Study On Chemical Constituents Of The Combined Decoction Of Corni Fructus/Rehmanniae Radix Preparata/Poria

Learning is the process of behavior change arising from the Individual contact with the environment and gain experience.Memory is the cognitive side of learning.It is generally divided into three processes:the process of memory,maintenance and reconstruction.Corni Fructus is the dry,ripe fruit of Cornus officinalis Sieb.et Zucc.Rehmanniae Radix Preparata is the root's processed products of Rehmannia glutinosa Libosch.Poria is the dry sclerotia of Poria cocos?Schw.?Wolf.The active index of this subject is the ability of learning and memory.Through the analysis of the changes of the quantity of the index components in the combined decoction,the separation,purification and structural identification of the chemical constituents of the combined decoction,etc,the effective materials of the combined decoction to enhance the learning and memory ability of the mice was discussed.Section 1 The preliminarily study of enhancing the ability of learning and memory in mice of the combined decoction of Corni Fructus/Rehmanniae Radix Preparata/Poria.Objective:A preliminary study of the decoction of Corni Fructus/Rehmanniae Radix Preparata/Poria in enhancing the ability of learning and memory in mice.Methods:30 mice were randomly divided into 5 groups:blank control group,single decoction of Corni Fructus,Rehmanniae Radix Preparata or Poria groups,combined decoction group.The mice of combined decoction group and the single decoction groups were administrated continuously for 6 days,1times·day^-1,and the dose was 0.02g·mL^-1(according to the original material:0.012g·g^-1).The mice of blank control group were provided with normal saline.1 hour after administration in the sixth day,the mice were familiar with the water environment for 3 mins.1h after administration in the seventh day,water maze experiment was conducted for each group of mice.Recording the incubation and the number of times entering blind end.Each mouse was tested2 times a day.The determinations were carried out for 4 days.Experimental data were processed by statistical software.Results:Compared with the blank control group,the combined decoction can obviously enhance the ability of learning and memory in mice,and the incubation of mice in the combined decoction group and the number of times entering blind end were decreased obviously?P<0.01?.Compared with each single decoction,after 4 days of determination,the incubation of the mice reaching the platform was reduced by 37.5%?comparison with the group of the single decoction of Corni Fructus?,34.4%?comparison with the group of the single decoction of Rehmanniae Radix Preparata?,53.4%?comparison with the group of the single decoction of Poria?.The number of times entering blind end of mice in the combined decoction group were reduced by 37.5%?comparison with the group of the single decoction of Corni Fructus?,40.0%?comparison with the group of the single decoction of Rehmanniae Radix Preparata?,50.0%?comparison with the group of the single decoction of Poria?.Conclusions:The ability of learning and memory in mice in the combined decoction group was better than that of the blank control group and the single decoction group.Section 2 Study on the contents of index components of the combined decoction of Corni Fructus/Rehmanniae Radix Preparata/Poria and each single decoctionObjective:To study the contents of the index components in the combined decoction.Methods:High performance liquid chromatography conditions of Loganin,morroniside,5-hydroxymethyl furfural:Chromatographic column:ZORBAX Eclipse XDB-Phenyl C₁₈?4.6mm×250mm,5?m?;Mobile phase:acetonitrile?A?-0.3%aqueous solution of phosphoric acid?B?;Current Speed:1mL·min^-1;Detection wavelength:240nm;Column temperature:35?;Sampling quantity:10?L;Gradient elution?0²0min,7%A;20⁵0min,7%A?20%A?.High performance liquid chromatography conditions of verbascoside:Chromatographic column:ZORBAX Eclipse XDB-Phenyl C₁₈?4.6mm×250mm,5?m?;Mobile phase:acetonitrile-0.1%aqueous solution of phosphoric acid?17:83?;Current Speed:1 mL·min^-1;Detection wavelength:334nm;Column temperature:30?;Sampling quantity:20?L.High performance liquid chromatography conditions of gallic acid:Chromatographic column:ZORBAX Eclipse XDB-Phenyl C₁₈?4.6mm×250mm,5?m?;Mobile phase:acetonitrile-0.3%aqueous solution of phosphoric acid?7:93?;Current Speed:1mL·min^-1;Detection wavelength:240nm;Column temperature:35?;Sampling quantity:10?L.Results:Compared with the single decoction of Corni Fructus,the contents of loganin and 5-hydroxymethyl furfural were decreased?P<0.01?,the contents of morroniside were increased?P<0.01?;The effect of Poria on the opposite effect of Rehmanniae Radix Preparata?P<0.01?;For the combined decoction,the contents of loganin and 5-hydroxymethyl furfural were reduced?P<0.01?,the contents of morroniside were increased?P<0.01?.The contents of gallic acid in the decoction were lower than that of the single decoction of Corni Fructus?P<0.01?.Compared with the single decoction of Rehmanniae Radix Preparata,the contents of verbascoside of Corni Fructus and Rehmanniae Radix Preparata decoction or Corni Fructus,Poria and Rehmanniae Radix Preparata decoction were increased?P<0.01?,the contents of verbascoside of Poria and Rehmanniae Radix Preparata decoction were decreased?P<0.01?.Conclusions:Compared with the single decoction of Corni Fructus,the contents of morroniside were increased,while the contents of gallic acid,loganin and5-hydroxymethyl furfural were decreased.Compared with the single decoction of Rehmanniae Radix Preparata,the contents of verbascoside were increased.It is speculated that morroniside and verbascoside may be the bioactive compounds of the decoction.Section 3 Separation,purification and structure identification of the chemical constituents of the combined decoction of Corni Fructus/Rehmanniae Radix Preparata/PoriaObjective:To separate the constituents from the combined decoction and identify the structure of the compounds.Methods:The n-butanol extraction of the decoction was eluted with 30%,60%and95%ethanol respectively by macroporous resin column chromatography.After the 30%ethanol eluent was separated by silica gel column chromatography,the semi-prepared HPLC method was used to getting the compounds.Finally,structures of obtained compounds were elucidated by physicochemical properties and spectral data.Results:Constituents from the combined decoction were isolated and purified,the structures were identified as 7?-O-methylmorroniside,loganin and cornusideConclusions:All the compounds are known compounds,derived from Corni Fructus.