| Objective This study was conducted in vivo and in vitro.The correlation between the "time(curing time)-effect-toxicity" of Polygonum multiflorum and different processed products was discussed.Using HPLC technology,cell flow meter and blood biochemical indicators,immunohistochemistry and PCR technology,the raw materials of Polygonum multiflorum and The main components of the processed products at different times and the changes of some related indicators were studied,and theoretical support was provided for the clinical correct use of drugs.Method 1.HPLC method using a column: HYPERSIL GOLD AQ 5UM(250*4.6MM)COLUMN.Methanol is phase A,0.1% formic acid water is gradient elution of phase B,0~19min,10%~36% A,90%~63% B;19~30min,36%~71% A,63%~ 29% B;30~50min,71%~100% A,29%~0% B;50~60min,100% A.Column temperature: 35 ° C;detection wavelength: 254 nm;flow rate: 1.0 ml / min;injection volume of 10 ?L method for the detection of terpenoids,gallic acid and stilbene glycoside in different processed products of Polygonum multiflorum.2.48 SD rats were randomly divided into blank group,Shengshouwu group,steamed for 8h,steamed for 16 h,steamed for 32 h,steamed for 48 h,and steamed for 72 h.There were 7 groups in each group,6 in each group.7 days in a row,2 times a day,2 hours after the last gavage,blood was taken from the abdominal aorta,centrifuged at 3000 r after 30 minutes,serum was inactivated at 37°C for 30 min,and the microporous membrane was filtered to prepare CCK-containing serum.8 and detection of apoptosis.3.Liver injury group: 70 SPF mice were randomly divided into blank group,Shengshouwu group,steamed 8h,steamed for 16 h,steamed for 32 h,steamed for 48 h,steamed for 72 h,7 groups,10 in each group,weighing,mark The dosage of each administration group was 12g/kg(corresponding to 60 times of the maximum dosage of adults),0.2ml/50 g,and the blank group was given the same amount of normal saline.After 4 weeks,serum and liver tissues were taken.Detection of indicators.Baogan group: 90 SPF mice were randomly divided into blank group,model group,positive drug group,Shengshouwu group,steamed 8h,steamed 16 h,steamed 32 h,steamed 48 h,steamed 72 h group,9 groups.10 only.Except for the blank group,each group of mice was subcutaneously injected with 10% carbon tetrachloride peanut oil solution,and the first dose was 0.2 ml/10 g,and then 0.1 ml/10 g per stomach was administered once every two days.After the model,the drug was administered at a dose of 6 g/kg(equivalent to 30 times the maximum dosage of the adult),0.2 ml/10 g,and the blank group and the model group were given the same amount of physiological saline,and the positive drug was given biphenyl.Ester dropping pills(200mg/kg),after 4 weeks,serum and liver tissue were taken for testing.Result 1.The results of HPLC showed that the content of anthraquinones and quinone gallic acid in Radix Scutellariae was lower than that of Shouwu,while the content of stilbene was higher.The content of emodin,emodin and gallic acid in the steamed group was generally increased with the prolongation of processing time,and only the content of stilbene was decreased with the increase of processing time.2.The results of CCK-8 showed that the survival rates of each drug-administered group were 98.67%,95.46%,48.90%,56.60%,63.13%,65.39%,74.18%,78.41%,respectively.Compared with the group,the serum containing Shengshou group,steamed for 8h,steamed for 16 h,steamed for 32 h,steamed for 48 h,and steamed for 72 h,the serum containing HepG2 cells had obvious inhibitory effect;Compared with the Shengshouwu group,the steaming group also had statistical difference(P<0.05)in the 16 h,32h,48 h and 72 h groups.3.The results of cell flow test showed that there were different degrees of early and late apoptosis and cell necrosis in each group.There was no significant difference between the blank rat serum group and the fetal bovine serum group(P>0.05).10% fetal calf serum and 15% blank rat serum have similar efficacy.The apoptotic rates of the Shouwu group,the steamed steamed 8h,16 h,32h,48 h and 72 h groups were significantly different from those of the blank group(P<0.01),Compared with the Shengshouwu group,the steaming group had statistical difference(P<0.01).mainly in the AV+ and PI-regions,indicating the steaming group and the raw group.The drug-containing serum of the Shouwu group caused different degrees of apoptosis in HepG2 cells.4.The results of blood biochemical indicators in the liver injury group showed that the AST,ALT and