-sample t tests.@*RESULTS@#Petasin significantly inhibited the proliferation of human colon carcinoma cell lines, induced apoptosis, and suppressed migration and invasion in SW-620 cells. Western blotting results showed that petasin decreased the phosphorylation of Akt (1.01 ± 0.16 vs. 0.74 ± 0.06, P = 0.042), mTOR (0.71 ± 0.12 vs. 0.32 ± 0.11, P = 0.013), and P70S6K (1.23 ± 0.21 vs. 0.85 ± 0.14, P = 0.008), elevated the expression of caspase-3 (0.41 ± 0.09 vs. 0.74 ± 0.12, P = 0.018) and caspase-9 (1.10 ± 0.27 vs. 1.98 ± 0.22, P = 0.009), decreased the Bcl-2 protein (2.75 ± 0.47 vs. 1.51 ± 0.36, P = 0.008), downregulated the expression of matrix metalloproteinase (MMP)-3 (1.51 ± 0.31 vs. 0.82 ± 0.11, P = 0.021) and MMP-9 (1.56 ± 0.32 vs. 0.94 ± 0.15, P = 0.039) in SW-620 cell. In vivo, 10 mg/kg petasin inhibited tumor growth in Balb/c nude mice (924.18 ± 101.23 vs. 577.67 ± 75.12 mm at day 28, P = 0.001) and induced apoptosis (3.6 ± 0.7% vs. 36.0 ± 4.9%, P = 0.001) in tumor tissues.@*CONCLUSIONS@#Petasin inhibits the proliferation of colon cancer SW-620 cells via inactivating the Akt/mTOR pathway. Our findings suggest petasin as a potential candidate for colon cancer therapy.


Subject(s)
Animals , Antineoplastic Agents , Therapeutic Uses , Apoptosis , Caco-2 Cells , Cell Line, Tumor , Cell Proliferation , HT29 Cells , Humans , In Situ Nick-End Labeling , Matrix Metalloproteinase 3 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Phosphorylation , Proto-Oncogene Proteins c-akt , Genetics , Metabolism , Sesquiterpenes , Therapeutic Uses , Signal Transduction , TOR Serine-Threonine Kinases , Genetics , Metabolism
15.
Article in Chinese | WPRIM | ID: wpr-771502

ABSTRACT

To investigate the active fraction from Bletilla striata in Caco-2 cell monolayer,so as to explore its absorption mechanism of oral administration preliminarily.Active fraction from B.striata in Caco-2 cell monolayer was analyzed by UPLC-Q-TOF and detected by UPLC-MS/MS,and the effects of different concentrations,pH and P-glycoprotein inhibitors on Caco-2 cells Monolayer were investigated.Six compounds were isolated from the active fraction of B.striata in Caco-2 cell monolayer by UPLC-Q-TOF,and identified as B6,B12,B14,B17,B19 and B23,with concentration dependence.Within the 0-180 min,the uptake of B12 and B14 had a time dependence,while B6,B17,B19 and B23 tended to saturate after 60 min.All of the components had a good absorption in an acidic environment.B6 had a good absorption at pH 6.0,while the other components B12,B14,B17,B19 and B23 had a good absorption at pH4.0.The absorption of the 6 main components of B.striata were not be affected by P-glycoprotein inhibitors(verapamil/cyclosporin A).Compared with the control group,there was no difference in the absorption of B6 and B12,and the absorption of B14,B17,B19 and B23 increased,but with no significant difference.The absorption characteristic of B.striata extract across the Caco-2 cell monolayer is probably passive diffusion,and the absorption process was not affected by P-glycoprotein.


Subject(s)
Biological Transport , Caco-2 Cells , Chromatography, Liquid , Humans , Intestinal Absorption , Orchidaceae , Chemistry , Plant Extracts , Pharmacology , Tandem Mass Spectrometry
16.
Article in English | WPRIM | ID: wpr-762687

