ABSTRACT
Pelodiscus sinensis meat is a nutritional food and tonic with angiotensin-converting enzyme (ACE) inhibitory activities. To identify the bioactive substances responsible, several bioinformatics methods were integrated to enable a virtual screening for bioactive peptides in proteins identified within a water-soluble protein fraction of Pelodiscus sinensis meat by Shotgun proteomics. The peptides were generated from the identified proteins by in silico proteolysis using six proteases. A comparison of the numbers of proteins suitable for digestion with each enzyme and the iBAQ (intensity-based absolute quantification) values for these proteins revealed that bromelain and papain were the most suitable proteases for this sample. Next, the water solubility, toxicity, and ADMET (absorption/distribution/metabolism/excretion/toxicity) properties of these peptides were evaluated in silico. Finally, a novel ACE inhibitory peptide IEWEF with an IC50 value of 41.33 µM was identified. The activity of the synthesized peptide was verified in vitro, and it was shown to be a non-competitive ACE inhibitor. Molecular docking revealed that IEWEF could tightly bind to C-ACE, and N-ACE with energies less than 0 kJ mol-1, and the peptide IEWEF can form hydrogen bonds with C-ACE and N-ACE respectively. These results provide evidence that bioactive peptides in the water-soluble protein fraction account for (at least) some of the ACE inhibitory activities observed in Pelodiscus sinensis meat. Furthermore, our research provides a workflow for the efficient identification of novel ACE inhibitory peptides from complex protein mixtures.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors , Molecular Docking Simulation , Peptides , Protein Hydrolysates , Solubility , Angiotensin-Converting Enzyme Inhibitors/chemistry , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Angiotensin-Converting Enzyme Inhibitors/metabolism , Protein Hydrolysates/chemistry , Protein Hydrolysates/metabolism , Animals , Peptides/chemistry , Peptides/pharmacology , Peptides/metabolism , Water/chemistry , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Papain/metabolism , Papain/antagonists & inhibitors , Papain/chemistry , Fish Proteins/chemistry , Fish Proteins/metabolismABSTRACT
Carapax Trionycis (the shell of the turtle Pelodiscus sinensis) was hydrolyzed by six different commercial proteases. The hydrolysate prepared from papain showed stronger inhibitory activity against angiotensin I-converting enzyme (ACE) than other extracts. Two noncompetitive ACE inhibitory peptides were purified successively by ultrafiltration, gel filtration chromatography, ion exchange column chromatography, and high-performance liquid chromatography (HPLC). The amino acid sequences of them were identified as KRER and LHMFK, with IC50 values of 324.1 and 75.6 µM, respectively, confirming that Carapax Trionycis is a potential source of active peptides possessing ACE inhibitory activities. Besides, both enzyme kinetics and isothermal titration calorimetry (ITC) assay showed that LHMFK could form more stable complex with ACE than KRER, which is in accordance with the better inhibitory activity of LHMFK.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/chemistry , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Peptides/pharmacology , Turtles , Amino Acid Sequence , Angiotensin-Converting Enzyme Inhibitors/metabolism , Animals , Chromatography, Gel , Chromatography, High Pressure Liquid , Drug Evaluation, Preclinical/methods , Hydrolysis , Inhibitory Concentration 50 , Peptide Hydrolases/metabolism , Peptides/chemistry , Peptides/isolation & purification , Protein Hydrolysates/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Structure-Activity RelationshipABSTRACT
Objective: To establish the pre-column derivatization combining HPLC to determine the contents of the amino acids of the raw and processed products of Turtle shell. Methods: The samples were prepared by the acidic hydrolysis method, derivatized by OPA and FMOC, and analyzed by C18 column with gradient elution. Results: 18 kinds of amino acids were separated within 38 min, which showed a good linear relationship( r ≥0. 9991). The average recoveries of 16 amino acids in the samples were between 90. 43% ~110. 69%,and the RSD were between 0. 76% ~ 5. 89%( n = 6). Conclusion: This method can be used to determine the contents of the amino acids in the raw and processed products of Turtle shell.
Subject(s)
Turtles , Amino Acids , Animals , Chromatography, High Pressure LiquidABSTRACT
OBJECTIVE: To study the volatile constituents of Loranthus delavayi. METHODS: The volatile constituents were extracted by steam distillation and supercritical CO2 fluid extraction, and the chemical compositions were analyzed by GC-MS techniques. RESULTS: Sixty-one peaks were separated and 41 of them were identified, accounting for 81.15% of the total content from the extracts of steam distillation; Ninty-tuo peaks were separated from the extracts of supercritical CO2 fluid extraction, and 46 of them were identified, accounting for 63.31% of the total content. CONCLUSION: There are great differences between the chemical compositions of the volatile constituents extracted by steam distillation and supercritical CO2 fluid extraction.
