Search

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.

Two new phenylpropanoid derivatives ( 1-2 ), together with eight known compounds ( 3-10 ) were isolated from the stems of Dendrobium sonia . The structures of these compounds were elucidated on the basis of spectroscopic analyses, including HRESIMS, 1 D and 2 D NMR experiments. All of the isolated compounds were tested for their Nitric Oxide (NO) Inhibitory Activities. The results of bioactive screening showed that compounds 2 , 8 , 9 and 10 exerted inhibitory effects on NO production with IC 50 values in the range of 26.3 to 31.6 μM. Compound 8 and 9 exhibited stronger anti-inflammatory activities with IC 50 values 26.3 and 27.7 μM, comparable to that of the positive control.

Fifteen bibenzyls were isolated and purified from the ethyl acetate extract of the stems of Dendrobium officinale by macroporous resin, MCI, silica gel, Sephadex LH-20, and ODS column chromatographies, as well as preparative thin-layer chromatography and preparative HPLC. The structures of compounds were identified according to the spectra data of ~1H-NMR, ~(13)C-NMR, and MS, and the physical and physiochemical properties: dendrocandin X(1), 3,4'-dihydroxy-4,5-dimethoxybibenzyl(2), 6″-de-O-methyldendrofindlaphenol A(3), 3,4-dihydroxy-4',5-dimethoxybibenzyl(4), dendrosinen B(5), 3,4,4'-trihydroxy-5-methoxybibenzyl(6), 3,3'-dihydroxy-4,5-dimethoxybibenzyl(7), 3,4'-dihydroxy-5-methoxybibenzyl(8), moscatilin(9), gigantol(10), 4,4'-dihydroxy-3,5-dimethoxybibenzyl(11), 3,4',5-trihydroxy-3'-methoxybibenzyl(12), 3-O-methylgigantol(13), dendrocandin U(14), and dendrocandin N(15). Compound 1 was a novel compound. Compound 2 was isolated from Dendrobium species for the first time. Compounds 3-7 were isolated from D. officinale for the first time.

A novel HPLC method with the quantitative analysis of multi-components by single marker( QAMS) combined with the dual-wavelength method was developed for simultaneous determination of six flavonoids in Dendrobium officinale stems from different producing areas,cultivation and processing methods to clarify the main factors contributing to the different composition of flavonoids.The separation of six flavonoids was performed on a Shiseido Capcell PAK MGⅡ C18 column( 4. 6 mm×250 mm,5 μm) using a linear gradient elution system of acetonitrile-0. 1% formic acid aqueous solution. Schaftoside,isoschaftoside,vicenin-2,and glucosylvitexin were simultaneously analyzed using rutin as a reference standard at detection wavelength of 340 nm,and naringenin was determined at290 nm. The credibility and feasibility of QAMS method were validated and the results demonstrated that no significant differences were observed as compared with the external standard method. Finally,a total of 82 batches of D. officinale samples were analyzed and principal component analysis( PCA) and discriminant analysis were applied to distinguish and compare D. officinale samples from different producing areas,cultivation and processing methods. The results showed that the total flavonoid content of D. officinale stems cultivated in the simulated wild( attached tree cultivation or attached stone cultivation) was significantly higher than that in greenhouse bed cultivation. The content of flavonoids in simulated-wild D. officinale stems was higher in Jiangxi,Guizhou,Zhejiang,and Fujian provinces,while that in greenhouse bed cultivation was higher in Fujian and Zhejiang provinces. The content of naringenin was positively correlated with processing temperature,and that of the other five flavonoids was negatively correlated with processing temperature. PCA showed that wild-simulated D. officinale and greenhouse bed-cultivated D. officinale could be roughly divided into two clusters. The samples cultivated in the greenhouse bed were divided into four categories according to the geographical habitats. Wild-simulated D. officinale samples from Guizhou gathered together,and there was no obvious rule in samples from other producing areas. The established method simplified the determination method of flavonoids in D. officinale,and could provide the basis for effective quality control,cultivation and processing of D. officinale.