ABSTRACT

PURPOSE: Preoperative chemoradiation therapy (CRT) has become the standard treatment for patients with locally advanced rectal cancer, 15%–30% of patients still progress while being treated with CRT. The aim of this study was to identify as important biomarker of poor response and evaluate the mechanism associated with CRT resistance. METHODS: This study included 60 human colon tumour pre-irradiation specimens. Expressions of epidermal growth factor receptor (EGFR), p53, Krüppel-like factor 5 (KLF5), C-ern, Ki67 were assessed and correlated with tumor regression grades and complete remission. We added in vitro study with biomarker which has been identified as important biomarker of poor response to evaluate the mechanism associated with CRT resistance. RESULTS: Pathologic complete remission (pCR) was achieved by 9 patients (18%). EGFR and KLF5 were significantly associated with pCR (P = 0.048, P = 0.023, respectfully). And multivariate analysis showed high KLF5 intensity was worse factor for pCR (P = 0.012). In vitro study, radiation or chemotherapy therapy stabilized KLF5 protein levels in a time- and dose-depended manner in HCT116 and Caco-2 cells. KLF5 overexpression in HCT116 stable cell line showed significantly better cell viability by increasing cyclinD1 and b-catenin compared to control cells in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, suggesting that KLF5 mediates cell survival. CONCLUSION: KLF5 was significantly associated with the presence of KRAS mutations, and KLF5 was an independent poor response predictor of CRT in rectal cancer. Our study is pilot study and more research will be needed in the future.


Subject(s)
Caco-2 Cells , Cell Line , Cell Survival , Chemoradiotherapy , Colon , Drug Therapy , Humans , In Vitro Techniques , Multivariate Analysis , Pilot Projects , Polymerase Chain Reaction , Prognosis , ErbB Receptors , Rectal Neoplasms
17.
Frontiers of Medicine ; (4): 83-93, 2019.
Article in English | WPRIM | ID: wpr-771264

ABSTRACT

Colorectal cancer (CRC) is a common malignant tumor in the digestive tract, and 30%-85% of CRCs express epidermal growth factor receptors (EGFRs). Recently, treatments using cetuximab, also named C225, an anti-EGFR monoclonal antibody, for CRC have been demonstrated to cause an S492R mutation in EGFR. However, little is known about the biological function of S492R EGFR. Therefore, we attempted to elucidate its biological function in CRC cells and explore new treatment strategies for this mutant form. Our study indicated that EGFR and S492R EGFR accelerate the growth of CRC cells in vitro and in vivo and monoclonal antibody CH12, which specifically recognizes an EGFR tumor-specific epitope, can bind efficiently to S492R EGFR. Furthermore, mAb CH12 showed significantly stronger growth suppression activities and induced a more potent antibody-dependent cellular cytotoxicity effect on CRC cells bearing S492R EGFR than mAb C225. mAb CH12 obviously suppressed the growth of CRC xenografts with S492R EGFR mutations in vivo. Thus, mAb CH12 may be a promising therapeutic agent in treating patients with CRC bearing an S492R EGFR mutation.


Subject(s)
Animals , Antibodies, Monoclonal , Pharmacology , Antineoplastic Agents, Immunological , Pharmacology , Caco-2 Cells , Cell Proliferation , Colorectal Neoplasms , Therapeutics , ErbB Receptors , Genetics , Allergy and Immunology , Female , HT29 Cells , Humans , Mice , Mice, Inbred BALB C , Mutation , Xenograft Model Antitumor Assays
18.
Braz. j. med. biol. res ; 51(1): e6472, 2018. graf
Article in English | LILACS | ID: biblio-889011

ABSTRACT

Cetuximab is widely used in patients with metastatic colon cancer expressing wildtype KRAS. However, acquired drug resistance limits its clinical efficacy. Exosomes are nanosized vesicles secreted by various cell types. Tumor cell-derived exosomes participate in many biological processes, including tumor invasion, metastasis, and drug resistance. In this study, exosomes derived from cetuximab-resistant RKO colon cancer cells induced cetuximab resistance in cetuximab-sensitive Caco-2 cells. Meanwhile, exosomes from RKO and Caco-2 cells showed different levels of phosphatase and tensin homolog (PTEN) and phosphor-Akt. Furthermore, reduced PTEN and increased phosphorylated Akt levels were found in Caco-2 cells after exposure to RKO cell-derived exosomes. Moreover, an Akt inhibitor prevented RKO cell-derived exosome-induced drug resistance in Caco-2 cells. These findings provide novel evidence that exosomes derived from cetuximab-resistant cells could induce cetuximab resistance in cetuximab-sensitive cells, by downregulating PTEN and increasing phosphorylated Akt levels.


