Genetic Identification Of Dendrobium Officinale By SCoT Markers And Its Preparation And Pharmacodynamics Of Compound Hypoglycemic Capsules

ObjectiveTo a new kind of Dendrobium officinale Kimura et Migo with high content of polysaccharides,genetic polymorphism and cluster analysis of 11 Dendrobium species were performed using the target start codon（SCoT）molecular marker technique.Then the quality standard for molecular identification of these Dendrobium species was established.The D.officinale,as the king drug,in addition to other Chinese medicinal herbs such as Pueraria lobata,Schisandra chinensis and Astragalus membranaceus,were conducted to technological studies and prepared D.officinale assisted hypoglycemic capsules,then to establish the quality standards of particle preparations.According to the requirements of SFDA health care products,to conduct the studies of assisted hypoglycemic pharmacodynamic.Methods1.Genetic diversity,population structure and identification of D.officinale‘Ruishen No.2’using molecular markersThe target start codon（SCoT）molecular marker technique was used to analyze 11Dendrobium cultivars.The PCR reaction system and amplifications were optimized;36SCoT primers were screened and their annealing temperature was optimized;Genetic diversity and cluster analysis were conducted among 11 Dendrobium cultivars by SCoT;SCoT-SCAR marker and SCoT-Nested PCR marker were used,which were used as a tool for identifying the D.officinale‘Ruishen No.2’.2.The preparation technology of D.officinale compound capsuleOrthogonal experimental design method was used to optimize the extraction process with the content of puerarin,schisandrin,astragaloside and extract yield as comprehensive evaluation indexes;adopting wet granulation,and with the angle of repose,molding rate,and bulk density of the particles as the evaluation to Single factor optimize prescription formulation process and prepare D.officinale compound capsule.3.Study on Hypoglycemic Effect of Compound Capsules from D.officinaleDiabetic rat model was induced by streptozotocin（28 mg/kg）plus full high-fat diet for 3 weeks.The rats were divided into normal control group（constant volume of normal saline）,model group（constant volume of normal saline）,metformin hydrochloride group and compound capsules high-dose,medium-dose and low-dose groups（1.0,0.5,0.25g/kg）.They were given relevant medicine intragastrically for consecutive 28 days.Body weight was recorded weekly.Fasting blood glucose were determined at 4th weekend of intragastrical administration.The sugar tolerance test was carried out at the end phase of experiment.The levels of total cholesterol and blood serum insulin were determined.Results1.SCoT-PCR analysisThe optimal SCoT-PCR reaction system（20μL）was established:1×buffer（20 mM Tris（pH 8.0）,100 mM KCl,0.1 mM ethylenediaminetetraacetic acid（EDTA）and 1.0 mM dithiothreitol）,20 ng DNA template,2.0 mM Mg²⁺,200μM of each of the four deoxynucleotides,1.0 U Ex Taq DNA polymerase and 0.5μM primer.Amplification reactions were performed with the following steps:pre-denaturation at 94℃ for 3 min;35cycles of denaturation at 94℃ for 1 min,annealing at 52℃ for 1 min;elongation at 72℃ for 2 min;and a final elongation at 72℃ for 10 min.The optimum reaction system was further verified in 11 Dendrobium materials,which showed good stability and repeatability.2.Genetic diversity and cluster analysisUsing start codon targeted（SCoT）polymorphism molecular markers to analyse eleven Dendrobium cultivars.Using 13 selected SCoT primers,181 bands were generated,157（86.86%）of which were polymorphic.The clustering results showed that D.officinale cultivars of different cultivars were clustered together,but the other cultivars were differentiated,indicating that the SCoT marker has the characteristics of rich polymorphism and strong specific amplification ability.It is suitable for the identification of D.officinale germplasm.3.Two combinations of SCoT-SCAR and SCoT-Nested PCR markersThe SCoT-nested PCR markers indicated that the primer SC1,SC2,and SC3 could distinguish D.officinale‘Ruishen No.2’from the other cultivars;however,the SCAR primers amplified twenty-seven distinct bands in total,but not in the other two varieties（D.chrysotoxum and D.heterocarpum）,suggesting that the SCoT-SCAR marker cannot be used specifically for the identification of D.officinale‘Ruishen No.2’,but to distinguish between D.chrysotoxum,D.heterocarpum and other nine species.4.The quality standard for molecular identification of‘Long tou feng wei’D.officinale‘Ruishen No.2’（1）Extraction and examination of total genomic DNAGenomic DNA was isolated from the seedlings according to the manufacturer’s instructions for the Plant Genomic DNA Kit（DP305）.Subsequently,the concentration and purity of the extracted DNA were examined using an Ultramicro UV spectrophotometer,and the OD260/OD280 and OD260/OD230 ratios were calculated(A₂₆₀/A₂₈₀=1.8-2.0;A₂₆₀/A₂₃₀≥2.0).The integrity of the extracted DNA was examined by 0.8%agarose gel electrophoresis.After adjusting the DNA concentration to 20 ng/μL,the DNA samples were stored at-20℃ for future use.