Analysis of Differences in Secondary Metabolites from Dendrobium nobile Stems Cultivated on Epiphytic Rocks in Different Growth Years Based on Metabolomics / 中国实验方剂学杂志

ObjectiveTo study the changing characteristics of secondary metabolic compounds accumulated in Dendrobium nobile stems at different growth years， a simulated wild stone plant， in order to provide a theoretical basis for rational planning of the harvesting period of D. nobile. MethodUltra-high performance liquid chromatography-mass spectrometry（UPLC-MS/MS） was used to detect and analyze the secondary metabolites in the stems of 1-year-old， 2-year-old， and 3-year-old D. nobile. The mass spectrometry data were processed using Analyst 1.6.3 software， and all samples were subjected to principal component analysis（PCA）， cluster heat map analysis， partial least squares-discriminant analysis（PLS-DA）， and differential secondary metabolites were screened based on variable importance in projection（VIP） values>1， fold change（FC）≥2 and FC≤0.5. Then differential secondary metabolites were identified based on relative molecular weight， fragmentation ions and mass spectrometry database， and enriched pathways were identified based on the Kyoto Encyclopedia of Genes and Genomes（KEGG） database. ResultA total of 1 317 secondary metabolites were identified in the stems of D. nobile at three growth stages， with flavonoids， phenolic acids， alkaloids and terpenoids accounting for 76.55% of the total. Compared with the 1-year-old stems of D. nobile， 289 differential secondary metabolites were identified in the 2-year-old stems， of which 255 were up-regulated and 34 were down-regulated， 682 differential secondary metabolites were identified in the 3-year-old stems， of which 502 were up-regulated and 180 were down-regulated. Compared to the 2-year-old stems， the 3-year-old stems had 602 differential secondary metabolites， with 405 up-regulated and 197 down-regulated. As the growth stage of D. nobile increased， the top 10 up-regulated differential metabolites mainly included flavonoids， phenolic acids， phenylpropanoids and terpenoids， such as kaempferol derivatives， asperulosidic acid， apigenin derivatives， chrysoeriol derivatives， isorhamnetin derivatives， taxifolin derivatives， quercetin derivatives. KEGG enrichment analysis showed significant enrichment of secondary metabolites in the flavonoid biosynthesis， flavone， and flavonol biosynthesis， secondary metabolite biosynthesis， and phenylpropanoid biosynthesis pathways with the increase of growth years. ConclusionWith the increase of the growth years， the levels of secondary metabolites such as flavonoids， phenolic acids， phenylpropanoids and terpenoids in the wild-grown D. nobile have been significantly enhanced. In practical production， grading based on different growth years can be carried out to improve the medicinal and economic values of D. nobile.

A new allo-aromadendrane sesquiterpene from Dendrobium nobile / 中国中药杂志

To study the chemical constituents in the non-alkaloid part of stems of Dendrobium nobile. The macroporous adsorption resin, MCI, silica gel, RP-C_(18), and Sephadex LH-20 gel, preparative thin layer chromatography, and preparative high-performance liquid chromatography(HPLC) were used to isolate and purify the compounds. The structures of the compound were determined according to the spectra data, physicochemical properties, and relevant references. A total of 8 compounds were isolated from D. nobile, which were soltorvum F(1), p-hydroxyphenylpropionic acid(2), vanillic acid(3), p-hydroxybenzoic acid(4), N-trans-cinnamic acid acyl-p-hydroxybenzene ethylamine(5),(+)-(1R,2S,3R,4S,5R,6S,9R)-2,11,12-trihydroxypicrotoxane-3(15)-lactone(6), dendronobilin H(7), soltorvum E(8). Compound 1 was a novel compound, named as soltorvum F. Compound 8 was isolated from Dendrobium species for the first time.

