Mechanism of Dendrobium huoshanense Polysaccharide Against Neuroinflammation in Parkinson's Disease Model： Based on NLRP3 Inflammasome / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Dendrobium huoshanense polysaccharide （DHP） against inflammatory damage of neurons in Parkinson's disease （PD） model. MethodSH-SY5Y cells were randomized into blank group， model group， and DHP group. The survival rate of cells was measured by thiazole blue（MTT） assay， and the levels of lactate dehydrogenase （LDH）， reactive oxygen species （ROS）， malondialdehyde （MDA）， and superoxide dismutase （SOD） were measured by colorimetric analysis. BV-2 microglia were classified into blank group， model group， DHP group， and MCC950 group （positive control group）， and enzyme-linked immunosorbent assay （ELISA） was applied to detect the levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and interleukin-18 （IL-18）. The expression of NOD-like receptor protein 3 （NLRP3）， adaptor protein apoptosis-associated dot protein （ASC）， cysteine aspartic protease-1 （Caspase-1）， and IL-1β was measured by Western blot. A total of 50 C57BL/6 mice were randomized into blank group， model group， DHP low-dose （100 mg·kg-1） group， DHP equivalent-dose （350 mg·kg-1） group， and MCC950 group （positive control group）， 10 mice in each group. The motor balance and coordination of C57BL/6 mice were observed by beam walking test， tail suspension test and rotarod test. The levels of Iba-1 and tyrosine hydroxylase （TH） were detected by immunofluorescence staining. The damage of dopaminergic neurons in the substantia nigra was detected by FJB staining. The levels of inflammatory factors such as IL-1β， IL-18， and TNF-α in mouse midbrain tissues were detected by ELISA and the protein levels of NLRP3， ASC， Caspase-1， and IL-1β protein were measured by Western blot. ResultCompared with the blank group， the SH-SY5Y model group showed decreased cell survival， increased levels of LDH， ROS， and MDA （P<0.05）， and decreased levels of SOD （P<0.05）. Compared with the model group， the DHP group demonstrated increased cell survival， decreased levels of LDH， ROS， and MDA （P<0.01）， and increased level of SOD （P<0.01）. Compared with the blank group， BV-2 model group had high levels of IL-1β， IL-18， and TNF-α （P<0.05） and high protein expression of NLRP3， Caspase-1， IL-1β， and ASC （P<0.05）. Compared with the model group， DHP and MCC950 groups demonstrated low levels of IL-1β， IL-18， and TNF-α （P<0.01） and low protein expression of NLRP3， Caspase-1， IL-1β， and ASC （P<0.01）. Compared with the blank group， the C57BL/6 model group displayed long time to pass the balance wood （P<0.05）， short time spent on the rod in the rotarod test （P<0.05）， high levels of IL-1β， IL-18， and TNF-α （P<0.05） and expression of Iba-1 in the midbrain substantia nigra （P<0.05）， low TH expression （P<0.05）， more positive neurons in the FJB staining （P<0.05）， and high expression of NLRP3， Caspase-1， ASC， and IL-1β proteins （P < 0.05）. Compared with the model group， the mice in the DHP and MCC950 groups had short time to pass the balance beam （P<0.01）， long time spent on the rod （P<0.01）， low levels of IL-1β， IL-18， and TNF-α （P<0.01）， low Iba-1 expression in midbrain substantia nigra （P<0.01）， high TH expression （P<0.01）， and small number of positive neurons in the midbrain substantia nigra （P<0.01）. The expression of NLRP3， ASC， and IL-1β proteins was lower in the MCC950 group （P<0.01）， and the expression of NLRP3， ASC， Caspase-1 and IL-1β proteins was lower in the DHP equivalent-dose group （P<0.01） than in the model group. ConclusionDHP has anti-oxidative stress effect. It regulates the expression of NLRP3 inflammasome and inhibits the overactivation of microglia， thereby alleviating the neuroinflammatory injury in PD and exerting the neuroprotective effect.

