Systematic Analysis Of Danshen Seeds And Its Adulterants

Danshen is root and rhizome of Labiatae plants Salvia miltiorrhiza Bge. There are many other kinds of local substitute of Danshen in Hebei, such as Salvia przewalskii Maxim, Salvia umbratica Hance, Salvia plebeia R. Br. At present the medicinal substances source mainly comes from cultivation, but the origin of species is very confusion. It's necessary to identify seed of Danshen and its adulterants. Since the four kinds of plant belong to the same species, their seeds are similar in exterior morphous characters. We identify them by their Macroscopic Characters, Microscopical Features, Physical-chemical Characters and by molecular biology technology. Through experiments we can prevent planting Adulterants and choose better variety of Danshen. The methods are established. It is not only a basic applying but also a deep study. We can establish standards for identification of Danshen.Part 1 Identification from Macroscopic Characters, Micro scopical Features and Physical-chemical CharactersObjective: To establish identification standards for Salvia miltiorrhiza Bge., Salvia przewalskii Maxim, Salvia umbratica Hance, Salvia plebeia R. Br. 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 long and put them into choral hydrate solution. (2) Tabletting and observation. 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 2 h to 3 h. (3) Transparention: Used mixture of ethanol and xylene (1:1). Changed solutions at every 2 h to 3 h. (4) Put samples in the constant temperature oven with 65℃and added paraffin every 30 min. (5) Embedment and slice up with sliding microtome, the thickness was 12μm. (6) Pasted the slices of samples and melted paraffin. (7) Dyeing: Use Safranine and Fast Green double dyeing. (8) Dehydrated and sealed the slices with neutral gum. 4 Seed coat observation with scanning electron 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 min. Double dehydration in every grade. (3) Fixation: fix materials with Glutaral in 30 min. (4) Observation. 5 Study on the characters of ultraviolet spectrum: Prepare solutions extracted with petroleum benzene in apparatus, Soxhlet's for 3 h, then extracted with acetic ether in apparatus, Soxhlet's for 3 h, last extracted with methanol in apparatus, Soxhlet's for 3 h. (2) Scanning wavelength was 200~400 nm. (3) Results analysis. 6 Identification by TLC characters: (1) Prepare solutions extracted with petroleum benzene in apparatus, Soxhlet's for 3 h, then extracted with acetic ether in apparatus, Soxhlet's for 3 h. (2) Dropped sample solutions on the lamella and spread them out. (3) Coloration, observation and took photos. 7 HPLC: extracted principle from seeds by methanol in apparatus, Soxhlet's. Chose suitable chromatographic column, adjusted the composition, pH and proportion of mobile phase, flow rate and wavelength to separate the component peaks well.Results: 1 Macroscopic identification: The five kinds of seeds were nealy cambiform, seeds of Salvia miltiorrhiza Bge., Salvia umbratica Hance and Salvia przewlskii Maxim, were a little bigger than Salvia plebeia R. Br.. But there had not obvious differences between the four seeds. 2 Preparation of discolored surface section: There were obvious differences in exterior and interior view of seed coat, cellwall of thickening and content of the four seeds. 3 Preparation of the paraffin transverse sections: The seed had obvious difference in heave, seedcase layers and cutin. 4 Seed coat observation with scanning electron microscope: There were differences in characters of hila, circumjacent area of hila and side surface of four seeds when observed with scanning electron microscope. 5 Ultraviolet scanning spectrums identification: The number and position of absorbing peaks and its absorbability were different in the four kinds of acetic ether and methanol extractive. 