Discrimination of cultivation modes of Dendrobium nobile based on content of mineral elements and ratios of nitrogen stable isotopes / 中国中药杂志

This study explored the feasibility of mineral element content and ratios of nitrogen isotopes to discriminate the cultivation mode of Dendrobium nobile in order to provide theoretical support for the discrimination of the cultivation mode of D. nobile. The content of 11 mineral elements(N, K, Ca, P, Mg, Na, Fe, Cu, Zn, Mn, and B) and nitrogen isotope ratios in D. nobile and its substrate samples in three cultivation methods(greenhouse cultivation, tree-attached cultivation, and stone-attached cultivation) were determined. According to the analysis of variance, principal component analysis, and stepwise discriminant analysis, the samples of different cultivation types were classified. The results showed that the nitrogen isotope ratios and the content of elements except for Zn were significantly different among different cultivation types of D. nobile(P<0.05). The results of correlation analysis showed that the nitrogen isotope ratios, mineral element content, and effective component content in D. nobile were correlated with the nitrogen isotope ratio and mineral element content in the corresponding substrate samples to varying degrees. Principal component analysis can preliminarily classify the samples of D. nobile, but some samples overlapped. Through stepwise discriminant analysis, six indicators, including δ~(15)N, K, Cu, P, Na, and Ca, were screened out, which could be used to establish the discriminant model of D. nobile cultivation methods, and the overall correct discrimination rates after back-substitution test, cross-check, and external validation were all 100%. Therefore, nitrogen isotope ratios and mineral element fingerprints combined with multivariate statistical analysis could effectively discriminate the cultivation types of D. nobile. The results of this study provide a new method for the identification of the cultivation type and production area of D. nobile and an experimental basis for the quality evaluation and quality control of D. nobile.

Dendrobium nobile protects against ovalbumin-induced allergic rhinitis by regulating intestinal flora and suppressing lung inflammation / 中国天然药物

Antibiotic exposure-induced dysbiosis of the intestinal flora increases the risk of developing allergic rhinitis. Hence, regulating the balance of intestinal flora may be useful for preventing and treating allergic rhinitis. However, the underlying mechanism is unclear. Dendrobium nobile (Shihu) exhibits anti-inflammatory and immune activities. Hence, in this study, we investigated the mechanism via which Shihu may improve allergic rhinitis. Mouse models of allergic rhinitis with intestinal flora dysbiosis (Model-D, antibiotics induce intestinal flora dysbiosis with ovalbumin-induced allergy) and normal intestinal flora with allergic rhinitis (Model-N, ovalbumin-induced allergy) were established. The effect of Shihu on intestinal flora and inflammation caused during allergic rhinitis were analyzed. Allergic symptoms, infiltration of hematoxylin and eosin in the lungs and nose, and the release of various factors [interleukin (IL)-2, IL-4, IFN-γ, IL-6, IL-10, and IL-17] in the lungs were evaluated. The results indicate that intestinal flora dysbiosis exacerbated lung and nose inflammation in allergic rhinitis. However, treatment with the Shihu extract effectively reversed these symptoms. Besides, the Shihu extract inhibited the PI3K/AKT/mTOR pathway and increased the level of Forkhead box protein in the lungs. Additionally, the Shihu extract reversed intestinal flora dysbiosis at the phylum and genus levels and improved regulator T cell differentiation. Furthermore, in the Model-D group, the Shihu extract inhibited the decrease in the diversity and abundance of the intestinal flora. Screening was performed to determine which intestinal flora was positively correlated with Treg differentiation using Spearman's correlation analysis. In conclusion, we showed that Shihu extract restored the balance in intestinal flora and ameliorated inflammation in the lungs of allergic rhinitis mice and predicted a therapeutic new approach using Traditional Chinese Medicine to improve allergic rhinitis.

Dendrobium nobile Lindl. Alkaloids Inhibit the Malignant Biological Behavior of Cervical Cancer SiHa Cells / 中国生物化学与分子生物学报