ALP of the Shouwu group and the steamed 8h group were significantly higher than those of the blank group(P<0.01 or P<0.05),and steamed for 16 h.The ALP at 32 h and 48 h was statistically significant compared with the blank group(P<0.05).There was no significant difference in ALP between the ALT,AST and steaming 16 h,32h,48 h and 72 h groups in the 16 h,32h and 48 h groups(P>0.05);Compared with the Shouwu group,the ALT levels in the 16 h,32h,48 h and 72 h groups were statistically significant(P<0.05),and the ASTs in the 32 h,48h and 72 h groups were significantly different(P<0.01).The ALP of the 72 h group was statistically significant(P<0.05).The blood biochemical indicators of the liver-protecting group showed that the AST,ALT and ALP of the model group were significantly different from the blank group(P<0.01),and the positive drug was statistically significant compared with the model group(P<0.05).There was no significant difference in AST,ALT and ALP between the drug-administered groups of Polygonum multiflorum and the model group(P>0.05),indicating the protective effect of different processed products of Polygonum multiflorum on liver damage caused by carbon tetrachloride.Not obvious.5.The results of immunohistochemistry showed that the expression levels of Caspase-3,Bcl-2 and NF-kbp65 in the Shouwu group were significantly different from those in the blank group(P<0.01).The expression of Bcl-2 was significantly different from that of the blank group(P<0.01),while the expression of Caspase-3 and NF-kbp65 was significantly different from that of the blank group(P<0.05);steaming for 16 h,steaming for 32 h The expression of Caspase-3 in the 48 h group was significantly higher than that in the blank group(P<0.05).The expression of Bcl-2 in the steamed 16 h and 32 h group was significantly different from that in the blank group(P<0.01);Compared with the Shouwu group,the expression levels of Caspase-3,Bcl-2 and NF-kbp65 in the 8h,16 h,32h,48 h and 72 h groups were statistically significant(P<0.01,P<0.05).The results of immunohistochemical examination showed that the expression levels of Caspase-3,Bcl-2 and NF-kbp65 in the model group were significantly different(P<0.01,P<0.05).Compared with the model group,the group also had significant increase and decrease,which was statistically significant(P<0.01,P<0.05).The expression levels of various indicators in the steaming group and the Shengshouwu group were not significantly different from those in the model group(P>0.05).6.The results of drug metabolism enzyme index in liver injury group showed that the expression levels of CYP2E1,CYP3 A and CYP1A2 in the drug-administered group decreased gradually according to the group of the Shouwu group>8h group>16h group>32hgroup>48hgroup>72hgroup.There were significant differences in the blank group comparison(P<0.01,P<0.05);Compared with the Shouwu group,the relative expression of CYP1A2 enzyme in the 48 h and 72 h groups of the steaming group was also statistically significant(P<0.05).The results of drug metabolism enzyme index in Baogan group showed that there were significant differences between the three subtypes of enzyme model group and the blank group(P<0.01),and the positive drugs were statistically different from the model group(P<0.05).However,the relative expression levels of the three enzymes in other drug-administered groups decreased,but there was no significant difference compared with the model group(P>0.05).Conclusion 1.The content of anthraquinones and gallic acid in Polygonum multiflorum and different time processed products will gradually decrease with the prolonged processing time,and only the content of stilbene will decrease with the increase of processing time.Therefore,it can be inferred that the processing time can affect the content of the main components in the steamed product of Polygonum multiflorum.2.The component of Polygonum multiflorum that causes liver damage may be stilbene glycosides.3.The liver damage caused by Polygonum multiflorum was negatively correlated with the time of processing.4.The mechanism of liver damage caused by Polygonum multiflorum may be related to the pathway regulating drug metabolism enzymes.5.The liver damage of Polygonum multiflorum and the dose of liver-protecting effect on administration have a certain correlation,and the dose is positively correlated with toxicity.The liver protection effect may be negatively correlated with the dose. |
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