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OBJECTIVE： To optimize the proteolytic enzymes for enzymolysis technology of degreasing ointment from Periplaneta americana， and to improve the extraction rate and activity of anti-liver fibrosis active part from P. americana. METHODS： Using degreasing ointment of P. americana as control， ninhydrin method and folin-ciocalteu method were used to investigate the hydrolysis degree of trypsin （TR）， pepsin （PE）， alkaline protease （AL）， papain （PA） and neutral protease （NE） to the degreasing ointment. Macroporous resin isolation and purification method was used to investigate the yield of elution part from hydrolyzate， with 50％， 60％， 70％， 95％ ethanol as eluting solvents. Inhibition test in vitro of rat hepatic stellate cells HSC-T6 was performed， and anti-liver fibrosis activity of elution part from hydrolyzate was investigated. RESULTS： The hydrolysis degree of PA and NE were 14.15％ and 15.70％， showing strong enzymatic hydrolysis ability. The yield of 95％ ethanol elution part from PA， NE and AL hydrolyzate were （0.73±0.04）％，（0.65±0.01）％ and（0.64±0.05）％， improving 30.36％， 16.07％， 14.29％ compared with degreasing ointment without enzyme. Results of inhibition test in vitro showed that inhibitory rate of 50％， 60％， 70％ ethanol elution parts isolated and purified from hydrolyzate had a low inhibition rate or a growth-promoting effect on HSC-T6 cells. Inhibition rates of 95％ ethanol elution parts to HSC-T6 cells were all more than 20％. IC50 of 95％ ethanol elution part isolated and purified from PA and NE hydrolyzate for 24-72 h were 94.5-112.3 and 117.1-120.0 μg/mL， which were lower than that （116.1-123.0 μg/mL） of degreasing ointment without enzyme. CONCLUSIONS： PA is the best hydrolyzate for enzymolysis technology of active parts against liver fibrosis in degreasing ointment from P. americana， followed by NE and AL； PE and TR， which have poor effect， are not suitable for the enzymatic hydrolysis technology.
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Objective: To analyze the dynamic changes of components in the enzymolysis process of raw products of Raphani Semen. Method: HPLC was employed to analysis of characteristic spectra of Raphani Semen at different enzymolysis time with mobile phase of acetonitrile-0.1% phosphoric acid aqueous solution for gradient elution and detection wavelength at 225 nm.The characteristic peaks were calibrated, meanwhile, the UV spectra of characterstic peaks were extracted, and the difference between UV spectra and the changes of peak areas were compared, and the dynamic changes of characterstic components in Raphani Semen were analyzed. Result: Eleven characteristic peaks were marked from the characteric spectra of raw products of Raphani Semen at different enzymolysis time, and glucoraphenin and sinapine thiocyanate were assigned.Glucoraphenin was enzymatically hydrolyzed fastly by myrosinase, and an intermediate was generated, and then continue to be decomposed into other components.Sinapine thiocyanate did not change significantly during the enzymolysis process, and sinadiosides was also enzymatically degraded. Conclusion: The enzymolysis of Raphani Semen is not only the glucoraphenin, but also the sinadiosides.This paper can provide reference for the property change of Raphani Semen in processing.
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Background: Astaxanthin from natural sources is typically esterified with fatty acids; hence, it must be hydrolyzed to remove esters before identification and quantification by conventional HPLC. Alkaline-catalyzed saponification and enzyme-catalyzed enzymolysis are the most commonly used de-esterification methods. However, information on the efficiency and isomerization during de-esterification of natural astaxanthin esters by these two methods remains scarce. Therefore, we conducted two HPLC-based experiments to determine which method is better for hydrolyzing astaxanthin esters. Results: To assess the effect of enzymolysis (0.67 U/mL cholesterol esterase, at 37°C) and saponification (0.021 M NaOH, at 5°C) conditions on free astaxanthin recovery and destruction or structural transformation of astaxanthin, we varied the total treatment time across a range of 195 min. The results showed that enzymolysis and saponification were complete in 60 min and 90 min, respectively. After complete hydrolysis, the maximum free astaxanthin recovery obtained by enzymolysis was 42.6% more than that obtained by saponification. The identification of by-products, semi-astacene and astacene, during the process of saponification also indicated that a more severe degradation of astaxanthin occurred during saponification. Moreover, the composition of astaxanthin isomers during saponification was similar to that of the isomers during enzymolysis between 30 min and 75 min (all-trans:9-cis:13-cis = 21:3:1, approximately) but dramatically changed after 90 min, whereas the composition in the enzymolysis treatment remained relatively stable throughout. Conclusion: Compared with saponification, enzymolysis with cholesterol esterase was recommended as a more accurate method for de-esterification of natural astaxanthin esters for further qualitative and quantitative HPLC analysis.