Dendrobium nobile Lindl. alkaloids (DNLA) promote the apoptosis of breast cancer and colon cancer cells, but whether they affect the malignant biological behavior of cervical cancer cells is unknown. Herein we explored the effects and possible mechanisms of DNLA on the proliferation, apoptosis, migration and invasion of cervical cancer SiHa cells. SiHa cells were transfected with si-NC, siKCNQ1OT1, miR-NC, miR-487a-3p mimics, pcDNA-NC or pcDNA-KCNQ1OT1. Different doses (15, 30, 60 ng/mL) of DNLA were applied. The CCK-8 method was used to detect cell proliferation; Tran-swell was used to detect cell migration and invasion; flow cytometry was used to detect cell apoptosis; Western blotting was used to detect the expression of MMP2, MMP9 and Cleaved-Caspase-3 genes at the protein level; RT-qPCR was used to detect the expression of KCNQ1OT1 and miR-487a-3p. The dual luciferase reporter gene experiment verified the regulatory relationship between KCNQ1OT1 and miR-487a-3p. The results showed that different doses (15, 30, 60 ng/mL) of DNLA reduced the absorbance value, migration number, invasion number, the protein level of MMP2 and MMP9, reduced the expression of KCNQ1OT1, and increased the apoptosis rate, the abundance of Cleaved-Caspase-3 and the expression of miR-487a-3p (P<0. 05). Low expression of KCNQ1OT1 or high expression of miR-487a-3p reduced the absorbance value, migration number, invasion number, and the protein level of MMP2 and MMP9, but increased the apoptosis rate and the abundance of Cleaved-Caspase-3 (P<0. 05). KCNQ1OT1 negatively regulated the expression of miR-487a-3p. The effects of high expression of KCNQ1OT1 on the proliferation, apoptosis, migration and invasion of SiHa cells were opposite to that of low expression of KCNQ1OT1, and high expression of KCNQ1OT1 reduced the effects of 60 ng/mL DNLA on the proliferation, apoptosis, migration and invasion of SiHa cells. In summary, DNLA may reduce the proliferation, migration and invasion of cervical cancer SiHa cells and promote SiHa cell apoptosis by regulating the KCNQ1OT1/miR-487a-3p axis.

Correlation analysis and breeding application of agronomic traits and quality indexes in Dendrobium nobile / 中国中药杂志

The present study aimed to explore the correlation between agronomic traits and quality indexes of Dendrobium nobile and its application value in agricultural breeding. The cultivated strains of D. nobile in Hejiang-Chishui producing areas were extensively collected,and the main agronomic traits and quality indexes were measured. The agronomic traits with significant correlation with quality indexes were screened out by the correlation analysis,and then the parental lines and self-bred F_1 generation plants were furtherverified. Among 96 lines of D. nobile,the content of soluble polysaccharides showed a significant negative correlation with dendrobine( P < 0. 01),and no significant correlation with agronomic traits in stems and leaves. The content of dendrobine exhibited a significant positive correlation with the stem width-thickness ratio( at the largest cross section; P < 0. 01),and no significant correlation with other agronomic traits. Regression analysis further verified the positive correlation between dendrobine content and stem width-thickness ratio( R2> 0. 9). Two lines,JC-10 and JC-35,with significant differences in stem width-thickness ratio were screened out( P <0. 05). The corresponding F1 generation plants by self-pollination both showed that the dendrobine content was higher with greater stem width-thickness ratio( P < 0. 01). The experimental results suggested that within a certain range,the dendrobine content was higher in D. nobile with flatter stem. Therefore,in the breeding of D. nobile,this specific trait could be used for screening plants with high content of quality indexes such as dendrobine.

Establishment of HPLC Fingerprints of Ethanol Extract of Dendrobium nobiles from Different Habitats and Spectrum Effect Relationship Study of Its Anti-inflammatory Effect / 中国药房

OBJECTIVE：To establish the HPLC fingerprints of ethanol extracts of Dendrobium nobile from different habitats ， and to study spectrum effect relationship of its anti-inflammatory effect. METHODS ：The ethanol extracts from 12 batches of D. nobile from different habitats （S1-S12）were prepared. The fingerprints of ethanol extracts from 12 batches of D. nobile were established by HPLC and TCM Fingerprint Similarity Evaluation System （version 2012）. Common peaks were identified and the similarity was evaluated. Using the contents of inflammatory factors （IL-4，IL-6）as anti-inflammatory indexes ，ELISA was used to investigate the effects of ethanol extracts from 12 batches of D. nobile on RAW 264.7 macrophages in inflammatory model induced by lipopolysaccharide. Grey correlation analysis was used to analyze the correlation between common peaks and anti-inflammatory indexes in the fingerprint of ethanol extract from D. nobile . RESULTS ：There were 18 common peaks in ethanol extracts from 12 batches of D. nobile ，and No. 12 peak was identified as dendrophenol. The similarity of each sample was 0.911-0.996. The content of IL- 4 in cell culture medium was significantly reduced by ethanol extracts of D. nobile S1-S12（P＜0.05 or P＜0.01）；the content of IL- 6 in cell culture was significantly reduced by ethanol extracts of D. nobile S1-S8，S10（P＜0.05 or P＜0.01）. Grey correlation analysis found that except for peaks 4 and 18，the correlation degree between the other common peaks and the content of IL- 4 was greater than 0.6，and the correlation degree between peaks 5 and 7 and the content of IL- 4 was greater than 0.8；except for peaks 9，14，4，3 and 18，the correlation degree between the other common peaks and the content of IL- 6 was greater than 0.6，and the correlation degree between peaks 1，12 and 13 and the content of IL- 6 was greater than 0.8. CONCLUSIONS ：HPLC fingerprints of ethanol extracts from 12 batches of D. nobile ； from different habitats have high similarity and good consistency in composition ； the chemical constituents represented by peaks 1，5，7，12（dendrophenol）and 13 may 769096783@qq.com be potential anti-inflammatory components of D. nobile .

