| Dendrobium officinale is the fresh or dried stem of Dendrobium officinale Kimura et Migo, which is a kind of perennial herbaceous of Orchidaceae. It’s mainly processed in two methods, one is heating while twisting into a spiral or the spring like, named "Tiepi Fengdou", another is cut the stem into sections while fresh, heat or low temperature drying, named "straight Fengdou".In order to improve the methods of true and false identification of Dendrobium officinale and prefect the current quality control system, this study systematically study the quality standard system,through the following five chapters of research work.Chapter 1 Documents about the research progresses in bencaological study, pharmacognostical study, chemical constituents and detect methods study, pharmacological activites study and the history and current situation of Dendrobium officinale in last twenty years were summed up and analyzed by cite70 referencesChapter 2 Investigate the ecology environment situationof Dendrobium officinale and collected the 129 samples of Dendrobium officinale from 12 provinces in China and 11 species 23 affinitive Dendrobium SW common.Chapter 3 Study on the identification of origin and perfective of standard.The origin of Dendrobium officinale samples andcommon affinitive Dendrobium SW samples were identified by synthetically analyzes of phytomorphology, molecular identification by internal transcribed spacer Ⅱ (ITS2) DNA barcoding, morphology and microscopic characteristics.DNA barcode offer a believable proof in identification of origin. Through the improvement and purification of the protein extraction by 3 steps, add a quantity of 10% PVP to sample and grinding together in the crushing step; adjust the dissociation condition to 56 degrees 8-10 hours, using chloroform-isoamyl alcohol(24:1) to precipitate protein, the total DNA is purity enough for PCR and sequencing accurate. The origin of Dendrobium officinale are the same genus and the origin of affinitive Dendrobium SW are identification accurate.Complement the description of the current starndard of Dendrobium officinale. The whole stem of straight Fengdou long 20-40 cm, diameter 0.2-0.3 cm, slightly wavy, length of internodes 0.5-1.8cm, with ash white membranous sheath on the stipes, gray green section. The color of the stem is divided into two categories by green and red.The green stem is dark green with brown spots or not on the surface of the stem and membranous sheath. The red stemis purple green to purple red, with dense brown spots on the surface of the stem and the membranous sheath.The internodeslength of Tiepi Fengdou is 0.5-1.8cm while pull straight.The sample cross section was dyed by benzene III phenol solution, epidermal cells 1 layer was dyed parenchyma cells was not dyed, columns of fiber bundle cells around the vascular bundle has been dyed. The 3 kinds characters can distinguish some of Dendrobium SW, such as D. chrysanthum, D. crepidatwn, D. nobile, D. Devonianum.Chapter 4 Study on the compands and improve and perfect the current standard.TLC method based on low polar component parts was set up to suitable for represent the Dendrobium officinale and to distinguish the common affinitive Dendrobium SW. Method verification proved to be good operability by application, durability, repeatability. The method is the powder was abstract by chloroform-methanol (9:1), ultrasonic treatment for 20 minutes, filtration liquid is the test solution. Use Dendrobium officinale reference drug the same manner as the reference drug solution. Using silica G as the coaing substance, and a mixture of toluene-ethyl formate-formic acid (6:3:1) as the mobile phase, spraying 10% sulfuric acid ethanol solution,95 degrees heat for 3 minutes, detected under the UV (365nm). The spots in the chromatogram obtained with the test solution correspond in position and color to thefluorescent spots obtained with the reference drug solution.Chromatography (GC) methods has been set up to detect and determine the distribute of dendrobine in Dendrobium officinale and the common affinitive Dendrobium SW. The results showed that 60 batches of Dendrobium officinale is dendrobineis negative, and positive in 6 affinitive species. They’re D. nobile, D. Chrysotoxum, D. brymerianum. D. crystallinum, D. crepidatum. The detetion limit of dendrobine is l.Ong. Follow this result, dendrobine is used as marker compound to discern the false from the genuine Dendrobium officinale by GC. The method is the powder is abstract by ammonia and chloroform, ultrasonic 30min, the supernatant is the test solution. GC conditions is using DB-1 Silica column, temperature program is first temperature 80℃, heating rate is 20℃per minute to 218 degrees, hold for 3 minutes, heating rate is 20℃per minute to 250℃, after 5 minutes, the inlet temperature was 250℃, detector’s temperature was 250℃. Method validation results showed good accuracy and repeatability.linear equation:The calibration curve are linear at ranges of 0.95 ng-74.6 ng, R=0.999, (n=8), Recory of Dendrobium officinaleis is 100.75%, RSD=2.29%, Recory of Dendrobium nobil is 96.2%, RSD=1.14%.Content of extract was dected by hot dip process.with alcohol, content of polysaccharide was determined by phenol-sulphuric acid method, content of seminose and the rate of seminose and glucose were dectected by HPLC in 114 Dendrobium officinale and 20 common affinitive Dendrobium SW.The result is that the content of extract, polysaccharide, and seminose were variation between 12 provinces, polysaccharide and seminose were positive correlation. The change of extract is not significant with the growth time. Polysaccharide and seminose are reach the highest in 2 growth year sample. The rate of seminose and glucose is found stable in different place and growth year samples, but significant variety in different harvest time. So the best harvest time of Yunnan is between December to the next February of a year.The content of Pb, Cd, As, Hg and Cu are five common heavy metals and hazardous elements that harm human body. It wil be overtaken than standard dose when Dendrobium Stem use as health products. microwave digestion and ICP-MS determination method was established to determine the contents of Pb, Cd, As, Hg and Cu in 20 Dendrobium Stem samples from 12 producing areas, and were evaluate refer the foreign and the domestic relevant standards are within the range of drug safety.Chater 5 This study systematically study the Dendrobium quality standard system by the study of the origin identification and component analysis. To established and perfected a number of authenticity research methods of Dendrobium officinale in quality control system, and accumulated a large amount of experimental data for Dendrobium officinale forward research.The main innovation:1. Improve the TLC of Dendrobium Stem in current quality standard.2. Detect method was established by gas chromatography and found Dendrobium Stem dendrobine negative, identify Dendrobium Stem by using dendrobine as reference.3. Perfect a series of description and microscopic identification characteristics of Dendrobium Stem.4. Improved extraction method of the total DNA in sequencing procedure.
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