Mechanism of Dendrobium huoshanense in Treatment of Gastric Ulcer： Based on Network Pharmacology and in Vivo Experiment / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Dendrobium huoshanense in the treatment of gastric ulcer （GU） based on network pharmacology and in vivo experiment. MethodThe active components of D. huoshanense were searched from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） and literature， and the targets of the components were screened from TCMSP and SwissTargetPrediction. GU-related genes were retrieved from GeneCards， Online Mendelian Inheritance in Man （OMIM）， and DisGeNET. Thereby， the common targets of the disease and the medicinal were yielded and the protein-protein interaction （PPI） network was constructed， followed by Gene Ontology （GO） term enrichment and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment. According to the predicted results， hematoxylin-eosin （HE） staining， immunohistochemistry， enzyme-linked immunosorbent assay （ELISA）， Western blot， and real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） were used to validate the effects of D. huoshanense on acetic acid-induced GU in rats. ResultA total of 63 active components of D. huoshanense and 37 target genes of D. huoshanense for the treatment of GU were screened out. PPI network analysis yielded several possible core anti-GU targets of D. huoshanense. They influenced the development of GU by acting on signaling pathways such as phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）， hypoxia inducible factor-1 （HIF-1）， tumor necrosis factor （TNF）， and nuclear factor-κB （NF-κB）， and various biological processes. The in vivo experiment showed that D. huoshanense significantly reduced the levels of inflammatory factors such as interleukin-1β （IL-1β）， interleukin-6 （IL-6）， and TNF-α in the serum of model rats （P<0.05， P<0.01）， increased gastric blood flow （GBF） at the ulcer margin， raised the expression of epidermal growth factor （EGF） and epidermal growth factor receptor （EGFR） at the ulcer margin （P<0.01）， significantly down-regulated protein and mRNA expression of PI3K and Akt， and up-regulated protein and mRNA expression of phosphatase and tensin homolog deleted on chromosome ten （PTEN） in the gastric tissues of GU rats （P<0.01）. ConclusionThrough regulating EGFR/PI3K/Akt signaling pathway， D. huoshanense can inhibit tissue inflammation， increase gastric microcirculatory blood flow at the ulcer margin， and promote cell proliferation and repair of damaged gastric mucosa.

Comparison of active ingredients and protective effects of Dendrobium huoshanense of different growth years on acute liver injury / 中国中药杂志

The differences of the active ingredients in Dendrobium huoshanense of different growth years and their protective effects on acute liver injury were studied to provide evidence for optimizing harvest time. The contents of polysaccharides, total flavonoids and total alkaloids in D. huoshanense of different growth years were determined by UV spectrophotometry, and the contents of gigantol in D. huoshanense were determined by HPLC. C57 BL/6 mice were randomly divided into blank control group(saline), modeling group(saline), high-dose(7.5 g·kg~(-1)) and low-dose(1.25 g·kg~(-1)) groups of D. huoshanense of different growth years. Each group was intragastrically administered every day for 2 weeks. 500 mg·kg~(-1) paracetamol was injected intraperitoneally 2 h after last treatment except the control group. After 12 hours, the serum and liver tissues were collected to detect the activities of ALT and AST, and the levels of SOD and MDA. The hepatic histopathological examination was performed. The results showed that the chemical constituents of D. huo-shanense of different growth years were significantly different(P<0.05). The contents of polysaccharide and gigantol of D. huoshanense of 2 growth years were the highest. The contents of flavonoids and alkaloids of D. huoshanense of 3 growth years were the hig-hest, followed by the D. huoshanense of 2 growth years, and the lowest were that of 1 growth year. Compared with the modeling group, D. huoshanense of different growth years could decrease the activities of ALT and AST in serum. Meanwhile, the levels of MDA reduced significantly, while those of SOD increased markedly. Histopathological results suggested that all D. huoshanense samples were effective in the reduction of the necrosis of hepatocytes in different degrees. The results of the multi-component SPSS paired tests showed that polysaccharide and gigantol probably played a leading role in the liver protection effects, while D. huoshanense of 2 growth years showed the best efficacy. The optimal harvesting time of D. huoshanense is 2 growth years.