6 Identification by the TLC characters: There were differences in the seeds of Salvia miltiorrhiza Bge., Salvia umbratica Hance and Salvia przewlskii Maxim, Salvia plebeia R. Br.by TLC. 7 HPLC: There were owned by both absorption band and characteristic absorption band between the four kinds of seed.Conclusion: From differences of appearance characters, microscopical features, physical-chemical characters and ultrastructure of seeds, we can identify Salvia miltiorrhiza Bge., Salvia przewalskii Maxim, Salvia umbratica Hance and Salvia plebeia R. Br. seeds. The methods were simple, convenient, feasible and accurate .Part 2 Cell biology and Molecular Biology Studies Objective: Using chromosome analysis technology and protein PAGE and SDS-PAGE technology distinguish Danshen and its adulterants.Methods: 1 Preparation of root-tip cell chromosome sections: (1) Seeds were sprouted at 25℃, root-tip with 0.5 cm to 1.0 cm long were cut down and treated in distilled water at 4℃for 24 hours. (2) Fixed the root-tips with Carnoy's fixative for 1.0~1.5 h. (3) Decomposed the root-tips with 95% ethanol: hydrochloric acid (1:1) in 10~15 min. (4) Dropped the root-tips into double-distilled water 8~15 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) To prepare sample solutions. (2) To prepare separation gel and concentrating gel. (3) Electrophoresis: constant voltage was 120~180 V. (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 Study on the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) features. (1) To prepare sample solutions. (2) To prepare separation gel and concentrating gel. (3) Electrophoresis: constant voltage was 80~120 V. (4) When electrophoresis was over, fast dyed the albumin gel with coomassie brilliant blue and decolored the gel to clear background. (5) Result analysis: Took photos and drew pictures of the tapes on gel and measured the molecular mass of protein of characteristic tapes at the same time.Results: 1 The differences of root-tip body cell karyotye: Salvia miltiorrhiza Bge. seed's chromosomal total number was 16, length in all was 26.25μm, average length was 1.64μm, variation coefficient was 1.72; brachi-relative value was 1.11~1.74; Salvia przewalskii Maxim seed's chromosomal total number was 32, length in all was 42.88μm, average length was 1.34μm, variation coefficient was 1.60; brachi-relative value was 1.06~1.82; Salvia umbratica Hance seed's chromosomal total number was 16, length in all was 24.94μm, average length was 1.56μm, variation coefficient was 2.03; brachi-relative value was 1.00~2.10; Salvia plebeian R. Br seed's chromosomal total number was 16, length in all was 14.34μm, average length was 0.90μm, variation coefficient was 1.69; brachi-relative value was 1.00~2.09. 2 PAGE feature: From the photos we can find that Salvia miltiorrhiza Bge. seed had 2 primary tapes and 2 secondary tapes; Salvia przewalskii Maxim seed had 2 primary tapes , 1 secondary tape and 1 thirdly tape; Salvia umbratica Hance seed had 2 primary tapes, 3 secondary tapes and 2 thirdly tapes; Salvia plebeia R. Br seed had 1 primary tape, 2 secondary tapes and 1 thirdly tape. There were different tapes between the four kinds of Danshen seeds which can be used in identification. 3 SDS-PAGE feature: From the photos we can find that the molecular mass of protein of Salvia miltiorrhiza Bge. seed were 24.8, 30.4, 37.3, 71.0, 89.7, 143.2 KD; the molecular mass of protein of Salvia przewalskii Maxim seed were 20.2, 22.7, 24.8, 30.4, 37.3, 71.0, 89.7, 143.2 KD; the molecular mass of protein of Salvia umbratica Hance seed were 20.2, 22.7, 24.8, 27.9, 30.4, 37.3, 71.0, 89.7, 143.2 KD; the molecular mass of protein of Salvia plebeia R. Br seed were 24.8, 29.6, 34.2, 37.3, 54.6, 79.8, 89.7, 143.2 KD. There were different the molecular mass of protein between the four kinds of seeds which can be used in identification.Conclusion: From studying on the root tip body cell chromosome number and karyotype characters, PAGE tape feature and SDS-PAGE tape feature, we can distinguish Danshen and its adulterants from cell and molecular level. It is helpful for study of species of Salvia.