| Objective:Combine with the modern analysis techniques, the present study was conducted to investigate the protective role and efficacious material basis of the Smilax glabra Roxb. against the mercury-induced damage in vivo and vitro,to provide foundation and scientific basis for the antimercurialism activities of the Smilax glabra Roxb.Methods:Study1:The mechanism and efficacious material basis of Smilax glabra Roxb in antimercurialism.Mercury(HgCl2,2.5mg·kg-1) resulted in injury of rats, the Smilax glabra water extract and the other type of extraction was conducted to investigate the protection、mechanism and effective fractions of antimercurialism function for mercury-induced injury; Then we examined the effects of the effective fractions against the damage of HgCl2-induced oxidative stress in rats.Study2:Spectrum-effect relationships of antimercurialism fractions of the Smilax glabra Roxb.The preparation of effective fractions was made by the rhizome of Smilax glabra collected from different sources.The suitable concentration of HgCl2and11effective fractions were determined by MTT method with the liver LO2cells; The inverted microscope was used to observe the form of cells,and the cell activity of all groups were tested with MTT method; The ALT and AST leakage were detected with instrument-kit method.To establish fingerprints by HPLC-UV for the detoxification pharmacological activities fractions of the Smilax glabra Roxb.;The ultraviolet-visible spectrophotometry was used for the determination of content of total flavonoids; To establish a HPLC method for simultaneous determination of Astilbin,Taxifolin, Resveratrol, Engelitin, Caffeic acid and Naringin the detoxification pharmacological activities fractions of the Smilax glabra Roxb.;The correlation analysis were used for the LO2cells test results combined with fingerprints、determination of main active constituents.Study3:Purification of the antimercurialism fractions of the Smilax glabra Roxb.4.Based on the results of Spectral correlation analysis,to establish a best method for the purification of the antimercurialism fractions of the Smilax glabra Roxb.with the uv-vis、 HPLC、Macroporous resin and other techniques. Results:1preliminary find out the protency、mechanism and antimercurialism fractions of Smilax glabra Roxb in HgCl2-induced damage1.1The results showed that the mercury levels of model group were higher than the untreated group in the liver, kidney and urine; the urine protein content、ALP and LDH activities,blood urea nitrogen levels were all significantly increased with the untreated group (p<0.01), These indicated the mercury accumulated in liver and kidney cause the injury of the renal function; However, the mercury levels and urine protein content、ALP and LDH activities,blood urea nitrogen levels were all decreased more or less after treatment with the Smilax glabra Roxb. water extraction and acetidin extraction.The water extraction (dosage,1、2g/mL crude drug)、acetidin extraction (dosage,1g/mL crude drug) were had statistical significance (p<0.01or p<0.05) in all the index;Besides, The water extraction (dosage,2g/mL crude drug) can release the HgCl2-induced renal damage.1.2The later study for the acetidin extraction of Smilax glabra Roxb showed a definite preventive effect on the HgCl2-induced rats,the GSH、CAT、SOD level were significantly decreased (p<0.01), The MDA level was significantly increased (p<0.01); However, the acetidin extraction of Smilax glabra Roxb (dosage,1、2g/mL crude drug) can increase the GSH、CAT、SOD level and decrease MDA level of the liver and renal (p<0.01or p <0.05)2Spectrum-effect relationships of antimercurialism fractions of the Smilax glabra Roxb.2.1The protection of effective fractions from Smilax glabra Roxb in HgCl2-induced liver LO2cells2.1.1MTT assay results reveal that with increasing dose and the extension of time, the inhibitory effect of HgCl2on LO2cells showed a gradually increasing trend, The effects of HgCl2and effective fractions in LO2cells, while its inhibition slows down to a certain concentration and time, where the largest concentration inhibitory effect within24h-36h.Refer to the relevant documents, the concentration of3.0μmol/L HgCl2with24h would be adaptted to be the best mode;MTT assay results showed with increasing dose and the extension of time,the inhibitory effect of11sources of effective fractions on LO2cells showed a gradually increasing trend.when the concentration turned up to250μg/mL, the cells just begin to get hurt which reflected in a lower cytotoxic with the LO2cells.2.1.2Compared with the control group,AST、ALT contents of the model group were increase remarkly (p<0.01), MTT assay results showed that the cell activity of model LO2cells had kept at about50%; However, just after interfereing with the effective fractions, The AST, ALT contents of cells were decrease at different level.Most Drugs (dosage,100,250μg/mL) had statistical significance (p<0.01or p<0.05) except for the T3(dosage,50、100、250μg/mL) and part of low (dosage,50μg/mL). MTT assay results showed a very different potency bwteen all. the11effective fractions from11sources of Smilax glabra Roxb, Drugs (dosage,50、100、250μg/mL) of T1、T2、T4、T7、T10、T11all