Comparison of planting modes of Dendrobium huoshanense and analysis of advantages of simulated cultivation / 中国中药杂志

Dendrobium huoshanense is a precious medicinal plant belonging to Dendrobium of Orchidaceae. It is a special medicinal material and extremely scarce in Huoshan county, Anhui province. At present, D. huoshanense has been greatly protected, which also makes it possible to industrialize relying on tissue culture and artificial cultivation technology. Three main planting methods were utilized for cultivating D. huoshanense including facility cultivation, under forest cultivation and simulative habitat cultivation. Firstly, the three cultivation modes and technical characteristics of D. huoshanense were compared and analyzed, and it was found that the ecological environment of D. huoshanense cultivated in the simulated environment was closer to that of wild D. huoshanense. Secondly, based on comparing the characters and quality of three cultivation modes, the results showed that the shape of D. huoshanense cultivated in simulated environment was more similar to that of "grasshopper thigh" recorded in Bencao Jing Jizhu, and its quality was better than that of facilities and under forest cultivation. The comprehensive benefit comparison of three modes showed that the simulated cultivation had high income, the lowest input-output ratio and significant economic benefit. The quality of cultivated D. huoshanense was further evaluated from four aspects of "excellent environment" "excellent shape" "high quality" "excellent effect", which summarized the comprehensive advantages of simulative habitat cultivation of D. huoshanense as follows: the original habitat and site environment of simulated wild D. huoshanense, the closer shape to the wild, the more content of main medicinal components, and higher economic benefit and better efficacy. The quality of D. huoshanense was improved by the use of simulative habitat cultivation, which has practical significance to guide its large-scale cultivation.

Study on chemical constituents of Dendrobium huoshanense stems and their anti-inflammatory activity / 中国中药杂志

Three bibenzyls 1-3 and six other compounds 4-9 were firstly isolated from Dendrobium huoshanense stems. They were identified as 3',4-dihydroxy-3,5'-dimethoxybibenzyl(1), batatasin Ⅲ(2), 3,4'-dihydroxy-5-methoxy bibenzyl(3), dihydroconiferyl dihydro-p-coumarate(4), syringaresinol(5), 3-(4-hydroxyphenyl)-propionic acid ethyl ester(6),(3-ethylphenyl)-1,2-ethanediol(7),(S)-5-hydroxy-3,4-dimethyl-5-pentylfuran-2(5H)-one(8) and loliolide(9). Anti-inflammation assay showed that bibenzyls 1-3 could significantly inhibit the production of nitric oxide(NO) and the expression of tumor necrosis factor α(TNF-α) and interleukin 1β(IL-1β) mRNA in LPS-induced RAW264.7 macrophages. Mechanism study exhibited that the phosphorylation of nuclear factor kappa B(NF-κB) p65, inhibitor of κB(IκB), extracellular regulatedprotein kinase(ERK), c-Jun N-terminalkinase(JNK), p38 and Akt of LPS-induced RAW264.7 macrophages could be remarkably reduced by 1. These results suggested that the inflammatory response of LPS-induced RAW264.7 macrophages could be significantly inhibited by 1-3. Additionally, the anti-inflammatory effect of 1 might be contributed to its ability on the regulation of NF-κB, MAPKs and Akt signaling pathways.

Comparative analysis of active components and transcriptome between autotetraploid and diploid of Dendrobium huoshanense / 中国中药杂志

In this study, the roots, stems and leaves of diploid and autotetraploid Dendrobium huoshanense were used as materials to compare their contents of polysaccharides and alkaloids, and the transcriptome sequencing analysis was carried out. The results showed that the contents of polysaccharides and alkaloids in the roots, stems and leaves of tetraploid were 7.6%, 34.5%, 17.2%, 0.01%, 0.024% and 0.035% higher than those of diploid D. huoshanense, respectively. The contents of active components in different tissues were significantly different. There were 3 687 differentially expressed genes in diploid and tetraploid D. huoshanense, of which 2 346 genes were up-regulated and 1 341 down regulated. Go functional analysis showed that these genes were mainly involved in growth and development, stress resistance and other related functions. KEGG pathway analysis showed that most of the differential genes were concentrated in the processes of carbon metabolism, signal transduction, carbohydrate metabolism, amino acid metabolism and energy metabolism. The differential expression of key genes involved in the metabolism of polysaccharides, terpenes and polyketones, amino acid metabolism, hormone synthesis and signal transduction in diploid and tetraploid plants may be the main reason for the high energy content, the increase of active components and the growth potential of tetraploid plants.