The violanthin, a specific component, was separated and identified from the stems of Dendrobium officinale by chromatographic technique and spectroscopic method for the first time. The microscopic characteristics of D. officinale powder were examined under a microscopy and described. Thin layer chromatography (TLC) method was used for qualitative analysis of the violanthin from D. officinale stems with a mixture of ethyl acetate, butanone, formic acid and water (4∶3∶1∶1) as the developing solvent on high performance silica gel precoated plate (SGF254) and using aluminium trichloride as a chromagenic agent. The results showed significant characteristics of violanthin from D. officinale stems on TLC, with certain specificity, and could be used to distinguish it from other easily confusing processed medicinal stems of D. devonianum, D. gratiosissimum and D. aphyllum. The content of naringenin, an active ingredient in D. officinale stems was determined by HPLC analysis on a Bischoff Chromatography HIPAK NC-04 ODS AB column (4.4 mm×250 mm, 5 mm) with acetonitrile-0.1% phosphoric acid solution as the mobile phase for gradient elution. The wavelength was set at 226 nm and column temperature was 25 ℃. The HPLC method showed good linearity within the range of 3.90-250.00 g•mL⁻¹ (r = 0.999 9) for naringenin. The average recovery of naringenin was 99.20% with 0.17% of RSD. The mass fraction of 20 batches of D. officinale stems was between 0.190 and 0.498 mg•g⁻¹. The established qualitative and quantitative method was simple and rapid with good repeatability and accuracy, providing experimental basis for improving the quality standard of D. officinale, with a very important significance to ensure its quality and clinical effect., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Isolation of high-quality genomic DNA from dried and processed crude drug is the key for the DNA identification of Dendrobii Caulis. The DNA extract of Dendrobii Caulis was firstly compared using different method to isolate genomic DNA from dried and processed crude drug, including commercial DNA extracted kit and CTAB method. Using modified CTAB method (extracted from large samples), the genomic DNA was successfully isolated from Dendrobii Caulis, including Huangcao and Fengdou. The ITS regions were amplified using the purified DNA as template, and then cloned and sequenced. These ITS sequences were compared with data from Genbank database and our lab, 14 Dendrobium species and five similar species were identified from "Huangcao" and "Huangcao" slice, while six species and three similar species from "Fengdou" according to their sequence similarity. The study demonstrated that the dried Dendrobii Caulis could be identified using DNA molecular method, which could overcome deficiencies and limitations of traditional identification method and has a certain application prospects.

Diabetic retinopathy (DR) is the most common and serious complication of diabetes mellitus (DM). The present study investigates the amelioration of ethanol extract of Dendrobium chrysotoxum Lindl (DC) on streptozotocin (STZ)-induced DR and its engaged mechanism. Retinal immunofluorescence staining with cluster of differentiation 31 (CD31) demonstrated that DC (30-300 mg/kg) decreased the increased retinal vessels in STZ-induced diabetic rats. Retinal histopathological observation also showed that retinal vessels were decreased in DC-treated diabetic rats. DC decreased the increased retinal mRNA expression of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) in diabetic rats, and DC also decreased the elevated serum VEGF level. Immunohistochemical staining further evidenced that DC decreased VEGF and VEGFR2 expression in retinas. Retinal mRNA expression of matrix metalloproteinase (MMP) 2/9 was decreased in DC (300 mg/kg)-treated diabetic rats. Serum levels of MMP 2/9, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) A/B, insulin-like growth factor 1 (IGF-1), interleukin 1β (IL-1β), and IL-6 were all decreased in DC-treated diabetic rats. In addition, DC decreased the increased phosphorylation of p65 and the increased expression of intercellular adhesion molecule-1 (ICAM-1). In conclusion, DC can alleviate retinal angiogenesis during the process of DR via inhibiting the expression of VEGF/VEGFR2, and some other pro-angiogenic factors such as MMP 2/9, PDGF A/B, bFGF, IGF-1. In addition, DC can also ameliorate retinal inflammation via inhibiting NFκB signaling pathway., (Copyright © 2014 Elsevier Inc. All rights reserved.)

To establish the local quality standard for Dendrobii devoniani caulis from Longling, Yunnan, the pharmacognostic characteristics microscopic characteristics and TLC identification were developed. Sulfuric acid-phenol method was used to determine the content of polysaccharide. An HPLC method was adopted to determine the content of mannose, and extractives were determined according to the procedures recorded in the Appendix of Chinese Pharmacopoeia(2010). The results showed a strong characteristics microscopic of Dendrobii devoniani caulis, and its TLC identification had a good resolution with clear spots; the content of polysaccharide is 35.7% -52.1% (average 42.7%), mannose 27.8%-46.1% (average 35.8%), and extract 4.5%-10.6% (average 7.38%). The method is simple, accurate and reliable, with good reproducibility. The established standard is acceptable for quality evaluation of Dendrobii devoniani caulis from Longling, Yunnan.

