RESUMO
Satiety hormone cholecystokinin (CCK) plays a vital role in appetite inhibition. Its secretion is regulated by dietary components. The search for bioactive compounds that stimulate CCK secretion is currently an active area of research. The objective of this study was to evaluate the ability of buckwheat (Fagopyrum esculentum Moench) protein digest (BPD) to stimulate CCK secretion in vitro and in vivo and clarify the structural characteristics of peptides stimulating CCK secretion. BPD was prepared by an in vitro gastrointestinal digestion model. The relative molecular weight of BPD was <10 000 Da, and peptides with <3000 Da accounted for 70%. BPD was rich in essential amino acids Lys, Leu, and Val but lacked sulfur amino acids Met and Cys. It had a stimulatory effect on CCK secretion in vitro and in vivo. Chromatographic separation was performed to isolate peptide fractions involved in CCK secretion, and five novel CCK-releasing peptides including QFDLDD, PAFKEEHL, SFHFPI, IPPLFP, and RVTVQPDS were successfully identified. A sequence length range of 6-8 and marked hydrophobicity (18-28) were observed among the most CCK-releasing peptides. The present study demonstrated for the first time that BPD could stimulate CCK secretion and clarify the structural characteristics of bioactive peptides having CCK secretagogue activity in BPD.
Assuntos
Colecistocinina , Fagopyrum , Colecistocinina/metabolismo , Fagopyrum/metabolismo , Peptídeos , Proteínas , DigestãoRESUMO
CONTEXT: Earthworm Eisenia foetida (Lumbricus rubellus), a traditional Chinese medicine, is used for treating many diseases, and its coelomic fluid has extensive biological functions. OBJECTIVE: The hemolytic, antibacterial and antitumor activities of an earthworm protein purified from coelomic fluid were investigated in vitro. MATERIALS AND METHODS: We used ultrafiltration, gel chromatography, and ion exchange chromatography in sequence to isolate and purify an earthworm protein from coelomic fluid (ECFP), and ECFP was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Hemolytic assay and antibacterial tests were applied to determine the cytolytic activity of ECFP. The MTT method was carried out to evaluate the antitumor effect of ECFP on HeLa cells and LTEP-A2 cells. RESULTS: ECFP, with molecular weight determined to be approximately 38.6 kilodaltons (KDa), was shown to possess significant hemolytic activity to chicken red blood cells (CRBC) (minimal hemolytic concentration 0.39 µg/mL). Antibacterial effect of ECFP obviously tested against Escherichia coli (minimal bactericidal concentration, MBC 180 µg/âmL) and Staphylococcus aureus (MBC 90 µg/mL) were observed. Moreover, ECFP notably inhibited the proliferation of HeLa cells (IC50 77 µg/mL) and LTEP-A2 cells (IC50 126 µg/mL) both in a time- and dose-dependent manner. DISCUSSION AND CONCLUSION: ECFP could serve as a component of the innate defense system of earthworms against foreign organisms, and thus it has potential pharmaceutical application in the future.
Assuntos
Antibacterianos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Líquidos Corporais , Hemólise/efeitos dos fármacos , Oligoquetos , Proteínas , Animais , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Células HeLa , Hemólise/fisiologia , Humanos , Medicina Tradicional Chinesa/métodosRESUMO
Treating sepsis remains challenging at present. Bacterial lipopolysaccharide (LPS) and bacterial DNA/CpG DNA are important pathogenic molecules and drug targets for sepsis. It is thus a promising strategy to treat sepsis by discovering agents that neutralize LPS and CpG DNA simultaneously. In this study, we present evidences of the biosensor based screening and isolation of active anti-sepsis fractions and monomers from traditional Chinese herbs using dual targets (LPS and CpG DNA) guided drug discovery strategy. Firstly, LPS or CpG DNA was immobilized on surfaces of cuvettes in the biosensor to establish a screening platform. Then, Cortex lycii with both highest affinities was selected out from one hundred and fourteen traditional Chinese herbs. In subsequent experiments, chromatography was utilized and coupled with the biosensor to purify fractions with a higher affinity for LPS and CpG DNA. In line with affinity assay, these fractions were shown to neutralize LPS and CpG DNA and inhibit their activity in vitro and in vivo. Lastly, the contributing monomer Kukoamine B (KB) was purified. KB neutralized LPS and CpG DNA in vitro. It inhibited TLR4, TLR9 and MyD88 mRNA expressions up-regulated by LPS and CpG DNA, and also attenuated the LPS and CpG DNA elicited nuclear translocation of NF-κB p65 protein in RAW264.7 cells. It also protected mice from lethal challenge of heat-killed E. coli, a mixture of LPS and CpG DNA. In conclusion, we presented a dual target guided discovery of a novel anti-sepsis agent KB from traditional Chinese herbs via combination of biosensor technology and chromatography methods.