Content Determination of 3 Saccharides in 3 Kinds of Medicinal Dendrobii Caulis by HPLC-CAD / 中国药房

OBJECTIVE：To establish the content determination me thod of 3 mono/disaccharides in 3 kinds of medicinal Dendrobii Caulis. METHODS ：HPLC-CAD method was established. The determination was performed on Shodex Asahipak NH2P-50 4E column with mobile phase consisted of acetonitrile-water （75 ∶ 25，V/V）at the flow rate of 1.0 mL/min. The column temperature was set at 30 ℃，and sample size was 10 µL. CAD detection condition included that data acquisition frequency was 5 Hz，filter constant was 5 s，atomization temperature was 35 ℃ ，gas source was nitrogen with pressure of 4.012 × 105 Pa. RESULTS：The linear range of fructose ，D-anhydrous glucose and sucrose were 0.156 2-1.873 8 mg/mL（r＝0.999 5），0.012 7- 0.152 4 mg/mL（r＝0.999 7），0.277 6-3.331 2 mg/mL（r＝0.999 8），respectively. The limits of quantification were 0.002 61，0.004 24 and 0.005 12 mg/mL，and the limits of detection were 0.000 78，0.001 27 and 0.001 54 mg/mL，respectively. RSDs of precision ， stability，reproducibility and durability tests were all lower than 3％. The recoveries were 95.98％-98.15％（RSD＝0.83％，n＝6）， 95.64％-98.62％（RSD＝1.10％，n＝6）and 97.53％-98.94％（RSD＝0.53％，n＝6）. The contents of them were 0.28％-1.12％， 0.02％-0.13％，0.76％-2.67％，respectively. The total content was 1.38％～3.10％. The order of saccharide content in 3 kinds of Dendrobii Caulis was sucrose ＞fructose＞D-anhydrous glucose ；the order of sucrose content and total content were Dendrobium huoshanense＞D. moniliforme ＞D. officinale ；the order of D-anhydrous glucose content was D. huoshanense ＞D. officinale ＞D. moniliforme； the order of fructose content was D. moniliforme ＞D. officinale ＞D. huoshanense . CONCLUSIONS ：Established method is sensitive ，reproducible and simple in operation ，and can be used for content determination of 3 saccharides in 3 kinds of medicinal Dendrobii Caulis. There are differences in the contents of saccharide s among 3 kinds of Dendrobii Caulis.

Diversity and difference of endophytes in Dendrobium huoshanense with different growth years / 中国中药杂志

In order to explore endophytes diversity and difference in Dendrobium huoshanense,in this paper,the metagenomics method was used to analyze the endophytic bacteria and fungi community of 5 groups include 30 samples in different growth years. The results indicate that 3 540 bacterial OTUs were identified from D. huoshanense,and there are 138 OTUs in 5 groups simultaneously;2 168 fungal OTUs were identified,and 143 OTUs exist in 5 groups simultaneously. The dominate endophytic bacteria community are Sphingomonas sp.,Acinetobacter sp.,Burkholderia sp.,Methylobacterium sp.,Enterococcus sp.,Bacillus sp.,the difference endophytic bacteria community are Oceanobacillusd sp.,Actinomycetospora sp.,Paenibacillus sp.. The dominate endophytic fungi community are Zasmidium sp.,Zymoseptoria sp.,Alternaria sp.,Cladosporium sp.,Fusarium sp.,the difference endophytic fungi community are Cyphellophore sp.,Fusarium sp.. The results of clustering revealed that both the endophytic bacteria and the endophytic fungi,ⅢY2 and ⅢY3 are complete clustered,and ⅡY1 and ⅢY1 are also cluster completely. These enriched the species and resources of endophytic bacteria and fungi in D. huoshanense,and provided a theoretical reference for the reasonable harvest of D. huoshanense.