Subject(s)
Humans , Colonic Neoplasms/drug therapy , PTEN Phosphohydrolase/drug effects , Proto-Oncogene Proteins c-akt/drug effects , Exosomes/drug effects , Cetuximab/pharmacology , Antineoplastic Agents, Immunological/pharmacology , Tetrazolium Salts , Time Factors , Blotting, Western , Analysis of Variance , Caco-2 Cells , Cell Line, Tumor
19.
São Paulo; s.n; 2018. 108 p.
Thesis in Portuguese | LILACS | ID: biblio-916041

ABSTRACT

Introdução: O diabetes mellitus (DM) está associado a complicações que comprometem a qualidade de vida e a sobrevida dos indivíduos. Além disso, acarreta elevados custos para o controle metabólico e o tratamento de suas complicações, sendo assim caracterizado como um problema de saúde pública. A regulação da digestão e da absorção intestinal dos carboidratos, com vista a manter a homeostase da glicose plasmática, constituem importantes estratégias de proteção em condições clínicas como o diabetes tipo 2 (DM2), obesidade e síndrome metabólica. Os compostos fenólicos compreendem um grupo complexo de fitoquímicos bioativos presentes nos vegetais. Estudos in vitro e in vivo têm demonstrado que os compostos fenólicos inibem a atividade de carbohidrases (α-amilase e α-glicosidase) e o transporte intestinal de glicose mediado pelos transportadores SGLT1 e GLUT2. O cerrado brasileiro compreende uma larga biodiversidade, porém, apesar de muitas espécies terem sido identificadas, o seu potencial nutritivo e funcional ainda é pouco conhecido. Dentre estas espécies nativas é destacado o jatobá-do-cerrado. O jatobá-do-cerrado é uma leguminosa nativa brasileira, cuja a polpa farinácea que envolve suas sementes apresenta quantidades significativas de compostos fenólicos, podendo ter um potencial efeito sobre o metabolismo da glicose. Objetivos: Verificar os efeitos dos compostos fenólicos da farinha de jatobá-do-cerrado na digestão de carboidratos e na captação de glicose em células intestinais Caco-2. Metodologia: Os compostos fenólicos da farinha de jatobá foram obtidos por extração sequencial com as soluções de etanol (60%) e acetona (70%). Em seguida, o extrato foi digerido utilizando enzimas (α-amilase, pepsina e pancreatina) em pH fisiológico. Os compostos fenólicos presentes no extrato antes e após a digestão foram identificados por cromatografia líquida de ultra performance - espectrômetro de massas (UPLC-MS/MS). Foi avaliada a capacidade de inibição dos extratos de jatobá digeridos em relação à atividade das enzimas α-amilase e α-glicosidase. Células intestinais Caco-2 foram incubadas com diferentes concentrações (0,05 mg/mL - 0,1 mg/mL) de extratos de farinha de jatobá digeridos em diferentes tempos (30 min, 2h e 12 h) para a avaliação da captação de glicose e da expressão gênica dos transportadores de glicose SGLT1 e GLUT2. Resultados: 44 compostos fenólicos foram identificados, dentre eles, a principal classe presente são os flavonoides. Compostos como o ácido cafeico, o kaempferol, quercetina-3- rutinosideo e a quercetrina estavam presentes no extrato antes da digestão. O conteúdo de compostos fenólicos do extrato foi reduzido após a digestão, entretanto o mesmo ainda apresentou compostos de relevância biológica como o ácido p-cumárico, ácido 3-o-feruloilquinico, theaflavina, crisina e grandinina que já apresentaram efeito positivo sobre o metabolismo da glicose in vitro em outros trabalhos. Os extratos fenólicos de jatobá após a digestão in vitro inibiram significativamente a atividade das enzimas α-amilase (76 e 91%) e α- glicosidase (53 e 77%). Os extratos também demonstraram inibir significativamente tanto a captação de glicose independente de sódio quanto a expressão gênica dos transportadores de glicose SGLT1 e GLUT2 de maneira dose-dependente. Conclusão: Este é o primeiro trabalho que identificou os compostos fenólicos presentes na farinha de jatobá. A partir do exposto, podemos concluir que a farinha de jatobá apresenta potencial benefício a saúde devido ao seu conteúdo de compostos fenólicos e a capacidade destes compostos de regular a digestão e a absorção de carboidratos in vitro