（2）Amplification of SCoT-PCR（the first time）The genomic DNA were amplified by PCR using the primers S8（5’-CAACAATGGCTACCACGT-3’）,The total reaction volume of 20μL contained 1×buffer（20 mM Tris（pH 8.0）,100 mM KCl,0.1 mM ethylenediaminetetraacetic acid（EDTA）and 1.0 mM dithiothreitol）,20 ng DNA template,2.0 mM Mg²⁺,200μM of each of the four deoxynucleotides,1.0 U Ex Taq DNA polymerase and 0.5μM primer.Amplification reactions were performed using a 96-well Arktik^TM thermal cycler with the following steps:pre-denaturation at 94℃ for 3 min;35 cycles of denaturation at 94℃ for1 min,annealing at 52℃ for 1 min;elongation at 72℃ for 2 min;and a final elongation at 72℃ for 10 min.The primary SCoT-PCRs（concentration of about 400-500 ng/μL）,which were diluted 1000-fold（concentration of about 0.4-0.5 ng/μL）,were used as templates to conduct a nested PCR.（3）Amplification of SCoT-Nested PCR（the second time）The primary SCoT-PCRs,diluted 1000-fold,were used as templates to conduct a nested PCR using the optional 1 pair of specific primers:Primer SC1（523 bp）:Upstream（5’-TGGCTACCACGTGATAGATGT-3’）Downstream（5’-GTTTCTTAGGAGGAATTGCTCAG-3’）Primer SC2（1017 bp）:Upstream（5’-TCCAGCGGAAAGGGAGATAC-3’）Downstream（5’-ACTTTCCCTCCCAAGCTGAG-3’）Primer SC3（1178 bp）:Upstream（5’-CTCGCCATATCGTCGAGCA-3’）Downstream（5’-ACGTTCTGTTATGGGGTCCT-3’）The 20-μL nested PCR system contained 10μL Ex Taq PCR Master Mix,2μL 2.5mM of each SCAR primer,1μL templates and double-distilled water.The PCR was performed using a 96-well Arktik^<sup>TMthermal cycler,with an initial pre-denaturation for 4min at 94℃ followed by 30 cycles of denaturation at 94℃ for 50 s,annealing at 58℃ for 45 s,and extension at 72℃ for 60 s.The final extension step was performed at 72℃ for 8 min.A 2-μL aliquot of 6×loading buffer was added to the samples,and the amplicons were separated in 1%agarose gel,stained with SYBR （?） Safe DNA Gel Stain,The amplicons were visualized and photographed using a gel documentation system.（4）Result determinationIf the clear target bands were amplified at the corresponding size in the SCoT-Nested PCR,the seedlings can be further cultivated;otherwise,the mutant seedlings are removed.5.The preparation process of D.officinale compound capsule:All the three Chinese medicines are mixed together with ten times 30%ethanol and decocted three times where each period is sustauned for 1 hour.The decoctions are combined and filtered.The decoctions are concentrated under reduced pressure and vacuum-dried.D.officinale ultrafine powder is added and mixed,wet granulation.The granules are weted and prepared with 70%ethanol,and being drying for about 2 hours at50-60℃passing the dried granules.And then he granules are sized through a 20-mesh sieve,and directly filling the capsules.The pellet forming rate was 96.15%,the angle of repose a was 35.21°,and the bulk density was 0.2758.All of the items are in compliance with the requirements.6.Study on Hypoglycemic Effect of Compound Capsules from D.officinaleFour weeks after the administration,compared with normal control group,the level of blood glucose,the area under curve of the blood glucose,the content of TC was increased significantly in model group（P<0.01）;the serum insulin was decreased significantly（P<0.01）.Compared with model group,the level of blood glucose,the area under curve of the blood glucose,the content of TC were decreased significantly（P<0.05）,while the serum insulin and the body weight of mice were increased significantly（P<0.05）in compound capsules high-dose and medium-dose groups.The results showed that high-dose and medium-dose of compound Capsules from D.officinale decreases the level of blood glucose on streptozotocin plus high-fat diet-induced insulin resistance diabetes rats of sugar/lipid metabolic disorder,which can increase insulin secretion,decrease serum cholesterol content.ConclusionGenetic diversity and cluster analysis were conducted among 11 Dendrobium cultivars by SCoT;SCoT-SCAR marker and SCoT-Nested PCR marker were used,which were used as a tool for identifying the D.officinale‘Ruishen No.2’.Then the quality standard for molecular identification of these Dendrobium species was established.Orthogonal experimental design and Single factor method was used to determine the extraction process.The efficacy test showed that compound Capsules from D.officinale has hypoglycemic effect and can be developed as an auxiliary hypoglycemic functional food.This study can improve the quality standards of this species of Dendrobium,and its preparation products have a good hypoglycemic effect.