Cloning and quantitative expression analysis of GMPP gene from Dendrobium huoshanense / 中国中药杂志

In order to understand the function of GDP-mannose pyrophosphorylase(GMPP) function and its regulation in polysaccharide biosynthesis mechanism in Dendrobium. D. huoshanense was used to clone GMPP gene. GMPP gene expression in D. huoshanense,D. officinale and D. moniliforme was also determined by qPCR. The results showed that the length of D. huoshanense GMPP gene c DNA sequence is 1 867 bp,containing 1 245 bp open reading frame(ORF),encoding 415 amino acids. Phylogenetic tree analysis showed that D. huoshanense,D. officinale and D. moniliforme are closely related with GMPP taken into consideration. Bioinformatics analysis demonstrated that GMPP sequence similarity among the three species reached as high as 99%. qPCR results indicated that GMPP genes was highly expressed in stem of D. huoshanense compared with its leaf,flower and root. According to GMPP gene expression profile in D. huoshanense,D. officinale and D. moniliforme grown in Huoshan area,it was clear that GMPP in D. huoshanense showed the highest expression level. Furthermore,our findings of GMPP gene expression profile will facilitate future researches into its polysaccharide biosynthetic mechanism.

Cloning and bioinformatics analysis of β-1,4-xylosyltransferase IRX9 gene from Dendrobium huoshanense / 中草药

Objective To clone IRX9 gene from Dendrobium huoshanense and perform the bioinformatics and codon bias analysis. Methods The full-length cDNA sequence was cloned from the IRX9 gene based on transcriptome sequencing data of D. huoshanense generated in our previous study by using RT-PCR and further analyzed by bioinformatic methods. Results The cloned IRX9 gene was 1 533 bp, containing a 1 047 bp open reading frame (ORF) which encoded 348 amino acids. The theoretical molecular weight was 39 350 and its isoelectric point was 7.26. IRX9 protein was a hydrophilic protein and had no signal peptide, which had multiple phosphorylation sites and glycosylation sites and might be located in the plasma membrane. Phylogenetic analysis indicated that sequence of amino acids had the highest homology with D. candidum and the conserved region “DXD” of the GT-A superfamily. Codon bias analysis showed that IRX9 gene prefered to use A/T ending codon, with 27 skewed codons. Nicotiana is the most suitable host for exogenous expression of IRX9 gene. Conclusion The IRX9 gene was cloned from Dendrobium huoshanense successfully and it provided a theoretical basis for regulating the growth and development of D. huoshanense and improving the yield and quality of D. huoshanense.

Therapeutic effect and mechanism of three kinds of Dendrobium on constipation in rats with spleen Yin deficiency / 中国中药杂志

Books on Chinese herbal medicines have shown that Dendrobium has the effect of nourishing Yin and reinforcing Yin,usually used for constipation induced by spleen Yin deficiency in clinical application. D. huoshanense,as an independent species among many species of Dendrobium,has no experimental studies about its effects on spleen Yin deficiency-type constipation. The purpose of this experiment was to illustrate the therapeutic effect of D. huoshanense on the constipation of spleen Yin deficiency type in rats,investigate its preliminary mechanism,and compare it with the D. officinale and D. nobile contained in the Chinese Pharmacopoeia to clarify its characteristics. The spleen Yin deficiency model was replicated in 70 rats by the composite factor method,and then the model rats were randomly divided into 7 groups: model group,Liuwei Dihuang Pills group( LWDHP),D. huoshanense high( DHS-H),medium( DHS-M),low( DHS-L) dose groups,D. nobile group( DNS),and D. officinale group( DOS),and another 10 rats were used as normal group( Normal). After 7 continuous days of administration,the fecal water content and intestine propulsion rate of each group were detected. HE staining was used to observe the pathological damage of ileum and colon in each group. Immunohistochemistry and Western blot were used to detect aquaporin 3( AQP3) expressions,while the expression levels of the somatostatin( SS) and motilin( MTL) in the ileum of each group were detected by enzyme-linked immunosorbent assay. The results showed that as compared with the model group,the rats in each drug-administered group had increased number of fecal pellets,increased fecal water content,and the increased intestinal propulsion rate( P<0. 01),while the pathological damage of the ileum and colon was significantly reduced; the expression of AQP3 protein was significantly decreased( P<0. 01); the level of MTL was significantly increased and the level of SS was decreased( P<0. 01). All DHS groups showed a good dose-effect relationship,and the same dose treatment effect was equivalent to that of DOS,but it was superior to DNS. Therefore,DHS has a significant therapeutic effect on constipation of spleen Yin deficiency type,and its mechanism may be related to intestinal motility and water-liquid metabolism,with a good therapeutic effect.