Subject(s)
Xanthophylls/chemistry , Esters/chemistry , Carotenoids , Xanthophylls/metabolism , Alkalies , Enzymes/metabolism , Esters/metabolism , Hydrolysis , IsomerismABSTRACT
Objective To study the process of hyaluronidase enzymolysis under different condi tions and to provide the basis for clinical application.Methods The experiment of 15 hyaluronic acid products from 10 brands was conducted in vitro with 15 concentrations and 4 dosages of hyaluronidase at 37 ℃ and 24 C.The results of experiment were compared with the clinical observations.Results The duration of enzymolysis was inversely correlated with hyaluronidase concentration.The duration of enzymolysis process reduced 60％ when equal hyaluronidase was used as compared with that when half hyaluronidase was used.The enzymolysis duration could be 24 folds in different products.The enzymolysis time was 2.9 folds at 24 C [24 ℃ was (77.67±35.33) min],compared with that at 37 ℃[37 ℃ was (27.13±11.05) min].It took much more time of enzymolysis in vivo than in vitro for the same product.Conclusions Enzymolysis efficiency is positively correlated with the concentration and dosage of enzyme and inversely associated with the particle size and viscosity.
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Objective: To prepare and evaluate bionic enzymatic Epimedium flavonoids-loaded enteric-coated capsules, composed of Epimedium flavonoids as drug, snail enzyme as hydrolase and common formulation accessories, which is in favor of in vivo real-time enzymolysis and real-time absorption of Epimedium flavonoids. Methods: The UV method and single factor experiment were applied to investigating the effects of key factors such as filling agents species and dosage, disintegrating agents species and dosage, wetting agents species and drying temperature on the cumulative release degree of Epimedium flavonoids. Response surface method was used to optimize the prescription for bionic enzymatic Epimedium flavonoids-loaded enteric-coated capsules, and the enzymatic process of the four Epimedium flavonoids (icariin and epimendin A, B, C) was also evaluated by HPLC. Results: The optimal formulation for the bionic enzymatic Epimedium flavonoids-loaded enteric-coated capsules was composed of 100 mg Epimedium flavonoids extract, 68 mg snail enzyme, 65 mg α-lactose, and 11.7 mg low-substituted hydroxypropyl cellulose. This enteric-coated capsules showed a cumulative Epimedium flavonoids release rate of 85.43% within 45 min in simulated intestinal fluid and a complete enzymolysis within the intestine emptying time (3-6 h). Conclusion: The bionic enzymatic Epimedium flavonoids-loaded enteric-coated capsules show an advantage in improving hydrolysis and anti-osteoporosis efficacy of Epimedium flavonoids, providing some research ideas for improving the oral bioavailability of other flavonoids.
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Two kinds of β2-agonistresidues in sheep plasma and urine were disposed by enzymolysis and organic solvent extraction pretreatment methods, and UPLC-MS/MS was used for the qualitative and quantitative analysis. Detection results were compared to study the influences of two pretreatment methods. The experimental results showed that more than 95% of Ractopamine and 40% of Salbutamol exist in the conjugated form in sheep plasma. The detection results of 2 kinds of β2-agonist residues were significantly enhanced when adding β-glucuronidase/aryl sulfatase. The experimental repeatability is very poor ( RSD>40%) when the enzymolysis was not carried out. There were 57% of Ractopamine and less than 1% of Salbutamol exists in the conjugated form in sheep urine. Enzymolysis pretreatment method was useful for the Ractopamine residues determination in urine, and Enzymolysis pretreatment method was useless for Salbutamol determination in urine. Matrix effect of plasma was less than the effects of urine. The influence of organic solvent extraction pretreatment method on the detection results was unremarkable, and there was the possibility that organic solvent extraction could lead partial loss of target compound in extraction process. However, it did not influence the detection results by using internal standard calibration.
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Objective To optimize the enzymolysis technology of ginsenoside Rg1.Methods Taking ginsenoside Rg1 content as the index,orthogonal design method was used for optimization and HPLC for determination.Results Cellulase enzymolysis was the best extracting process,and enzyme amount,enzymolysis time,and enzymolysis temperature had obvious effect on the extraction of ginsenoside Rg1.The optimum extraction technologies were as follows: cellulase amount was 1.4%,enzymolysis time 60 min,the enzymolysis temperature 45 ℃.Conclusion The optimization extraction technology is simple,steady,and the extracting rate is high.
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The technique of extracting and refining mucopolysaccharide from Dasyatis akajei cartilage obtained was studied and the content of mucopolysaccharide was measured.The results showed that when the content of NaOH was 6%,temperature 40℃, extracting time 6h,followed neutral protein enzyme dissolution for 7h under 40℃,protein precipitation with trichloroacetic acid and 4 times volume 95% C_(2)H_(5)OH for polysaccharide precipitation.the extraction rate was over 21%, the product was white powder and its purity was about (84.53%).Physical and chemical properties of the mucopolysaccharide were identified.
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Objective To optimize the enzymolysis condition for Acaudina leucoprocta to research lencoprocta the antioxidant activity of its hyoholysate in vivo.Method Response surface methodology (RSM) was employed to optimize the hydrolysis conditions with protamex.The ICR mice were given ig the hydrolysate of Acaudina lencoprocta obtained under optimal conditions.The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the content of MDA in blood serum and liver in mice were analyzed.Results and Conclusion The best hydrolysis conditions were:the amount of enzyme was 2.02%;enzymolysis temperature was 51.18 ℃;enzymolysis time was 127.81min,hydroxyl radical scavenging rate was 73.10%.The activities of SOD,GSH-Px in hydrolysate were significantly enhanced and the content of MDA was significantly lower,which showed that this hydrolysate had remarkable antioxidation activity.So the enzymolysis technology for Acaudina leucoprocta was optimal.