| Objective:To study and compare the chemical constituents of raw Pinellia ternata?SBX?,Pinelliae Rhizoma Praeparatum?FBX?,Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine?JBX?,Pinelliae Rhizoma Praeparatum cum Alumine?QBX?and their sleep-improving effects on mice.The antioxidant activities of Pinellia ternata and its processed products were also studied,and an on-line analytical method for rapid screening of antioxidant components in traditional Chinese medicine was established.Method:The Pinellia ternata and its processed products samples were prepared to obtain their ethanol extracts through heating reflux extraction,concentration and constant volume procedures.Experiment 1,study and compare the main chemical constituents of Pinellia ternate and its three processed products were analyzed via thin layer chromatography?TLC?,Infrared Spectroscopy?IR?,high performance liquid chromatography?HPLC?and high performance liquid chromatography-mass spectrometry?HPLC-MS?.Experiment 2,fifty-four healthy Kunming male mice?SPF grade?were randomly divided into 9 groups after one week of adaptive feeding,6 mice in each group.Control group?normal saline?,Pinellia ternata and three processed products low dose groups?8 g/kg,convert by the weight of the raw medicinal materials?,Pinellia ternata and three processed products high dose groups?16 g/kg,convert by the weight of the raw medicinal materials?.Mice were fasted 8 hours with free access to water before intragastric administration of sample drugs.Normal saline or Pinellia ternata extracts were intragastric administrated according to the body weight of each mouse.After drug administration,mice were placed into the mouse autonomic activity tester box individually to record their movements within 1 h period of time,then systematical analysis were conducted for drug activity evaluation and comparison.Experiment 3,chromatographic analysis of antioxidant active components using HPLC-UV-DPPH/ABTS technology was performed on in house combined equipment that contains HPLC system with UV detector and DPPH/ABTS evaluation component.Agilent Extend C18?250 mm×4.6 mm,5?m?column with gradient elution of acetonitrile-0.1%formic acid at the flow rate of 1.0 mL/min,and the detection wavelength was at set 265 nm.The column temperature was maintained at 25?.Flow rate of DPPH/ABTS solution was 0.5 mL/min,The detection wavelengths was at 517 and 734 nm,were selected to obtain DPPH and ABTS free radical scavenging profiles,respectively.The on-line HPLC-UV-DPPH/ABTS method was verified by analyzing the ethanol extracts of Rehmannia Radix and Rehmanniae Radix Praeparata,and then was applied for the screening and identification of antioxidant compounds in four Pinellia ternate samples.Results:Experiment 1,the results of TLC analysis showed that the compositions of the Pinellia ternata and its processed products samples were significantly different.There were seven spots for SBX,six spots including one larger spot for FBX,six similar spots for QBX,while JBX showed only five spots.The result of IR analysis shows that Pinellia ternata and its processed products contain hydroxyl,carbonyl and other characteristic functional groups and fatty acid components,and can distinguish Pinellia ternata and its processed products.The results of HPLC-FTMS analysis showed that fourteen compounds were identified in Pinellia ternata and its processed products.Arginine,cyclo?valine-tyrosine?,tyrosine,adenosine,phenylalanine,cytidine,1,2,2,6,6-Pentaametylepipiridi-4-ylmethacrylate,and N-?1-?phenylacetamide?L-prolyl?glycineethyl ester are common components in Pinellia ternata and its processed products.Experiment 2,pharmacological results show that,the high and low dose group of Pinellia ternata and its processed products were compared with the control group,the differences have statistical significance?P<0.01?,the resting time of the mice in all the drug administration groups was higher than that in the control group.The high dose of JBX,low dose of QBX and low dose of SBX ethanol extracts significantly inhibited the spontaneous activity of the mice,and increased their resting time,while the resting time of the mice with high-dose of SBX was only 5%higher than that in the control group,which may cause by the gastric irritation side effect induced by SBX ethanol extract.Experiment 3,the analysis results of HPLC-UV-DPPH/ABTS showed that only one component possessed antioxidant activity in either the Pinellia ternate or its three processed products,but active peak areas were different.Nine antioxidants in Rehmanniae Radix extract were found by HPLC-UV-DPPH,while thirteen antioxidants in Rehmanniae Radix Praeparata were detected,and there were eight common antioxidants in the two above medical materials.Conclusion:?1?the composition of Pinellia ternata was changed after processing procedures by different process methods.TLC and IR could distinguish Pinellia ternata and its processed products.The main components of Pinellia ternata ethanol extracts were identified as amino acids,nucleosides and organic acids.?2?Pinellia ternata and its processed products showed the sleep-improving activities on mice,but they did not induce the mice enter the deep sleep stage,only extended resting time phenomenon were observed in all drug treated mice.The high dose of JBX,low dose of QBX and low dose of SBX ethanol extracts exerted better sleep-improving effect,while the sleep-improving effect of high-dose SBX was unconspicuous,which might be related to irritant toxicity of raw Pinellia ternate with excessive dose.?3?Antioxidant activity Pinellia ternata and its processed products were not strong due to their special compositions,and different processing procedures may lead to variation of the amount of antioxidant active component,and then causethevariationofantioxidantactivities.Thedeveloped HPLC-UV-DPPH/ABTS technology can be applied to screen the antioxidant components in Chinese herbal medicines with encouraging speed,accuracy and efficiency. |
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