Assuntos
Técnicas Biossensoriais , Ácidos Cafeicos , DNA Bacteriano/química , Descoberta de Drogas , Medicamentos de Ervas Chinesas , Lipopolissacarídeos/química , Espermina/análogos & derivados , Animais , Ácidos Cafeicos/isolamento & purificação , Ácidos Cafeicos/farmacologia , Linhagem Celular , Ilhas de CpG/efeitos dos fármacos , Ilhas de CpG/genética , DNA Bacteriano/efeitos dos fármacos , Descoberta de Drogas/métodos , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Sepse/tratamento farmacológico , Sepse/imunologia , Espermina/isolamento & purificação , Espermina/farmacologiaRESUMO
Bacterial DNA/CpG DNA is recognized as a key molecule during the pathogenesis of sepsis. Therefore, preventing CpG DNA from binding to its receptor is considered as the most promising strategy. In the present experiments, Radix et Rhizoma Rhei had the highest CpG DNA-binding ability among the seventy-eight traditional Chinese herbs. After the isolation of silica gel chromatography and high performance liquid chromatography (HPLC) and evaluation with affinity biosensor, the active fraction was confirmed and named Fraction D. It was found that in vitro, Fraction D bound to both CpG DNA and lipid A with high affinity, and strongly inhibited LPS- and CpG DNA-induced TNF-alpha release from RAW264.7 cells in a dose-dependent manner. Furthermore, Fraction D reduced the expression of TLR9 mRNA up-regulated by CpG DNA. In vivo, Fraction D protected mice challenged with lethal heat-killed E. coli. Using HPLC method, two monomers with high affinity for CpG DNA were isolated and identified as rhein and emodin. Rhein could significantly reduce CpG DNA- and LPS-induced TNF-alpha release, but emodin only reduced CpG DNA-induced TNF-alpha release. Rhein in combination with emodin could play synergistic inhibitory effect on both CpG DNA and LPS-induced TNF-alpha release, which contributed to the bioactivity of Fraction D. In conclusion, we successfully established the platform to screen anti-CpG DNA components of traditional Chinese herbs using affinity biosensor technology, got active Fraction D from Radix et Rhizoma Rhei and determined rhein and emodin as the main bioactive ingredients in Fraction D.
Assuntos
Aconitum/imunologia , DNA Bacteriano/química , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli/imunologia , Macrófagos/metabolismo , Fitoterapia , Sepse/tratamento farmacológico , Receptor Toll-Like 9/metabolismo , Animais , Antraquinonas/farmacologia , Técnicas Biossensoriais , Linhagem Celular , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Ilhas de CpG/genética , DNA Bacteriano/imunologia , DNA Bacteriano/metabolismo , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Emodina/farmacologia , Inibidores Enzimáticos/farmacologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Imunidade Inata/efeitos dos fármacos , Lipídeo A/imunologia , Lipídeo A/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos , Ligação Proteica , Sepse/genética , Sepse/imunologia , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To investigate the lipopolysaccharide (LPS) antagonizing biological activity of densefruit pattany root-bark extract (DPR-2) in vitro. METHODS: The effect of DPR-2 in neutralizing LPS (0.1 microg/L) was detected by kinetic turbidimetric limulus test. The effect of different concentrations of DPR-2 (0,8.0,16.0,32.0,64.0 mg/L) on binding of FITC-conjugated LPS (FITC-LPS,100.0 microg/L) to murine RAW264.7 cells was analyzed with laser scanning confocal microscopy. The expression of TNF-alpha and IL-6 mRNA in RAW264.7 cells after exposure to LPS (100.0 microg/L) were determined by real-time RT-PCR. RESULTS: DPR-2 could neutralize LPS (P < 0.05 or P < 0.01), and inhibit the binding of FITC-LPS to RAW264.7 cells in a dose-dependent manner when the concentration of DPR-2 was above 16.0 mg/L. Furthermore, DPR-2 could markedly inhibit the expression of TNF-alpha and IL-6 mRNA in LPS-stimulated murine RAW264.7 cells. CONCLUSION: DPR-2 exhibit an anti-LPS effect in vitro, which may be related to its capacity to neutralize LPS and inhibit binding of LPS for its receptors.