Does soaking time during disinfestation affect germination rates in Dendrobium? / O tempo de imersão durante a desinfestação influencia nas taxas de germinação de Dendrobium?

Asymbiotic germination is considered an efficient and viable technique that can increase germination rates. The effect of type and concentration of disinfestants, and the exposure time to disinfestants may differ according to the plant species. Therefore, species-specific standardization of disinfestation agent and procedure is necessary to achieve optimal germination rates. The objective of this study was to determine a disinfestation methodology to increase in vitro germination rates and the early development of seedlings of Dendrobium nobile and Dendrobium phalaenopsis, using different times for seed disinfestation and different culture media. Seeds were disinfected by soaking in a 0.8% sodium hypochlorite solution for 5 or 15 min under aseptic conditions, after which seed suspensions were either washed with water or left unwashed. Next, they were seeded in culture flasks containing four different culture media (MS, ½MS, K, and VW). The flasks were then transferred to a growth room under controlled photoperiod and temperature, where they remained under an irradiance of 20 µmol m-2 s-1. Germination rates of the species were evaluated 45 days after placement in the culture flasks. A higher germination rate was observed when the seeds were triple washed, regardless of the culture medium or soaking time. Seed soaking disinfestation for 5 min is also recommended. MS and ½MS media were the most effective culture media in promoting in vitro germination of the species under study.

A germinação assimbiótica é considerada uma técnica eficiente e viável resultando em elevados percentuais de germinação. Apesar do sucesso dessa técnica, o tipo, a concentração e o tempo de exposição do agente desinfestante diferem, necessitando padronização para cada espécie. Assim, a padronização do agente desinfestante e do procedimento são necessários para o aumento das taxas germinativas. Objetivou-se com este trabalho determinar metodologia que aumente a taxa de germinação in vitro e favoreça o desenvolvimento inicial de plântulas de Dendrobium nobile e Dendrobium phalaenopsis, utilizando diferentes métodos de desinfestação de sementes e diferentes meios de cultura. Sementes, sob condições assépticas, foram desinfestadas em solução de hipoclorito de sódio a 0,8%, por cinco ou quinze minutos. Após esses períodos, as suspensões de sementes receberam ou não a tríplice lavagem com água. Em seguida, foram semeadas em frascos de cultivo que continham quatro diferentes meios de cultura (MS, MS½, K e VW). Posteriormente, foram transferidos para sala de crescimento com fotoperíodo e temperatura controlados, onde permaneceram sob irradiância de 20 µmol m-2 s-1. Quarenta e cinco dias após a semeadura foi avaliada a porcentagem de germinação das espécies estudadas. Os resultados neste trabalho indicam que, independentemente do meio de cultura ou do tempo de desinfestação, as sementes quando submetidas à tríplice lavagem apresentaram porcentagem de germinação superior a àquelas que não receberam este procedimento. Recomenda-se a desinfestação das sementes por 5 minutos. Os meios MS e MS½ foram os mais efetivos em promover a germinação in vitro dessas espécies.