A method was developed for rapid identification of Dendrobium species by a dot blot hybridization assay. The dot blot system is based on species-specific amplified fragments derived from the internal transcribed spacer (ITS) region of different Dendrobium species as target DNA, which were blotted as dots on the nylon membrane. A small aliquot of the ITS amplified product from a selected species or mixed ITS DNA from several species was labeled by peroxidase and used as a probe for hybridization to the dot blot membrane. Simply visualizing the positive hybridization reaction between the probe DNA and the target DNA could identify the selected species. The D. officinal-specific and D. nobile-specific ITS-based probes could separately identify D. officinal and D. nobile. Eight Dendrobium species, including D. salaccense, D. brymerianum, D. ellipsophyllum, D. chrysanthum, D. officinale, D. thyrsiflorum, D. nobile and D. chrysotoxum could be simultaneously detected using their complex ITS amplified products as a probe, and the ITS DNA probe amount for detection by the dot blot membrane is very small, just 2.5 ng. The assay can also be used to identify the resource species of Fengdou Shihu and Huangcao Shihu from the commercial market. The identification based on the ITS sequence was superior to the conventional approaches in speed, sensitivity, and specificity. Therefore, the polymerase chain reaction (PCR)-dot blot hybridization assay can be considered a reliable method for general identification of commercial Shihu.

Five new compounds, dencryol A (1), dencryol B (2), crystalltone (3), crystallinin (4), and 3-hydroxy-2-methoxy-5,6-dimethylbenzoic acid (5), together with six known compounds, dendronobilin B (6), syringic acid (7), apigenin (8), isoviolanthin (9), 6'''-glucosyl-vitexin (10), and palmarumycin JC2 (11), have been isolated from the stems of Dendrobium crystallinum, of which compounds 9-11 were isolated from the genus Dendrobium for the first time, and all the other compounds were first obtained from this plant. Their structures were established on the basis of spectroscopic analysis and literature data.

Objective: To study the phenolic constituents of Dendrobium aphyllum., Method: Various chromatographic techniques were used to isolate and purify the constituents, their physico-chemical properties and spectral data were employed to elucidate their structures., Result: Nine bibenzyls and two benzylethanyl compounds were isolated and identified as: moscatilin (1), gigantol (2), batatasin (3), tristin (4), 3, 5, 4'-trihydroxylbibenzyl (5), 3, 5-dimethoxyl-4, 4'-dihydroxylbibenzyl (6), moscatin (7), 2, 4, 7-trihydroxyl-9, 10-dihydrophenanthrene (8), hircinol (9), 2-(4-hydroxyphenyl) ethyl-beta-D-glucopyranoside, salidroside (10) and p-hydroxylbenzylacetic acid (11)., Conclusion: All compounds were obtained firstly from the plant, and the compounds 10 and 11 were isolated in this genus for the first time.

Objective: To study the chemical constituents of Dendobium crystallinum., Method: Compounds were isolated and purified by silica gel and Sephadex LH-20 column chromatography. Their structures were identified by physicochemical properties and spectral analyses., Result: Nine compounds were obtained and identified as: 4, 4'-dihydroxy-3, 5-dimethoxybi-benzyl (1), gigantol (2), naringenin (3) , p-hydroxybenzoic acid (4), n-tetracosyl trans-p-cou-marate (5), n-octacosy trans-p-coumarate (6), n-hexacosyl trans-ferulate (7), stigmasterol (8), daucosterol (9)., Conclusion: All these compounds were obtained from this plant for the first time, compounds 1 and 4 were isolated firstly from the genus.

Objective: To study the active constituents of Dendrobium aphyllum., Method: Various chromatographic techniques were used to isolate and purify the constituents, their physico-chemical properties and spectral data are determinated to elucidate the structure., Result: Eight compounds were isolated and identified as: 4'-methoxyl-tricin (1), tricin (2), 7, 3', 5'-tri-O-methyl-tricetin (3), syringic acid (4), ( + )-syring-aresinol (5), D-allitol (6), sucrose (7), icariside D2 (8)., Conclusion: Compounds 1-3, 6-8 were isolated from genus Dendrobium for the first time, additionally, the others were obtained firstly from the plant.

