Systematic Analysis Of Huangqi Seeds And Its Adulterants

Huangqi is drying roots of pulse family plants Astragalusmembranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiao.And A. membranaceus (Fisch. ) Bge..It is a kind of commonlyused Chinese crude drug which can invigorate qi forstrengthening superficies. The quality of Astragalusmembranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiao. isbetter than A. membranaceus (Fisch. ) Bge. At present thematerials mainly come from cultivation, accordingly the betterseeds is the precondition. But there have seeds of Astragaluscomplanatus R. Br. and Medicago sativa L. for sale in market.So identifying Seeds of Huangqi and its Adulterants isnecessary by its Macroscopic Characters, MicroscopicalFeatures, Physical-chemical Characters and molecular biologytechnology through experiments to prevent plantingAdulterants and choose better variety of Huangqi. The methodsare not only basic but also fast, convenient and feasible.1 Identification from Macroscopic Characters,Microscopical Features and Physical-chemical CharactersObjective: To establish identification standards forHuangqi and its adulterants from their appearance characters,microscopical features and physical-chemical characters withfast, simple, feasible methods.Methods: 1. Macroscopic identification. 2. Preparation ofdiscolored surface section: (1) Cut samples into 1 mm to 2 mmlong and put them into choral hydrate solution. (2) Selectedmaterials pressed them into sections and sealed with neutralgum. 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 at every 2to 3h. (3) Transparention: Used mixture of ethanol and xylene(1:1), xylene. Change solutions once every 2 to 3 h. (4) Putsamples in the constant temperature oven with 65 ℃and addedparaffin every 30 min. (5) Embedment and slice up with slidingmicrotome, the thickness was 10 to 15μm. (6) Pasted the slicesof samples and melted paraffin. (7) Dyeing: Use Safranine andFast Green double dyeing. (8) Dehydrated and sealed the sliceswith neutral gum. 4. Seed coat observation with scanningelectron microscope. (1) Cleaning: Seeds were cleaned with 50% ethanol and ultrasonic cleaning instrument. (2) Dehydration:The concentration of ethanol solutions were 50 % ,60 % ,70 %,80 % ,90 % ,100 % and changed solutions at every 30 minutes.Double dehydration in every grade. (3) Fixation: fix materialswith Glutaral in 30 minutes. (4) Observation. 5. Fluorescenceanalysis: (1) Seeds were crushed to pieces and extracted withalcohol and ultrasonic cleaning instrument. (2) To observeunder uviol lamp. 6. Study on the characters of ultravioletspectrum: (1) Prepare solutions with two methods: One is toextract with alcohol then use saturate n-butyl alcohol to extractagain; the other is to extract with petroleum benzine inapparatus, Soxhlet's. (2) Scanning wavelength was 200-400 nm.(3) Results analysis. 7. Identification by TLC characters: (1)Prepared the sample solutions. (2) Drop sample solutions onthe lamella and spread them out. (3) coloration, observationand took photos.Results: 1. Macroscopic identification: The four kinds ofseeds were like kidney. seeds of Huangqi were bigger than itsadulterants . But there had not obvious differences between thetwo kind of Huangqi seeds. 2. Preparation of discolored surfacesection: There were obvious differences in exterior and interiorview of seed coats. 3. Preparation of the paraffin transversesections: bar stone cells of Huangqi were longer than itsadulterants and cell wall of Huangqi had obviously thickened.Alfalfa had characteristic serration cutin and light line. 4. Seedcoat observation with scanning electron microscope: Therewere differences in characters of hila, circumjacent area of hilaand side surface of Huangqi and its adulterants seeds whenobserved with scanning electron microscope. 5. Fluorescenceanalysis: Under uviol lamp,each extract of seeds had differentfluorescence:Extract of Astragalus membranaceus (Fisch.) Bge.appeared yellow-white, extract of A. membranaceus (Fisch.)Bge. Var. mongholicus (Bge.) Hsiao Appeared reddish yellow,extract of A.stragalus complanatus R. Br. Appeared crocus,extract of Medicago sativa L. appeared blue-white. 