Technical evaluation and principle analysis of simulative habitat cultivation of Dendrobium nobile / 中国中药杂志

The technique of "simulative habitat cultivation" is to preserve the quality of traditional Chinese medicine by simulating the original habitat and site environment of wild Chinese medicine resources. Dendrobium nobile is the most representative variety of traditional Chinese medicine which reflects the coordinated development of medicinal material production and ecological environment. In this paper, the main technical points of the simulated cultivation model of D. nobile were summarized as follows: rapid propagation of seedling tissue technology to ensure the genetic stability of provenance; line card+fermented cow manure+live moss method to improve the survival rate; epiphytic stone cultivation to improve the quality of medicinal materials; and the integration of mycorrhizal fungi to improve the quality stability of medicinal materials. On the basis of summarizing the ecological benefits, economical and social benefits generated by the application of the technology, the paper systematically analyzes the principle of the technology for the cultivation of D. nobile to promote the excellent quality, the light, gas, heat and fertilizer resources of the undergrowth niche are in line with the wild site environment of D. nobile. The rich and complex soil microbial community in the forest laid the foundation for the species diversity needed for the growth of D. nobile.The stress effect on the growth of D. nobile resulted in the accumulation of secondary metabolites. The symbiotic relationship between the symbiotic fungi such as bryophytes and D. nobile promotes the synthesis of plant secondary metabolites. The high quality D. nobile was produced efficiently by improving and optimizing the cultivation techniques.

A new cadalene sesquiterpene glycoside from Dendrobium nobile / 药学学报

Using column chromatographic and preparative HPLC technologies, we isolated a new sesquiterpene glycoside from the stem of Dendrobium nobile. With spectroscopic techniques including NMR and MS, the new compound was identified as cadalene-12-O-β-D-glucopyranoside. This type of compound was dehydrogenated from cadinane sesquiterpene to achieve a naphthalene ring, and it is rare from a natural resource.

Comparison of Effect of Fine Powder and Ultrafine Powder of Dendrobium nobile Stem on Intestinal Transporters of Rats / 中国实验方剂学杂志

Objective:To compare the contents of alkaloids from fine and ultrafine powder of Dendrobium nobile stem in rat plasma,and investigate the effect of D. nobile stem with different particle sizes on gene expression of intestinal transporters. Method:Rats were randomly divided into the blank group,fine powder group of D. nobile stem(0.25 g·kg-1) and ultrafine powder group of D. nobile stem(0.25 g·kg-1).The rats were gavaged every 6 h for 5 days.The samples of rat plasma and small intestine were collected.The plasma samples were detected with UPLC-MS.The chromatography separation was performed on a Hypersil Gold C18 column(2.1 mm×150 mm,1.9 μm) with acetonitrile-0.1%formic acid solution as mobile phase for gradient elution.Electrospray ionization (ESI) was applied and operated in positive ion mode.The mRNA expression of multidrug resistance protein 1(MDR1),oligopeptide transporter protein 1(PEPT1),organic cation transporter protein 2(OCT2),breast cancer resistance protein 1(BCRP1),monocarboxylate transport protein 1(MCT1) and multidrug resistance related protein 2(MRP2) in small intestine were quantified by real time fluorescence quantitative polymerase chain reaction. Result:After intragastric administration of fine and ultrafine powder of D. nobile stem,dendrobine,mubironine B and dendramine could be detected in rat plasma.The contents of dendrobine and dendramine in the ultrafine powder group were significantly higher than that in the fine powder group(PD. nobile stem(PPD. nobile stem(PConclusion:Compared with the fine powder group of D. nobile stem,the plasma concentrations of dendrobine and dendramine in the ultrafine powder group are significantly increased,it may be related to the intestinal transporters of MDR1 and BCRP1.These results can provide experimental basis for selecting particle size of D. nobile stem.

Inhibitory Effect of Dendrobium nobile Lindl Wall-broken Powder on Tumor Growth of Transplanted Human Liver Cancer Cell Line HepG2 in Nude Mice / 中国药学杂志