Effects of Dendrobium huoshanense on expressions and activities of hepatic microsomal cytochrome P450s in mice / 中国中药杂志

This study was carried out to investigate the effect of oral administration of Dendrobium huoshanense on the expressions and activities of hepatic microsomal cytochrome P450s in mice, and to provide a reference for the evaluation of drug-drug interactions between D. huoshanense and clinical drugs. The C57BL/6 mice were randomly divided into blank control group, D. huoshanense low dose group (crude drug 1.25 g·kg⁻¹), D. huoshanense high dose group (crude drug 7.5 g·kg⁻¹), and phenobarbital positive control group (0.08 g·kg⁻¹). Each group was intragastrically administered with drugs for 2 weeks. The mice were sacrificed and their liver microsomes were prepared. The expressions of major subtypes of P450 enzyme were determined by Western blot and the probe drugs were used to detect the enzyme activities of P450 subtypes with protein expression changes. Western blot analysis showed that the protein expressions of CYP1A1, CYP1A2 and CYP2B in liver tissues were up-regulated in D. huoshanense-treated group. In vitro enzyme activity tests showed that there were no significant difference in metabolism of 7-ethoxyresorufin (a probe drug for CYP1A1) and bupropion (a probe drug for CYP2B) between D. huoshanense group and control group. The metabolism of phenacetin (a probe drug for CYP1A2) showed a statistical difference in rate Vmax, and it was significantly increased by approximately 20% in D. huoshanense group as compared with the blank control group, and the clearance CLint in treated group was also increased by about 32%. Therefore, oral administration of D. huoshanense had no effects on the activities of most hepatic P450 enzymes in mice, with no drug-drug interaction related to the P450 enzyme system in most clinical drugs theoretically. However, oral administration of D. huoshanense may accelerate the metabolism of CYP1A2-catalyzed drugs, which needs to be considered in clinical practice.

Plastome-wide comparison reveals new SNV resources for the authentication of and its corresponding medicinal slice (Huoshan Fengdou)

species and their corresponding medicinal slices have been extensively used as traditional Chinese medicine (TCM) in many Asian countries. However, it is extremely difficult to identify species based on their morphological and chemical features. In this study, the plastomes of were used as a model system to investigate the hypothesis that plastomic mutational hotspot regions could provide a useful single nucleotide variants (SNVs) resource for authentication studies. We surveyed the plastomes of 17 species, including the newly sequenced plastome of . A total of 19 SNVs that could be used for the authentication of were detected. On the basis of this comprehensive comparison, we identified the four most informative hotspot regions in the plastome that encompass to , to , to and to . Furthermore, to established a simple and accurate method for the authentication of and its medicinal slices, a total of 127 samples from 20 species including their corresponding medicinal slices (Fengdous) were used in this study. Our results suggest that and its medicinal slices can be rapidly and unequivocally identified using this method that combines real-time PCR with the amplification refractory mutation system (ARMS).