6.Ultraviolet scanning spectrums identification: The number andposition of absorbing peaks and its absorbability were differentof the four kinds of extract. 7. Identification by the TLCcharacters: Materials had different spots when Rf value is0.04,0.42 and 0.45.Conclusion: from differences of appearance characters,microscopical features and physical-chemical characters ofseeds, we can distinguish not only seeds of Huangqi from itsadulterants but also the two kinds of Huangqi seeds. Themethods were simple, convenient, feasible and accurate andcan be used for identification of seeds of Huangqi and itsadulterants.2 Cell biology and Molecular Biology StudiesObjective: Using chromosome analysis technology,protein PAGE and RAPD technology distinguish two kinds ofHuangqi and its adulterants .Methods: 1. Preparation of root-tip cell chromosomesections: (1) Seeds were sprouted at 25 ℃, root-tip when it is1.5 cm to 2 cm long were cut down and treated in solution of0.03% colchicines diluted with 0.002mol/L 8-hydroxyquinolinefor 2-6 hours. (2) Fixed the root-tips with Carnoy's fixative. (3)Decomposed the root-tips with 1 mol/L hydrochloric acid in5-10 minutes at 60 ℃. (4) Dropped the root-tips intodouble-distilled water 5-15 minutes. (5) Dyed the root-tips inalkaline Carmine solution. (6) Pressed the root-tips intosections, unclose the cover at frozen condition and sealed withneutral gum. (7) Take photos with microscope, counted thenumber and measured the length of chromosome. 2. Study onthe polyacrylamide gel electrophoresis (PAGE) features. (1) Toprepare sample solutions. (2) To prepare separation gel andconcentrating gel. (3) Electrophoresis: constant voltage was120-180 V. (4) When electrophoresis was over, dyed the gelwith coomassie brilliant blue R-250 and decolored the gel toclear background. (5) Result analysis: Took photos and drewpictures of the tapes on gel and measure the move rates ofcharacteristic tapes at the same time. 3. RAPD technologyanalysis. (1) Extract and purify total DNA from seedling ofHuangqi. (2) Experiment of PCR and primer selecting. (3)DNA map analysis.Results: 1. The differences of root-tip body cell karyotype:A. membranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiao.seed's chromosomal total number was 16, length in all was35.6 μm, average length was 4.45μm, variation coefficient was1.53; Astragalus complanatus R. Br. seed's chromosomal totalnumber was 16, length in all was 33.04 μm, average length was4.13 μm, variation coefficient was 3.04. 2. PAGE feature: Fromthe photos we can find Astragalus membranaceus (Fisch. ) Bge.seed had 10 primary tapes and 3 secondary tapes; A.membranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiaoseed had 14 primary tapes and 5 secondary tapes; Astragaluscomplanatus R. Br. seed had 8 primary tapes and 3 secondarytapes; Medicago sativa L. seed had 11 primary tapes and 2secondary tapes. Seeds of Huangqi and its adulterants wereeasy to distinguish ,There were also 14 different tapes betweenthe two kinds of Huangqi seeds which can be used inidentification of the two types. 3. RAPD technology analysis:With the amplification of primer S4(GGACTGGAGT), twosample got 15 DNA band. the differences were that Astragalusmembranaceus (Fisch. ) Bge. Had one relucent band but A.membranaceus (Fisch.) Bge. Var. mongholicus (Bge.) Hsiaohad not the band at position where molecular weight was about1300. With the amplification of primer S27(GAAACGGGTG)two sample got 7 DNA band. the difference was that A.membranaceus (Fisch.) Bge. Var. mongholicus (Bge.) HsiaoHad one relucent band but Astragalus membranaceus (Fisch. )Bge. had not the band at position where molecular weight isabout 1200. it can differentiate the two kinds of Huangqi.Conclusion: From studying on the root tip body cellchromosome number and karyotype characters, PAGE tapefeature and RAPD map, we can distinguish Huangqi and itsadulterants from cell and molecular level. what's more, we can...