OBJECTIVE: To study the inhibitory effect of Dendrobium nobile Lindl wall-broken powder on tumor growth and angiogenesis of transplanted human liver cancer cell line HepG2 in nude mice. METHODS: The transplant tumor model in nude mice for HepG2 was established. All tumor-bearing nude mice were randomly divided into control group (normal saline), positive group (bevacizumab,5 mg•kg-1, intraperitioneally), and low-,medium-, high-dose Dendrobium nobile Lindl wall-broken powder-treated groups(50,100 and 200 mg•kg-1,intragastrically).The tumor xenografts were harvested and measured for their weights. Immunohistochemistry was used to detected microvessel density (microvessel density, MVD) and vascular endothelial growth factor (vascular endothelial growth factor,VEGF) expression. RESULTS: After the administration, TV (tumor volume), RTV (relative tumor volume), and T/C(%) in Dendrobium nobile Lindl wall-broken powder treatment group decreased significantly, T/C(%) was 60.56%, 48.59%, and 47.15%, respectively. SABC assay showed that MVD in Dendrobium nobile Lindl wall-broken powder treatment group was sparse, MVD value decreased from (65.67±12.05) to (51.53±10.75),(45.89±9.24)and(40.76±8.93), Dendrobium nobile Lindl wall-broken powder can significantly reduce the positive expression of VEGF. The positive expression rate reduced from (82.25±15.59)% to(65.27±13.88)%,(48.02±12.14)% and (44.07±10.34)%. CONCLUSION: Dendrobium nobile Lindl wall-broken powder can inhibit tumor growth of transplanted liver cancer cell line HepG2 in nude mice in varying degrees, which may be related to reducing MVD value and the expression of VEGF in tumor tissue.

Optimization of Water Extraction Technology of Total Polysaccharide of Shuanghu Capsules by Orthogonal Design / 中国药房

OBJECTIVE： To optimize the water extraction technology of total polysaccharide of Shuanghu capsules. METHODS： The total alkaloid was firstly extracted from Dendrobium nobile and Dendrobium officinale mixture of Shuanghu capsules with ethanol， and then total polysaccharide was extracted with water. Using glucose as control， total polysaccharide was treated with phenol-sulfuric acid method and its content was determined at 488 nm. Using comprehensive score calculated with the yield of the extract and the content of total polysaccharide as index， the effects of material-liquid ratio， extraction temperature， extraction time and times on the extraction were investigated by single factor test. Then L9（34） orthogonal test was used to optimize solid-liquid ratio，extraction temperature，extraction time and extraction times according to the results of single factor test. The optimized technology was validated. RESULTS： The linear range of glucose were 0.041 4-0.207 0 mg/mL（r＝0.999 9）. RSDs of intra-day and inter-day ranged 3.61％-8.24％ （n＝3，n＝5）， and RSD of repeatability test was 1.49％ （n＝6）. Average recovery rate was 98.65％（RSD＝1.45％，n＝6）. The optimal water extraction technology included solid-liquid ratio of 1 ∶ 25（g/mL），extraction temperature of 100 ℃，extracting for 90 min， extracting once. Results of validation tests showed that average content of total polysaccharide was 379.292 8 mg/g （RSD＝1.93％，n＝3） and average yield of the extract was 22.75％（RSD＝2.41％，n＝3）. CONCLUSIONS： Established phenol-sulphuric acid method is simple， precise and accurate. The optimal water extraction technology is stable and feasible， which can be used for the extraction of total polysaccharides from Shuanghu capsules.

Optimization of the Ethanol Extraction Technology of Total Alkaloids from Shuanghu Capsules by Orthogonal Design / 中国药房

OBJECTIVE： To optimize the ethanol extraction technology of total alkaloids from Shuanghu capsules. METHODS： Using dendrobine as control， the contents of total alkaloids from Dendrobium nobile and Dendrobium officinale in Shuanghu capsules were determined by acidic dyes colorimetry. Using comprehensive scores calculated by the yield of the extract and the contents of total alkaloids as evaluation indexes， the effects of soaking time， ethanol volume fraction， extraction time， solid-liquid ratio and extraction times were investigated with single factor tests. L9（34） orthogonal test was used to optimize ethanol volume fraction， extraction time， solid-liquid ratio and extraction times according to the results of single factor test. The optimized technology was validated. RESULTS： The linear range of dendrobine were 4.16-14.56 μg/mL （r＝0.999 2）. RSDs of repeatability and precision tests were all lower than 5％. Average recovery tests were 93.01％ （RSD＝1.97％， n＝6）. The optimal ethanol extraction technology included soaking for 12 h， ethanol volume fraction of 70％， solid-liquid ratio of 1 ∶ 12 （g/mL）， extracting for 28 min， extracting 3 times. Results of validation test showed that the average yield of extract was 12.80％ （RSD＝4.39％， n＝3）， and the content of alkaloids was 0.359 0 mg/g（RSD＝0.66％， n＝3）. CONCLUSIONS： Established acidic dyes colorimetry is simple， precise and accurate， which can be used for the content determination of total alkaloids. The optimized ethanol extraction technology is stable and feasible， and can be used for the extraction of total alkaloids from Shuanghu capsules.