Objective: To study the chemical constituents of Dendrobium gratiosissimum., Method: The constituents were isolated and purified by silica gel, Sephadex LH-20 colum chromatography, and the structures were identified by physicochemical properties and spectral data., Result: Nine compounds were obtained as: 3,5,4'-trihydroxybibenzyl (1), 3,4'-dihydroxy-5-methoxybibenzyl (2), 3,4-dihydroxy-4', 5-dimethoxybibenzyl (3), apigenin (4), p-hydroxybenzaldehyde (5), defuscin (6), n-octacostyl ferulate (7), beta-sitosterol (8) and daucosterol (9)., Conclusion: All these compounds were obtained from this plant for the first time, and compounds 2, 4 and 5 were isolated firstly from this genus.

The aim of this study was to establish a diagnostic PCR method for authentication of D. thyrsiflorum Rchb. f. Based on rDNA ITS sequences of the 164 samples from 109 Dendrobium species sequenced and quoted from GenBank, the allele-specific diagnostic primers QH-JB1 and QH-JB2 for authentication were designed. Using the primers, diagnostic PCR was performed with the total DNA of D. thyrsiflorum Rchb. f. and other D. species as templates to establish the positive PCR condition for D. thyrsiflorum Rchb. f. A DNA fragment about 300 bp was amplified from D. thyrsiflorum Rchb. f. with anneal temperature at 63.5 degrees C and anneal time at 1 min, whereas no other DNA fragment was amplified from the rest D. species. The allele-specific diagnostic PCR was proved to be a simple and quick identification technique, suitable for the authentication of both fresh and dried materials of D. thyrsiflorum Rchb. f.

Investigation of phenolic patterns from the stems of Dendrobium chrysanthum by HPLC-PDA-MS has led to the isolation of a new phenanthrene derivative with a spirolactone ring, dendrochrysanene (1), that proved to suppress the mRNA level of TNF-alpha, IL8, IL10, and iNOS in murine peritoneal macrophages. The structure of 1 was characterized on the basis of various NMR (1H, 13C, 1H-1H COSY, HMQC, and HMBC), mass spectrometry, and X-ray crystal diffraction data.

Objective: To investigate the chemical constituents of Dendrobium chrysotoxum., Method: The chemical constituents were isolated by various column chromatographic methods and structurally elucidated by spectral evidences., Result: Ten compounds were obtained and identified as (+)-syringare sinol (1), 5alpha, 8alpha-epidioxy-24( R)-methycholesta-6, 22-dien-3beta-ol (2), trans-3-(4-hydroxy-3-methoxyphenyl)-acrylic acid octacosyl ester (3), defusin (4), 3, 4-dihydroxy benzoic acid (5), 3, 4-dimethoxy-benzoic acid (6), vanillic acid (7), 3, 4-dimethoxy-benzoic acid methyl ester (8), 3, 5-dibromo-2-aminobenzaldehyde (9), heptadecanoic acid 2, 3-dihydroxy-propyl ester (10)., Conclusion: Compounds 1, 2 and 6-10 were isolated from this plant for the first time.

Aim: To establish a simple method for molecular identification of original plants of D. chrysanthum and D. fimbriatum using molecular marker rDNA ITS region., Methods: Restriction patterns of ITS fragments were obtained using PCR-RFLP method. The PCR products of D. chrysanthum and its morphologically allied species were digested at 37 degrees C by Cla I and Apa LI, those of D. fimbriatum and its morphologically allied species were digested by Sph I., Results: D. chrysanthum, D. fimbriatum and their morphologically allied species could be identified by predicted restriction profiles of PCR-RFLP. The botanical origin of twenty-five fresh samples of "Shihu" collected in markets was identified by this method., Conclusion: The results showed that PCR-RFLP analysis of the rDNA ITS region is a feasible, simple and inexpensive method for determining the botanical origin of the traditional Chinese medicine "Shihu".

