The Large Leaves ÉX Antidepressant Effective Part Of The Preparation Process, Quality Standards For Research And Big-leaf ÉX Volatile Oils

Piper laetispicum C.DC.,known as "Da Ye Ju" in China,is one of the species in Paperaceae family used in Chinese folk medicine,being administered for alleviating the symptoms of rheumatism.P.laetispicum primarily grows in Guangdong,Guangxi, Yunnan and Hainan provinces.Based on our previous practice,acetic ether extracts of alcohol extraction have been identified for antidepressant activity.In addition, laetispicine is the major effective ingredient.This paper lays strong foundations for new drugs application of P.laetispicum (The 5^th Category:Effective Parts Extracted from Natural Medicines),including 2 parts:drug preparation & quality specification.Simultaneously,we find there is a great quantity of oil in alcohol extracts.To be specific,the research works consist of 1).To separate the chemicals of effective sites for the following works.2).To establish assay determination of total amides.3).To optimize the process of extraction and purification.4).To assay of five Amide Compounds.5).To compare the essential oil of the different parts of P. laetispicum.1.Preparation of AmidesFive amides are obtained by column chromatography and preparative HPLC and identified by spectral data(MS,¹H-NMR,and ¹³C-NMR),which are N-isobutyl-(3',4'-methylenedioxyphenyl)-2E,4E-heptadienamide,laetispiamide A, laetispiamide B,laetispicine and Dihydropipercide separately.2.Assay Determination of Total AmidesFor the first time,the determination of total amides is investigated by UV Spectrophotometry & Kai-Nitrogen Determination Method.Both of them are available.3.Process of Extraction and Purification.A process of extraction and purification of antideprensant fraction is established for the first time.The content of total amides is up to 58%by Kai-Nitrogen Determination Method. The observation of the extracted term of the effective fraction:Effects on extraction rate of total alkaloids are examined,and the factors such as ethanol (extraction solvent) concentration,extraction time and ethanol volume are selected. The optimum extraction conditions are as follows:solvent,70%ethanol;ethanol volume,12-folds of herbs;Time,30 min for three times.Purification of total alkaloids:the processes which offere the highest total alkaloids contents are screened.The optimal conditions:100 mg/mL solution prepared with extracts & distilled water,3 times petroleum ether to degrease(V/V),ethyl acetate:water(4:1,V/V),solvent is evaporated at low pressure.4.Assay of Five Amide CompoundsHigh Performance Liquid Chromatography(HPLC) method is set up to determine N- isobutyl-(Y,4'-methylenedioxyphenyl)-2E,4E- heptadienamide, laetispiamide A,laetispiamide B,laetispicine and Dihydropipercide simutanously for the first time.This method lays a certain foudation for the development of the fingerprint of P.laetispicum.The running conditions:methanol/water=77/23(V/V);flow rate:1.0 mL/min; absorbance:259 nm.It's the first time to do the qualitative and quantitative research in five kinds of indicators of the initial composition.3 batches of P.laetispicum are determined for all amides compounds in different nutrient organs.Among them,fruits have the highest laetispicine,stems follow,and the roots are in the highest laetispiamide A(about the plant's 1.1‰0.2‰and 1.6‰).Laetispicine proposed for the first time to be a characteristic element of P. laetispicum,from the same family(Piper).Five- alkaloids contents of other plants (Piper) are also examined.No laetispicine,laetispiamide A,laetispiamide B is found. Furthermore,laetispicine isn't interfered with the other components.5.Essential Oil Composition of the different parts of P.laetispicumThe essential oils from the different parts(roots,stems,leaves,and fruits) of P. laetispicum and the fruits of Piper nigrum Linn.have been extracted by steam distillation and analyzed by gas chromatography coupled to mass spectrometry (GC-MS).The major components of the volatile oil are compared.189 constituents are identified(roots,64;stems,93;leaves,87;fruits,58;pepper,42),which all represent above 90%.Camphene,1,4(8)-p-Menthadiene,α-Cubebene,trans-Caryophyllene(roots 9.29%;stems 12.54%;leaves 13.45%;fruits 2.60%in P.laetispicum and 29.11%in P. nigrum).Copaene,Guaia-1(5),11-diene and Cadina-1(10),4-diene are owned by each part of P.laetispicum and P.nigrum.