had statistical significance (p<0.01or p<0.05); Drugs (dosage,100、250μg/mL) of T6、T8、T9and T5(dosage,250μg/mL) had statistical significance (p<0.01or p <0.05; But all dosage of T3showed no-effectiveness at the HgCl2-induced liver LO2cells cytotoxicity.2.2Spectrum-effect relationships of antimercurialism fractions of the Smilax glabra Roxb.2.2.1HPLC Fingerprints of effective fractions from11sources of Smilax glabra Roxb: The HPLC fingerprints of11batches of effective fractions were established,a total of16common peaks were identified,and the known compounds were Astilbin, Taxifolin, Resveratrol, Engelitin, Caffeic acid and Naringin.The RSD of relative peak in11effective fractions from different sources were all<3.0%.The similarities of each HPLC fingerprint with the reference spectrum were between0.850·0.990except for T3sample.2.2.2Determinate of antimercurialism parts and Active ingredient contentsin:The content of total flavonoids in11batches of effective fractions were between21.30%～30.95%,there were significant differences between each samples with different sources (P<0.05).Among them, The content of total flavonoids in T1from Guangdong was the highest but the T3from Guangxi was the lowest.The The content of Astilbin,Taxifolin, Resveratrol, Engelitin, Caffeic acid and Naringin in11batches of effective fractions were between2.43%-3.65%,1.16%～1.52%、1.79～2.10%、1.95%～2.47%、0.44%～0.65%、1.79～2.10%、0.81%～1.15%.Among all, The contents of all the monomers were at a lower level.2.2.3spectrum-effect relationships:The content of total flavonoids、Astilbin、 Resveratrol、Engelitin、Caffeic acid in11batches of effective fractions have significant direct correlation with the activity of HgCl2-induced liver LO2cells (P<0.01). The content of total flavonoids,Astilbin, Taxifolin, Resveratrol, Engelitin, Caffeic acid and Naringin have significant inverse correlation with AST leakage rate at-0.88、-0.901.-0.948.-0.814.-0.691.-0.916.-0.852;HPLC Fingerprints-effect relationships shows that8common peaks have significant direct correlation with the activity of HgCl2-induced liver LO2cells(P<0.05), and the known compounds were Astilbin, Resveratrol, Engelitin, Caffeic acid.3Purification technical of antimercurialism fractions from Smilax glabra Roxb.The optimum parameters of the ADS-7macroporous adsorptive resins were as following:loading sample density1mg/mL, pH5.0, the maximum sample volume0.75mg every ml macroporous,5BV·h-1flow rate to adsorption and the volume of70%(v/v) ethano as8BV with desorption flow2mL/min. With these conditions, more than40%of total flavonoids could be obtained from primary adsorption and purification,and content of Astilbin, Resveratrol, Engelitin, Caffeic acid could raise up to4.23%.2.54%.2.56%.0.87%。Conclusion:1The effective fractions and mechanism of Smilax glabra Roxb. in antimercurialismIt was comfirmed that Smilax glabra Roxb. have an significant and reasonable antimercurialism in vivo, the acetidin fractions based on water extraction of Smilax glabra Roxb. was confirmed to be the effective fractions in antimercurialism.This antimercurialism effect in Smilax glabra Roxb.was due to the synthetic action of diuresis affect、oxidative stability、liver and renal protection.2Spectrum-effect relationships of antimercurialism fractions of the Smilax glabra Roxb2.1The protection of the acetidin fractions from Smilax glabra Roxb.It was comfirmed that Smilax glabra Roxb. have an significant and reasonable antimercurialism in vitro, the results showed that the effective fractions could raise up survival rate of HgCl2-induced liver LO2cells obviously; The AST. ALT contents and the toxicity of HgCl2-induced cells could be reduced by the acetidin fractions from Smilax glabra Roxb. at the suitable dose.This showed that effective fractions had protection in HgCl2-induced liver LO2cells,and these protection were at the different level with different sources of Smilax glabra Roxb.2.2Spectrum-effect relationships of antimercurialism fractions of the Smilax glabra RoxbThe HPLC fingerprints of11effective fractions were established,a total of16common peaks were identified. The chemical constituent of effective fractions had stable quality except for T3; The content of total flavonoids11effective fractions were significant differenct (P<0.05) at21.30%～30.95%;There were significant differenct (P<0.05) in the content of Astilbin,Taxifolin, Resveratrol, Engelitin, Caffeic acid and Naringin; The content of total flavonoids、Astilbin、Resveratrol、Engelitin、Caffeic acid of effective fractions have significant direct correlation with the activity of HgCl2-induced liver LO2cells, Infering that total flavonoids、Astilbin、Resveratrol、Engelitin、Caffeic acid were the key material basis of Smilax glabra Roxb in the antimercurialism activity.3Purification technical of antimercurialism fractions from Smilax glabra Roxb.With the optimum technological parameter, more than40%of total flavonoids could be obtained from primary adsorption and purification,and content of Astilbin, Resveratrol, Engelitin, Caffeic acid could raise up to4.23%、2.54%、2.56%、0.87%;To provide reference for the study of Smilax glabra Roxb. in new antimercurialism medicine. |
PDF Full Text Request