One bi-bicyclic and two bi-tricyclic derivatives of coumarin-benzofuran, phenanthrene-phenanthrene and phenanthrene-phenanthraquinone, along with seven known compounds, were isolated from stems of Dendrobium thyrsiflorum Rchb.f. (Orchidaceae). On the basis of chemical, NMR (1H, 13C, HMQC, HMBC and NOESY) and mass spectrometry data, their structures were elucidated as denthyrsin [3-(5',6'-dimethoxybenzofuran-2'-yl)-6,7-dimethoxy-2H-chromen-2-one; 1], denthyrsinol (4,5'-dimethoxy-[1,1']biphenanthrenyl-2,5,4',7'-tetraol; 2), and denthyrsinone (7,4',7'-trihydroxy-2,2',8'-trimethoxy-[5,1']biphenanthrenyl-1,4-dione; 3). Compounds 1-3 and denthyrsinin (1,5,7-trimethoxyphenanthrene-2,6-diol; 4) showed significant cytotoxic activities against Hela (13.5, 9.3, 9.9 and 2.7 microM, respectively), K-562 (0.45, 1.6, 6.0 and 2.3 microM, respectively) and MCF-7 (18.1, not tested, 3.5 and 4.8 microM, respectively) cell lines.

Aim: To determine the location and relative quantity of coumarins in the stem of Dendrobium thyrsiflorum Rchb. f. , and to provide a scientific basis for evaluating and utilizing the famous medicinal plant., Methods: The stems of one, two and three years old, separately, were collected in February. Location and relative quantity of coumarins in the top, middle and basal parts of each stem sample were determined by using laser scanning confocal microscopy (LSCM). ANOVA and Tukey's test were employed in the statistical analysis., Results: The coumarins located mainly in vascular bundles, especially on the walls of the outer fiber cells. There was significant difference or great significant difference exist in every part of the stem from different ages. The values of significant difference were as follows: basal part P = 0.004 (< 0.01); middle part P = 0.009 (< 0.01); top part P = 0.036( < 0.05)., Conclusion: Two years old stem could be the best choice when collecting Dendrobium thyrsiflorum in Feburary.

Two new fluorenones have been isolated from the orchid Dendrobium chrysotoxum Lindl. and their structures established as 2,4,7-trihydroxy-5-methoxy-9-fluorenone and 2,4,7-trihydroxy-1,5-dimethoxy-9-fluorenone.

A DNA microarray for detecting processed medicinal Dendrobium species (Herba Dendrobii) was constructed by incorporating the ITS1-5.8S-ITS2 sequences of 16 Dendrobium species on a glass slide. Using fluorescence-labeled ITS2 sequences as probes, distinctive signals were obtained for the five medicinal Dendrobium species listed in the Chinese Pharmacopoeia. The established microarray was able to detect the presence of D. nobile in a Chinese medicinal formulation containing nine herbal components.

Aim: To investigate the chemical constituents of Dendrobium fimbriatum Hook., Methods: Various chromatographic techniques were employed for isolation and purification of the constituents. The structures were elucidated by IR, MS, 1HNMR, 13CNMR and 2D-NMR., Results: Eight compounds were obtained and identified by spectral analysis as fimbriatone, confusarin, crepidatin, physcion, rhein, ayapin, scopolin methyl ether and n-octacostyl ferulate., Conclusion: Fimbriatone is a new compound, physcion and rhein are firstly isolated from Genus Dendrobium. The others are found from this species for the first time. Fimbriatone showed potential inhibitory effects on BGC cell line.

Objective: To analyse the 1H-NMR finger-print of the stem of Dendrobium loddigesii., Method: Silica gel column chromatography was used to separate the chemical constituents of SCE A of the stem of D. loddigesii. The characteristic signals of the H-NMR finger-print were analysed after determining the structures of the compounds isolated from SCE A., Result: 1H-NMR finger-prints of the samples of D. loddigesii collected from different regions showed highly characteristic features and reproducibility. Four compounds predominant in SCE A were isolated and their structures were determined by spectral analysis as 1, 2, 3 and 4, respectively., Conclusion: Compound 3 and 4 were isolated from D. loddigesii for the first time. The 1H-NMR finger-print of CGE A of the stem of D. loddigesii showed mainly the characteristic signals of the above four compounds and might be utilized for the original authentication of this plant.