Subject(s)
Volatilization , Carbon Dioxide , Chromatography, Supercritical Fluid , Distillation , Gas Chromatography-Mass Spectrometry , SteamABSTRACT
OBJECTIVE: To study the chemical constituents of Viscum ovalifolium. METHODS: The chemical constituents from Viscum ovalifolium were isolated and purified by silica gel column chromatography, polyamide column chromatography and recrystallization methods. Their structures were elucidated by physicochemical properties and spectral analysis. RESULTS: Twelve compounds were isolated and their structures were identified as 1-octadecene (1), ethyl palmitate (2), 28-hydrxy-amyrone (3), betulinic acid (4), rutin (5), quercetin (6), beta-amyrinpalmitate (7), lupeol acetate (8), beta-amyrin (9), beta-sitosterol (10), lupeol (11) and oleanolic acid (12). CONCLUSION: Compounds 1 - 6 are obtained from this plant for the first time.
Subject(s)
Alkenes/isolation & purification , Palmitic Acids/isolation & purification , Triterpenes/isolation & purification , Viscum/chemistry , Alkenes/chemistry , Molecular Structure , Palmitic Acids/chemistry , Pentacyclic Triterpenes , Plant Leaves/chemistry , Plant Stems/chemistry , Quercetin/chemistry , Quercetin/isolation & purification , Triterpenes/chemistry , Betulinic AcidABSTRACT
Further study on steam-treated notoginseng, the roots of Panax notoginseng (Burk.) F.H. Chen (Araliaceae), which is a famous traditional Chinese medicine that is used both in raw and treated forms for a long time, led to the isolation of a new dammarane-type saponin, namely notoginsenoside ST-4. Its structure was elucidated to be 3ß,12ß,20(S)-tri-hydroxydammar-24-ene-3-O-ß-d-xylopyranosyl-(1 â 2)-ß-d-glucopyranosyl-(1 â 2)-ß-d-glu-copyranoside, based on the detailed analyses of the 1D and 2D NMR spectral data and acidic hydrolysis. Notoginsenoside ST-4 was investigated for its antiviral activity on herpes simplex type 1 (HSV-1) and type 2 (HSV-2) in vitro. The 50% effective concentration (EC(50)) values, determined by plaque reduction assay, were 16.47 ± 0.67 and 19.44 ± 1.16 µM for HSV-1 and HSV-2, respectively, whereas the 50% cytotoxic concentration (CC(50)) determined by the XTT test on Vero cells was 510.64 ± 4.56 µM. As analyzed by attachment assay and penetration assay based on plaque reduction assay, the antiviral activity of notoginsenoside ST-4 was principally due to the penetration inhibition effects, which was confirmed by fluorescence microscopy observation that notoginsenoside ST-4 blocked the penetration of virus. Therefore, notoginsenoside ST-4 might be a promising agent for herpes simplex virus infection.
Subject(s)
Antiviral Agents/pharmacology , Drugs, Chinese Herbal/pharmacology , Panax/chemistry , Saponins/pharmacology , Simplexvirus/drug effects , Triterpenes/pharmacology , Animals , Antiviral Agents/chemistry , Antiviral Agents/isolation & purification , Chlorocebus aethiops , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/isolation & purification , Molecular Structure , Plant Roots/chemistry , Saponins/chemistry , Saponins/isolation & purification , Triterpenes/chemistry , Triterpenes/isolation & purification , Vero CellsABSTRACT
Notoginseng, the root of Panax notoginseng (Burk.) F. H. Chen (Araliaceae), is a Chinese traditional medicine, which is used in both raw and processed forms due to their different pharmacological activities. Detailed chemical investigation on steamed notoginseng led to the isolation of 27 dammarane-type triterpenoids (1-27), including 4 new glycosides, namely, notoginsenosides ST-1-ST-3 and ST-5 (1-4), in addition to 3 other known compounds. Of the new compounds, 1-3 possess new aglycones. Their structures were elucidated on the basis of detailed analyses of their 1D and 2D NMR spectra and chemical reactions. The known compounds, koryoginsenoside-R1 (19), yesanchinoside D (20), 6''-O-acetylginsenoside Rg3 (24), and 3beta,6alpha,12beta-trihydroxydammar-20(21),24-diene ( 25), were isolated from notoginseng for the first time.