Chemical constituents from Dendrobium nobile / 中成药

AIM To study the chemical constituents from Dendrobium nobile Lindl..METHODS The 95％ ethnol extract from D.nobile was isolated and purified by silica and Sephadex LH-20,then the structures of obtained compounds were identified by physicochemical properties and spectral data.RESULTS Eight compounds were isolated and identified as erianin (1),gigantol (2),moscatin (3),confusarin (4),4,5-dihydroxy-2-methoxy-9,10-dihydro-phenanthrene (5),coumarin (6),hexadecanoic acid,methyl ester (7),dibutyl phthalate (8).CONCLUSION Compounds 1,3,5,6,7,8 are isolated from this plant for the first time.

Optimization of combined enzymatic extraction for alkaloids and polysaccharides from Dendrobium nobile / 中成药

AIM To optimize the combined enzymatic extraction for alkaloids and polysaccharides from Dendrobium nobile Lindl..METHODS With enzyme consumption,enzymolysis temperature,enzymolysis time and solidliquid ratio as influencing factors,contents of dendrobine,total alkaloids and polysaccharides as evaluation indices,orthogonal test was applied to optimizing the combined enzymatic extraction.RESULTS The optimal conditions for papain extraction were determined to be 0.10 g for enzyme consumption,45 ℃ for enzymolysis temperature,2 h for enzymolysis time,and 1 ∶ 50 for solid-liquid ratio,the contents of dendrobine,total alkaloids and polysaccharides were 3.495 5,4.341 8 and 35.898 7 mg/g,respectively.The optimal conditions for cellulase extraction were determined to be 0.30 g for enzyme consumption,50 ℃ for enzymolysis temperature,2 h for enzymolysis time,and 1 ∶40 for solid-liquid ratio,the contents of three constituents were 3.514 8,4.351 3 and 36.331 2 mg/g,respectively.The optimal conditions for pectinase extraction were determined to be 0.45 g for enzyme consumption,55 ℃ for enzymolysis temperature,2.5 h for enzymolysis time,and 1 ∶ 40 for solid-liquid ratio,the contents of three constituents were 3.524 4,4.452 8 and 26.324 2 mg/g,respectively.CONCLUSION This stable and reliable method can be used for the rapid combined enzymatic extraction for alkaloids and polysaccharides from D.nobile.

Separation and Extraction of Total Flavonoids from Dendrobium nobile Leaves and Study on Its Anti-Alzheimer's Disease Activity in vitro / 中国药房

OBJECTIVE: To separate and isolate total flavonoids from Dendrobium nobile leaves, and to investigate its anti-Alzheimer's disease (AD) activity in vitro. METHODS: Total flavonoids were obtained by ultrasonic extraction method and extracted by chloroform, ethyl acetate and butyl alcohol after the obtained extract was dispersed with water. Qualitative analysis was carried out with color reaction and TLC. The content of total flavonoids in extracts was analyzed quantitatively by Aluminum nitrate-sodium nitrite method. Antioxidant activity of extract was investigated by DPPH radical scavenging assay; the inhibitory effect of each extract on Aβ42 protein aggregation was investigated by Thioflavin T assay. Metals (Cu2+, Fe3+, Zn2+) chelating property was studied by UV-vis spectrum scanning to investigate the anti-AD activity in vitro. RESULTS: The flavonoids were found in ethyl acetate, butyl alcohol and aqueous extracts, and their flavonoids contents were 0. 03, 0. 12, 0. 05 mg/mL, respectively. IC50 of three extracts to DPPH free radicals were 0. 021, 0. 011, 0. 013 mg/mL. Inhibitory rates of them to Aβ42 protein aggregation were 43. 77％, 52. 28％, 38. 42％, respectively. Three extracts exerted metal chelating ability which was best in Cu2+. CONCLUSIONS: The total flavonoids from D. nobile leaves have good antioxidant activities, Aβ42 aggregation inhibitory activities and metal chelating activity, show certain anti-AD activity in vitro especially in butyl alcohol extract.