Aim: To establish the whole rDNA ITS region sequence database of various Dendrobium species of "Fengdou" and to authenticate exactly the inspected species of "Fengdou"., Methods: The rDNA ITS regions of various Dendrobium species of "Fengdou" were amplified and sequenced. The database of their rDNA ITS regions was established in order to authenticate the inspected species by means of the softwares of CLUSTRAL and MEGA which were used to analyze the rDNA ITS region., Results: A database of the rDNA ITS sequences of 21 species of Dendrobium has been established. The notable and stable differences of the interspecies of the rDNA ITS regions have been demonstrated. The numbers of transitions and transversions among 21 species are 11-122. The variable sites are 341 while the informative sites are 195. The ITS sequence differences between the outgroup species (Pholidota yunnanensis) and species of "Fengdou" are obvious. The numbers of transitions and transversions are 131-161. The population differences of the rDNA ITS region of various species of "Fengdou" are very small (0-6)., Conclusion: On the basis of the database of various Dendrobium species of "Fengdou" and two genetics software, the botanical origin of the inspected species of "Fengdou" has been authenticated successfully by sequencing the rDNA ITS regions.

Objective: To define molecular characters to distinguish D. chrysanthum from its allied species D. primulinum, D. lituiflorum, D. aphyllum, D. crepidatum., Method: The molecular characteristics of D. chrysanthum and its allied species were compared. The sequences of rDNA ITS regions were exploited to explore the evidence for authentication D. chrysanthum and its allied species., Result: Although the morphological difference was slight, the sequence difference of ITS regions among five rDNAs was obvious and stable. Fifteen sites of ITS region were defined as DNA character to identify D. chrysanthum from the other four allied species., Conclusion: The difference of rDNA ITS sequences can be used to authenticate accurately D. chrysanthum from three allied species of Dendrobium.

Objective: To study rDNA ITS sequence differences between F type and that of H type of Dendrobium officinale in main habitat of China., Method: The population differences of the rDNA ITS region (including ITS1, ITS2, 5.8S) sequences of D. officinale were studied by the method of DNA sequences analysis., Result: There were two different sites between the rDNA ITS sequence of F type and that of H type. One was in ITS1 region, and the other was in 5.8S region. It was proved that there was some relativity between the character of rDNA ITS region and the life type of the populations. The phenomenon of single nucleotide polymorphism (SNP) existed in 5.8S region of rDNA ITS region between F type and H type. The sequences of rDNA ITS region of D. officinale were reported for the first time, and the sequences of ITS region ranged 634 bp (ITS1 231 bp, ITS2 240 bp, 5.8S 163 bp)., Conclusion: The analysis of rDNA ITS of D. officinale deeply reveal the population differences of D. officinale of F type and H type.

Aim: To study the genetic diversity of ITS sequences of Herba Dendrobii (Huangcao) and analyze the utility of ITS sequences in molecular authentication of Herba Dendrobii (Huangcao) and phylogenetic of Dendrobium., Methods: The ITS gene fragment was amplified using a pair of primers. The PCR products were purified and sequenced by the methods of Sanger Dideoxy., Results: The DNA sequence of 228-233 bp ITS 1, 242-247 bp ITS 2 gene fragments and 5.8S rDNA were obtained from 14 samples of Dendrobium. The interspecific substitution varies from 11.79% to 31.58% at ITS 1 and 10.29% to 25.30% at ITS 2. The intraspecific substitution of D. nobile is 0.87% at ITS 1 and without difference at ITS 2. The substitution between Dendrobium and outgroup varies from 23.56% to 36.89% at ITS 1 and 26.52% to 33.31% at ITS 2. The phylogenetic tree based on ITS 1 and ITS 2 data was set up., Conclusion: The ITS 1 and ITS 2 gene fragments were highly conservative at intraspecific level in Dendrebium, while they were less conservative at interspecific level in D. nobile. They were least conservative between Dendrobium and outgroup. Hence, the sequence of this fragment is a good molecular marker for authentication of the Huangcao. But, further study is necessary for the phylogenetic of Dendrobium.

Objective: To set up a system for the culture of Dendrobium chrysotoxum in vitro., Method: Tissue culture, fire fly luminescence and phenol-H2SO4 method., Result: The embryo could germinate with or without light, the MS, 1/2MS, B5, N6 mediums are suitable to the growth and the differentiation of sprout with light, 0.5 mg.L-1 NAA and 1 mg.L-1 6-BA, and ATP have regular changes, the content of polysaccharide was 2.833% in plant and 7.254% in sprout., Conclusion: The light has no effects on the embryo germination, but the phytohormone, nitrogen source and organized elements are important to the growth and differentiation of the sprout which should be transferred to the MS, 1/2MS, B5, N6 mediums in time supplemented with NAA [symbol: see text] 6-BA, ATP may be served as the dynamic indication of nourishment demand in the plant. The content of polysaccharide in the sprout is higher and can be utilized.