ABSTRACT
Objective: To investigate the best extracting conditions of Polygonatum sibiricum polysaccharides by single and compound enzymatic methods, and to study on the change content of polysaccharide in processing of P. sibiricum by steamed and shined for nine times, which provide theoretical basis for processing of P. sibiricum and other Chinese herbal medicines. Methods: Response surface method (RSM) was used to optimize the extraction of polysaccharides in P. sibiricum. PMP precolumn derivatization and HPLC characteristic map technology were used to identify and analyze the changes of polysaccharide of P. sibiricum being steamed and shined for nine times and establish polysaccharide characteristic map of P. sibiricum by ancient processing and distillate. The effect of different enzymes on the extraction efficacy of P. sibiricum polysaccharides was also compared. Results: The extracting effect of cellulase in single enzyme was the best. Moreover, cellulose and pectinase were also the most significant factor for extracting polysaccharides from P. sibiricum in four complex enzymes. The best extracting conditions for polysaccharide obtaining by RSM as follows: Cellulase temperature was 51 ℃, cellulase dosage was 2%, and the total polysaccharide content was (675.34 ± 0.10) mg/g when the pH value of enzymolysis was 5.40. PMP-HPLC was used to detect four processed P. sibiricum, which contained mannose, ribose, rhamnose, glucose, and galactose. Glucose had a high proportion in P. odoratum, P. cyrtonema, and P. sibiricum polysaccharide, and P. filipes polysaccharide mainly composed of mannose. With the increase of processed times, the content of each monosaccharide increased first, then decreased, then increased and then tended to be stable. The content of mannose and galactose varied greatly. The content of monosaccharides in the distillate increased first and then stabilized, and the content changes of mannose and glucose were obvious. Conclusion: The extraction efficiency and purity of P. sibiricum polysaccharides can be improved by single and compound enzymatic extraction with cellulase, and the extraction yield of polysaccharide in P. sibiricum could be affected by steamed and shined for nine times. PMP-HPLC characteristic map technology can be used to determine the content of the polysaccharide in processing of P. sibiricum accurately and reproducibly, which can be used for the quality evaluation of P. sibiricum and other Chinese herbal medicines.
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Objective This paper reports a modified method for the isolation of rat thoracic aortic vascular smooth muscle cells based on combined enzyme double digestion.Methods An enzyme mixture containing collagenase II, soybean trypsin inhibitor and elastase was prepared and used to remove the aortic tunica intima and tunica adventitia, and then the tunica media was subjected to second digestion using the same enzyme mixture and to isolate vascular smooth muscle cells.Results The isolated VSMCs were cultured in vitro and the growing cells had an elongated spindle-shape, reached confluence within a week, and displaying a typical hill-and-valley pattern.After 1 week, the cells were passaged.Contractile smooth muscle markers(α-SMA, myosin-II and β-tubulin) were highly expressed in the isolated cells.More than 90% of the cells significantly expressed alpha smooth muscle actin and myosin-II.Conclusions Themethod established in this study has advantages of simple and easy to operate, and good reproducibility, with a high activity and purity of the separated cells.It can ensure to obtain a large amount of contraction-type vascular smooth muscle cells within a short time.
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Objective:To explore the clinical effect of four oral vitamins [vitamin E (Vit E) +folic acid (FA)+ vitamin B2 (Vit B2) + vitamin B12 (Vit B12)] combined with FE complex enzyme rash on the recurrent oral ulcer (ROU) and on the serum inflammatory factors levels.Methods:126 cases with ROU in our hospital from January 2014 to February 2016 were selected as research objectives and randomly divided into two groups.FE complex enzyme was provided to the control group,while FE complex enzyme and four Vietnam (Vit E+FA+Vit B2+Vit B12) were given to the observation group.The local efficacy,long-term efficacy,serum inflammatory factor levels before and after treatment as well as the incidence of adverse drug reactions were recorded and compared between two groups.Results:Compared with the control group,the pain index was significantly decreased on 30th treating day in the observation group(P＜0.01),and the average ulcer period was shorten (P＜0.01).After being treated for 6 months,the overall effective rate was 95.2％ in the observation group,which was significantly higher than that of the control group (81.0％,P＜0.05).The serum TNF-α and IL-17 levels on the 30th treating day was significantly lower in both groups after treatment than those before treatment (P＜0.01),but the serum 1L-2 level was significantly increased (P＜0.01),and the improvement of each above inflammatory factors in the observation group were more significant than those of the control group (P＜0.01).No significant difference was found in the incidence of adverse reactions between two groups (P＞0.05).Conclusions:Four oral Vietnam combined with FE complex enzyme could promote the ulcer wound healing,reduce the pain,regulate the body to promote/anti-inflammatory factor balance and improve the long-term efficacy in the treatment of patients with ROU with high safety.
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Objective: To optimize the extraction processes used for enzymatic hydrolysis of total sugar from Cynomorii Herba (Cynomoriumsongaricum) by employed quadratic orthogonal rotation regression combination design. Methods: Taking the extraction rate of total sugar as index, as well as enzyme ratio, enzyme concentration, extraction temperature, extraction time, and initial pH value as investigation factors to screen out main factors. The quadratic orthogonal rotation regression combination design was applied to optimize the process. And then, to employ response surface analysis method (RSM) for evaluating the impacts on the response values of the single factor effect and interaction effect of each factor level. Results: The optimum conditions were as follows: enzyme concentration was 1% (wt% of Cynomorii Herba powder), initial enzyme solution pH was 5.5, extraction temperature was 55℃, extraction time was 45 min, and pectinase and cellulose was 1∶1 proportion. The extraction yield of total sugar was (17.54 ± 0.35) %. The influence of response values of each factor presented the relationship of the parabolic surface with the opening downward. The results showed that the impact degree on total sugar yield of Cynomorii Herba were extraction temperature, extraction time, and enzyme ratio, respectively. Conclusion: This method is a simple and effective method to increase the extraction yield of total sugar from Cynomorii Herba. Quadratic orthogonal rotation regression combination design has a favourable predictability.