HPLC fingerprint for different growth period of Dendrobium huoshanense / 中草药

Objective: To establish a HPLC fingerprint method for assessing the quality of Dendrobium huoshanense, in addition to determining concentrations of syringic acid, rutin, dendrophenol and naringenin in this crude drug. Methods: Agilent C18 column (250 mm × 4.6 mm, 5 μm) was utilized with the mobile phase comprising methanol-0.1% phosphoric acid with the flow rate of 1 mL/min in a gradient elution manner. The detection wavelength was set at 240 nm and the column temperature was 30 ℃. The resultant chromatograms were imported to Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica (2012.1) to obtain retention time and peak area of samples. Similarity for 39 sets of samples were analyzed. The peak area data were processed by SPSS software for cluster analysis and the clustering effect was discussed. Results: The line relationship of this way was good (R > 0.999), with high precision regarding instrument used (RSD < 3.00%), the method showed good reproducibility (RSD < 3.00%), standard recovery was between 99.26% and 100.32% (RSD of 0.35%-1.67%). Different growth period and different planting patterns of D. huoshanense were distinct regarding the concentration of four compounds. Conclusion: The method is useful to evaluate and discriminate D. huoshanense at different growth period for the purpose of providing scientific reference on harvest, development and evaluation of D. huoshanense.

Specific PCR Identification between Dendrobium huoshanense and Its Adulterants / 中国药学杂志

OBJECTIVE: To design specific identification primers based on ribosomal DNA ITS sequences and accurate identification of Dendrobium huoshanensis and its adultments. METHODS: Phylogenetic tree of D. huoshanense and D. officinale and D. henanense by maximum parsimony (MP) and Bayesian inference (BI) were constructed. Specific primers SH-CP9s /SH-CP9a, SHCP25s / SH-CP25a, SH-CP29s /SH-CP29a for PCR identification of D. huoshanensis based on mutation sites on ITS sequences were designed. RESULTS: In the phylogenetic tree, D. huoshanense and D. officinale and D. henanense were clustered into monophyletic group. The specific PCR reaction procedure of D. huoshanense was obtained by investigating the annealing temperature and the amount of DNA template, and different types of PCR and Taq enzymes. In the PCR product, D. huoshanense appearance of the band, and adultments with no band. CONCLUSION: The ITS sequences can be used as DNA Barcoding identification of D. huoshanensis, and identify D. huoshanense and its adultments by the designed primers.

Identification of Dendrobium huoshanense, Dendrobium officinale and Dendrobium devonianum by multiplex allele-specific polymerase chain reaction / 药学学报

This study was designed to establish a multiplex allele-specific polymerase chain reaction method for simultaneous identification of Dendrobium huoshanense, D. officinale and D. devonianum, which may resolve identification problems of caulis dendrobii. Internal transcribed spacer sequences and trnL-trnF sequences of the Dendrobium species were aligned by BioEdit software, then specific SNPs of the three species were analyzed for designing allele-specific primers and the multiplex allele-specific PCR reaction system was established. The different origin of Dendrobium huoshanense, D. officinale and D. devonianum was amplified and identified by the sizes of respective band. The results showed that 584 bp, 397 bp and 211 bp bands could be amplified by D. devonianum, Dendrobium officinale and Dendrobium huoshanense respectively, when the annealing temperature was 61 ℃ and the number of cycles was 35. The limit of detection (LOD) of D. devonianum and D. huoshanense were both 1.2 ng, while D. officinale was low than 0.24 ng. The detection limit of adulterates in D. devonianum, D. devonianum and D. huoshanense mixture sample was 1%, 1% and 5% respectively. This result suggests that the method of multiplex allele-specific PCR is useful to identify D. huoshanense, D. officinale and D. devonianum is accurate and specific.

