Systematic Analysis Of Bupleurum, Seeds And Its Adulterants

Bupleurum, a kind of commonly used medicine, was first described in "Shennong's Herbal". Two kinds of Bupleurum, Bupleurum chinese DC and B. scorzonerifolium Willd. have been recorded in Pharmacopoeia 2005 version. However, according to historical records and field surveys, the majority of Bupleurum species are used for medicine and several species are always mixed in the origin place of growth. As the species and chemical composition of Bupleurum are confused and the content of active ingredients is different significantly, the quality of Bupleurum and its products is instable. The Bupleurum is one of medicinal substances in Hebei province, where mainly distribute Bupleurum chinese, Yantai Bupleurum, Baihuashan Bupleurum, Xing'an Bupleurum, Red Bupleurum and Black Bupleurum etc. Forthermore, Sandao Bupleurum is planted in our province in large area. To ensure the stability of germplasm resources, increase the controllability of medicine resources and provide a reliable basis for a standardized planting in large scale, this experiment made a comprehensive identification on several species of Bupleurum seeds by using traditional identification methods, techniques of cell biology and molecular biology. The experimental method is rapid, simple and practical, which can meet the primary needs and settle scientific basis for variety identification, development and application of the Bupleurum seeds.Part 1 Identification from Macroscopic Characters, Micro- scopical Features and Physical-chemical Characters Objective: To establish identification standards for Chaihu and its adulterants from their appearance characters, microscopical features and physical-chemical characters with fast, simple, feasible methods.Methods: 1 Macroscopic identification. 2 Preparation of discolored surface section: (1) Cut samples into 1 mm to 2 mm length and put them into choral hydrate solution. (2) Selected materials pressed them into sections and sealed with neutral gum. 3 Preparation of the paraffin transverse sections: (1) Selected samples and fix them with F.A.A. fixative liquid. (2) Dehydration: the concentration of ethanol solutions were 70 %, 80 % ,90 %,95 %,100 %,and changed solutions every 2 or 3h. (3) Transparention: immersed the seeds into the mixture of ethanol and xylene (1:1/v:v) for 1h, and then xylene until the seeds appear transparent. (4) Put samples in the oven with constant temperature of 65℃and added paraffin every 30 min. (5) Embedded and sliced up with sliding microtome, the thickness was 10 to 15μm. (6) Pasted the slices of samples and melted paraffin. (7) Dyeing: Used Safranine and Fast Green for double dying. (8) Dehydrated and sealed the slices with neutral gum. 4 Seed coat observation with scanning electron micros- cope. (1) Cleaning: seeds were cleaned with 50 % ethanol and ultrasonic cleaning instrument. (2) Dehydration: the concentra- tion of ethanol solutions were 50 %, 60 %, 70 % , 80 %, 90 %, 100 % and changed solutions at every 30 min. Double dehydrate in every grade. (3) Fixation: fix materials with Glutaral for 30 min. (4) Observation. 5 Study on the characters of ultraviolet spectrum: (1) Prepared solutions with the method: to extract with petroleum benzine in apparatus, Soxhlet's,then saturate Ethyl acetate,at last absolute methanol. (2) Scanning wavelength was 200~400 nm. (3) Results analysis. 6 Identifica- tion by TLC characters:(1) Prepared the sample solutions. (2) Dropped sample solutions on the lamella and spread them out. (3) colored, observed and took photos.Results: 1 Macroscopic identification: The Bupleurum seeds were carpadelium, brachy-circularcylinder, and schizoca rp oblong. The schizocarp is nearly oblong, and its tip remains yellowish-brown excrescent stylopodium. There were petty foot stalk with five edges on the base. But there were not obvious differences among the seven seeds of their entirety characters. 2 Preparation of discolored surface section: There were not obvious differences on carpodermis, inclusion, stoma type and tubing cell shape etc. 3 Preparation of the paraffin transverse sections: The seeds had obvious differences in seedcase layers, the number of tubing and its diameter. 4 Seed coat observation with scanning electron microscope: There were some differences in characters of hila, surface of Bupleur- um seeds and its adulterants when observed with scanning electron microscope. 5 Ultraviolet spectrums identification: The number and position of absorbing peaks and its absorbability were different in the four kinds of extract. 6 Identification by the TLC characters: Materials had different spots.Conclusion: From the differences of appearance characters, microscopical features and physical-chemical characters of seeds, we can identify the Bupleurum seeds. The methods were simple, convenient, feasible and accurate and can be used for identification of Bupleurum seeds.Part 2 Cell biology and Molecular Biology Studies Objective: Using chromosome analysis technology, protein PAGE and RAPD technology to distinguish two kinds of Bupleurum and its adulterants.Methods: 1 Preparation of root-tip cell chromosome sections: (1) Seeds were sprouted at 25℃. When the root-tip was 1.5 cm to 2 cm long, cut down and treated in distilled water at 4℃for 24 h. (2) Fixed the root-tips with Carnoy's fixative. (3) Decomposed the root-tips with 1 mol/L hydrochloric acid for 5~10 min at 60℃. (4) Dropped the root-tips into double-distilled water for 5~15 min. (5) Dyed the root-tips in alkaline Carmine solution. (6) Pressed the root-tips into sections, unclosed the cover at frozen condition and sealed with neutral gum. (7) Took photos with microscope, counted the number and measured the length of chromosome. 2 Study on the polyacrylamide gel electrophoresis (PAGE) features. (1) Prepared sample solutions. (2) Prepared separation gel and concentrating gel. (3) Electrophoresis: constant voltage was 120~180V. (4) When electrophoresis was over, dyed the gel with coomassie brilliant blue R-250 and decolored the gel to clear background. (5) Result analysis: took photos and drew pictures of the tapes on gel and measured the move rates of characteristic tapes at the same time. 3 RAPD technology analysis. (1) Extract and purify total DNA from seedling of Bupleurum. (2) Experiment of PCR and primer selecting. (3) DNA map analysis.Results: 1 The differences of root-tip body cell karyotype: Bupleurum seed's and its var. chromosomal total number is 2n=12, while B.falcatum L. chromosomal total number is 2n=26, B.sibiricum Vest. chromosomal total number is 2n=64. B.scorzonerifolum Willd. chromosomal total number is 2n=12, B.smithii Wolff. chromosomal total number is 2n=12. The seeds chromosome has obvious differences in average length, variation coefficient .2 PAGE feature: Bupleurum seeds and its var. have little difference in the band number and position. There were different tapes between the five kinds of Bupleurum seeds which can be used in identification. 3 RAPD technology analysis: we filtrated ten effective primers for identification. With the amplification of the primers, two samples were different not only in the number of bands but also in the position of the bands. It can differentiate the five kinds of Bupleurum.Conclusion: From studying on the root tip body cell chromosome number and karyotype characters, PAGE tape feature and RAPD map, we can distinguish Bupleurum and its adulterants from cell and molecular level. It is helpful for study on species of Bupleurum.