Introduction: Diabetes mellitus (DM) is associated with complications that decrease the quality of life and survival of individuals. In addition, it entails high costs for metabolic control and treatment of its complications, thus being characterized as a public health problem. The regulation of digestion and intestinal absorption of carbohydrates to maintain plasma glucose homeostasis are important strategies for protection in chronic diseases such as type 2 diabetes (DM2), obesity and metabolic syndrome. Phenolic compounds are a complex group of chemical substances present in plants. In vitro and in vivo studies have shown that phenolic compounds are able to inhibit the activity of carbohydrases (α-amylase and α-glycosidase) and the intestinal transport of glucose mediated by SGLT1 and GLUT2 transporters. Brazilian Cerrado present a large biodiversity, but although many species have been identified, its nutritional and functional potential is still little known. Among these native species is the jatobá-docerrado. Jatobá-do-cerrado is a brazilian native legume, whose farinaceous pulp that surrounds its seeds presents significant amounts of phenolic compounds and may have a potential effect on glucose metabolism. Objectives: To verify the effects of phenolic compounds from jatobá-do-cerrado flour in the digestion of carbohydrates and uptake of glucose in Caco-2 intestinal cells. Methods: Phenolic compounds of jatobá flour were obtained by sequential extraction with olutions of ethanol (60%) and acetone (70%). The extract was digested using enzymes (α-amylase, pepsin and pancreatin) at physiological pH. The phenolic compounds present in the extract before and after the digestion were identified by liquid chromatography of ultra-performance - mass spectrometer (UPLCMS / MS). The ability of inhibition of the extracts of jatobá digested in relation to the activity of α-amylase and α-glycosidase enzymes was evaluated. Caco-2 intestinal cells were incubated with different concentrations of jatobá flour extracts (0.1 mg / mL - 0.05 mg / mL) for different time (30 min, 2 h and 12 h) to the evaluation of facilitated uptake (sodium-free buffer) and gene expression of SGLT1 and GLUT2 glucose transporters. Results: 44 phenolic compounds have been identified, among them a major class present are flavonoids. Compounds such as caffeic acid, quercetin-3-rutinoside and quercetrine were present in the extract before in vitro digestion. The content of phenolic compounds of the extract after digestion was reduced. However, the extract presents compounds with biological activity such as p-coumaric acid, 3-o-feruloylquinic acid , theaflavin, chrysin and grandinine, which already presented positive effects on glucose metabolism in vitro in other studies. Phenolic extracts of jatobá after in vitro digestion inhibited the activity of α-amylase (76 and 91%) and α-glycosidase (53 and 77%). The extracts also shown to inhibit both glucose uptake and gene expression of glucose transporters SGLT1 and GLUT2 in a dose-dependent manner. Conclusion: This is the first work that identified the phenolic compounds present in jatobá flour. Thus, we can conclude that the jatobá flour presents potential health benefit by modulate digestion and the absorption of carbohydrates in vitro


Subject(s)
Absorption, Physiological/genetics , Caco-2 Cells , Diabetes Mellitus , Fabaceae , Glucose/pharmacology , Phenolic Compounds , Biological Availability , Biological Transport , Digestion , Flour
20.
Article in English | WPRIM | ID: wpr-812421

ABSTRACT

Ginsenoside Rh2 (Rh2) is one of the major bioactive ginsenosides in Panax ginseng. However, the oral bioavailability of Rh2 is low, with P-glycoprotein (P-gp) and CYP3A4 being reported to be the main factors. The purpose of the present study was to determine the enhancing effect of piperine on the oral bioavailability as well as bioactivity of Rh2. The inhibitory effect of piperine on P-gp and CYP3A4 was determined using a Caco-2 monolayer model and a recombinant CYP3A4 metabolic system, respectively. The pharmacokinetics of oral Rh2 (10 mg·kg) administered alone or in combination with piperine (10 and 20 mg·kg) was performed in rats. The immune boosting effect of Rh2 was assessed in rats by measuring IL-12 level after treated by Rh2 alone or co-administered with piperine. The results indicated that piperine significantly increased the permeability of Rh2 and inhibited the metabolism of Rh2. The pharmacokinetic study results showed that the AUC of Rh2 was significantly increased in combination with piperine at high dose (20 mg·kg) when compared to the control group, with relative bioavailability of 196.8%. The increase of Rh2 exposure led to increased serum levels of IL-12. In conclusion, piperine may be used as a bioenhancer to improve pharmacological effect of Rh2 when given orally.


Subject(s)
Administration, Oral , Alkaloids , Animals , Benzodioxoles , Biological Availability , Caco-2 Cells , Cytochrome P-450 CYP3A , Metabolism , Drugs, Chinese Herbal , Ginsenosides , Pharmacokinetics , Humans , Interleukin-2 , Metabolism , Panax , Chemistry , Piperidines , Polyunsaturated Alkamides , Rats , Rats, Sprague-Dawley
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