Study on secondary metabolites of endophytic fungus Fusarium lactis from Dendrobium huoshanense / 中草药

Objective To study the secondary metabolites of endophytic fungus Fusarium lactis in Dendrobium huoshanense. Methods Compounds were isolated from the EtOAc extract by chromatography technology and their structures were elucidated on the basis of comprehensive spectroscopic analysis. Results Twenty-one compounds were isolated and identified as N-phenethylacetamide (1), 1H-indole-3-carbaldehyde (2), thymidine (3), uracil (4), lignoren (5), uridine (6), hexahydrate (7), adenosine (8), cyclo-glycine-(L)-proline (9), cyclo (D)-proline-(L)-phenylalanine (10), cyclo (L)-proline-(L)-phenylalanine (11), 2-pyrrolidinone (12), N-methyl-2-pyrolidinone (13), cyclo-(L)-4-OH-proline-(L)-phenylalanine (14), brevianamide F (15), 3-methyl- piperazine-2,5-dione (16), 7,8-dimethylbenzo[g]pteridine-2,4 (1H,3H)-dione (17), cyclo (L)-tyrosine-(L)-phenylalanine (18), beer sterols (19), daidzein (20), and erythritol (21). Conclusion All compounds are isolated from Fusarium lactis for the first time.

Purification and cytotoxicity of glycoprotein isolated from Dendrobium huoshanense / 中国中药杂志

Dendrobium huoshanense is a rare traditional Chinese medicinal herb, and the anti-tumor activity of its polysaccharides is a research hotspot in traditional Chinese medicine resources domain. This study aims to explore the material basis for the anti-tumor activity of polysaccharide. D. huoshanense was used as raw material in the experiment, and the different protein components were obtained through low salt solution extraction and ammonium sulfate fractional precipitation. Then glycoprotein components were determined by SDS-PAGE electrophoresis staining, and were further isolated and purified by DEAE ion column and Sephadex gel column. At the same time, MTT assay was used in detecting the cytotoxicity of different products on HepG2 cells in vitro. As a result, three kinds of glycoprotein components RG1, RG2, RG3 with relative molecular mass of 22.5, 19.8, 15.6 kDa were gained, and the IC₅₀ of three compounds on human liver cancer cell HepG2 was 534.23 mg•L⁻¹, meanwhile IC₅₀ of single glycoprotein component RG1, RG2 was 432.96, 413.91 mg•L⁻¹ respectively, and glycoprotein component RG3 had no cytotoxicity on HepG2 cells. All in all, the experiment results suggested that two kinds of glycoproteins components with relative molecular mass of 22.5, 19.8 kDa may be one of the material basis for anti-tumor activity of D. huoshanense polysaccharide, and they had a synergistic effect.

Effect of growth regulators on rooting of regenerated shoots from Dendrobium huoshanense protocorm-like bodies / 中草药

Object To study the effects of different growth regulators and organic additives on rooting of regenerated shoots from Dendrobium huoshanense C Z Tang et S J Cheng protocorm like bodies Methods In order to induce root development, the regenerated shoots were cultured on MS medium containing 3% sucrose and 0 8% agar and supplemented with NAA, IBA, BA, KT, banana mud or amino acids at different concentrations During rooting induction, the development of roots was observed and the frequency of rooted shoots was scored Results The frequency and the process of root initiation were significantly influenced by different growth regulators or organic additives For initiation and development of roots, the suitable concentration of NAA, IBA, BA, KT, banana mud or amino acids was 0 2 , 0 1, 0 8, 0 8 mg/L, 20% and 0 1%, respectively Conclusion In all media tested, NAA, IBA or banana mud display greater effects on rooting of regenerated shoots of D huoshanense than others and KT enables shoots to keep green

Influence of subculture cycle on tube plantlet growth of Dendrobium huoshanense and medium composition / 中草药

Objective To optimize the best subculture cycle through studying the influence of subculture time on tube plantlet growth of Dendrobium huoshanense and the change of medium composition.Methods Height,tiller,fresh weight,and chlorophyll content of the tube plantlet and pH value,water content,and sugar content of the medium were measured after different cycles of the subculture,the cost of culture medium for subculture was calculated as well.Results The height of the tube plantlet increase 282.86%,the tiller increase by 3.5 times,fresh weight reaches its maximum,chlorophyll content of the tube plantlet almost reaches its maximum after 40 d subculture;while water content and sugar content of the medium are decreased to the lowest point,pH value of medium(