Comparison of Polysaccharide and Dendrobine Content in Hejiang Dendrobium nobile at Different Harvesting Time / 中国药房

OBJECTIVE:To determine quality indexes of Hejiang Dendrobium nobile at different harvesting time and months,and to provide scientific reference for reasonable harvesting period of Hejiang D.nobile.METHODS:The stem of annual,biennial and triennial D.nobile were collected.The drying rate,the contents of polysaccharides (colorimetry),dendrobine (GC) and total alkaloids (colorimetry),effective component total rate (dendrobine×drying rate) were measured.Biennial D.nobile were collected in autumn and winter (Oct.of the second year-Mar.of the third year,15th day a month) to determine drying rate,the contents of polysaccharides and dendrobine.RESULTS:The drying rate,the contents of polysaccharides and effective component total rate were higher than annual and triennial D.nobile.The content of total alkaloids and dendrobine were in descending order:annual D.nobile (0.52％,0.48％) ＞biennial D.nobile(0.48％,0.44％) ＞triennial D.nobile (0.32％,0.22％).From Oct.of the second year to Mar.of the third year,the drying rate of biennial D.nobile was increasing month by month;the contents of polysaccharides and dendrobine increased firstly and then decreased;the content of polysaccharide was the highest in Feb.(17.32％),and the content of dendrobine reached the highest level in Dec.(0.51％).CONCLUSIONS:The optimal harvesting period is biennial Hejiang D.nobile in Dec.and Jan.(third years before flowering),considering flowering characteristics,drying rate,the contents of polysaccharide and dendrobine.

Antitumor Phenanthrene Constituents of Dendrobium nobile / 中国药学杂志

OBJECTIVE: To study the antitumor phenanthrene constituents in Dendrobium nobile.METHODS: A variety of chromatographic techniques were applied to extract, isolate and purify natural products from extract of Dendrobium nobile, their structures were identified on the basis of spectral data. The antitumor activity of these monomers in MCF-7 breast cancer cells was studied by MTT assay in vitro. RESULTS: Nine compounds were isolated from ethyl acetate part in Dendrobium nobile, there were four phenanthrene compounds, which were densiflorol B(1), cypripedin(2), moscatin(3) and 2,4,8-trimethoxy phenanthrene-3,7-diol(4); three coumarin compounds: coumarin(5), moellendorffiline(6) and isopimpinellin(7); one flavanoid: naringenin(8); one phenol: (E)-4-(2-methoxyvinyl) benzene-1,2-diol(9). Compounds 1-3 had good antitumor activities, their IC50 values were 2.99, 5.01 and 72.68 μmol•L-1 respectively. CONCLUSION: The compounds 6, 7, 9 are isolated from the Orchidaceae family for the first time, compounds 1, 2, 4, 5, 8 are isolated from this plant for the first time. Compounds 1-3 can be used as candidates for anti-tumor drugs.

DNA marker-assisted selection of medicinal plants (Ⅰ) .Breeding research of disease-resistant cultivars of Panax notoginseng / 中国中药杂志

SSR is one of the most important molecular markers used in molecular identification and genetic diversity research of Dendrobium nobile. In order to enrich the library of SSR and establish a method for rapid identification of D. nobile, the SSR information was analyzed in the transcriptome of D. nobile. A total of 32 709 SSRs were obtained from the transcriptome of D. nobile, distributed in 26 742 unigenes with the distribution frequency of 12.90%. SSR loci occurred every 3 748 bp. Mono-nucleotide repeat was the main type, account for as much as 72.18% of all SSRs, followed by di-nucleotide (15.97%) and tri-nucleotide (11.19%). Among all repeat types, A/T was the predominant one followed by AG/CT. Finally a total of 62 157 primer pairs were designed for marker development. Randomly 20 pairs of primers were selected for PCR amplification, 17 amplified on clear and reproducible bands, the amplification rate was 85.0%.Thirteen pairs were polymorphic among the 3 Dendrobium plants. The results indicated that the unigenes generated from transcriptome sequencing in D. nobile can be used as effective source to develop SSR markers. The SSR loci in the transcriptome of D. nobile have the characteristics of type riches, high density and high potential of polymorphism, and these characteristics might applied in the study of molecular identification, genetic diversity and marker-assisted breeding of D. nobile and its closely related species.