Résumé
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.
Sujets)
Agriculture , Dendrobium/génétique , Amélioration des plantes , Feuilles de plante/génétique , PolyosidesRésumé
Alkaloids were regarded as important biological activity constituent in medicinal Dendrobium Sw., being characteristic pharmacodynamic components in plants from Dendrobium Sw. A total of 52 constituents of alkaloids were found in plants of Dendrobium Sw. They could be divided into five types, according to different structures. They were sesquiterpenoid alkaloids, octahydroindolizine alkaloids, amides alkaloids, pyrrolidine alkaloids, and imidazole alkaloids etc. Sesquiterpenoid alkaloids could be divided into dendrobine type, dendroxine type, nobiline type, etc. Over 19 Dendrobium species contain alkaloids, and the main species included D. nobile, D. chrysanthum, D. crepidatum, D. findleyanum, D. signatum, D. friedericksianum, etc. This paper reviews the structural classification of alkaloids in Dendrobium, and the species which containing alkaloids and the influencing factors of alkaloids in Dendrobium Sw., in order to provide reference for the in-depth study and the development of alkaloids in plants from Dendrobium Sw.
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OBJECTIVE:To establish a method for the determination of dendrobine and its metabolites M-250 and M-280 in mice plasma for the first time. METHODS:Mice were given dendrobine 60 mg/kg by intragastric administration,1 h later plasma were collected and treated. Using pseudoephedrine hydrochloride as internal standard and dendrobine reference substance as control, the plasma concentrations of dendrobine and its metabolites M-250 and M-280 were determined by UPLC-MS combined with quantitative analysis of multi-components by single marker. The separation was performed on Hypersil Gold C18 column with 0.1％formic acid-acetonitrile(gradient elution)at the flow rate of 0.3 mL/min. The column temperature was set at 40℃,and sample size was 5 μL. Heatable electrospray ionization (HESI) source, scan/ESI + were applied and operated in positive ion mode with atomization temperature of 300℃,ion transmission tube temperature of 350℃,the sheath gas velocity of 35 arb,the auxiliary air velocity of 15 arb,the spray voltage of 3.5 kV,the collision voltage of 30,40,50 eV. The mass-to-charge ratio of detection range were 100-1500. RESULTS:The endogenous substances of mice plasma had no interference with the content determination of dendrobine and its metabolites M-250 and M-280. The linear range of dendrobine were 9.13-912.94 ng/mL(r=0.9996). The limit of quantitation was 3.04 ng/mL. RSDs of intra-day and inter-day were all less than 7.5％(n=5 or n=3). The accuracy were 96.8％-107.5％(n=5). Matrix effects were 97.1％-106.0％(RSD=1.8％-4.7％,n=5). RSDs of the content of sample at 15℃ for 24 h,at -70 ℃ after three times freeze-thaw,at -70 ℃ for 15 d were lower than 12.8％ (n=3). The content of dendrobine in plasma sample of mice was (41.3 ± 5.7) ng/mL (n=12). The contents of its metabolites M-250 and M-280 were (493.0 ± 73.1) and (41.4 ± 3.0) ng/mL (n=12) with Relative correction factor of 1.0. CONCLUSIONS: The method is sensitive and accurate,and can be used for content determination of dendrobine and its metabolites M-250 and M-280 in mice plasma.
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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.
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OBJECTIVE@#To ascertain the influence of light intensity and water content of medium on the total dendrobine of Dendrobium nobile (D. nobile).@*METHOD@#The principal component analysis combined with total dendrobine accumulation was conducted to assess the yield and quality of D. nobile in all treatments. In the experiment, D. nobile plants were cultivated in greenhouse as tested materials, and complete test of 9 treatments was adopted with relative light intensities 75.02%, 39.74%, 29.93% and relative water content of medium 50%, 65%, 80%. The plants were treated in June and harvested till December. Indexes including agronomic traits, fresh weight and dry weight of stem and leaf, ash content, extract, and dendrobine were measured.@*RESULTS@#Under the light intensity treatments of 75.02% with 50%, 65%, 80% water content of medium, the basal stems of plants were comparatively thicker with more leaves, and the fresh weight and dry weight of stems and leaves were significantly higher than other 6 treatments. Leaves in all treatments contained dendrobine. Under the light intensity treatments of 75.02% with 50%, 65%, 80% water content of medium, dendrobine content of leaves was lower while dendrobine contents of other treatments were more than 0.60%. After comprehensive assessment through the principal component analysis and total dendrobine accumulation, the results showed that 3 treatments with relative light intensity of 75.02% ranked the top three.@*CONCLUSIONS@#In brief, the moderately strong light intensity and water content of medium from low to medium can facilitate the growth and yield of D. nobile plants, while light intensity from moderately weak to weak can enhance the dendrobine content.
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Objective To ascertain the influence of light intensity and water content of medium on the total dendrobine of Dendrobium nobile (D. nobile). Method The principal component analysis combined with total dendrobine accumulation was conducted to assess the yield and quality of D. nobile in all treatments. In the experiment, D. nobile plants were cultivated in greenhouse as tested materials, and complete test of 9 treatments was adopted with relative light intensities 75.02%, 39.74%, 29.93% and relative water content of medium 50%, 65%, 80%. The plants were treated in June and harvested till December. Indexes including agronomic traits, fresh weight and dry weight of stem and leaf, ash content, extract, and dendrobine were measured. Results Under the light intensity treatments of 75.02% with 50%, 65%, 80% water content of medium, the basal stems of plants were comparatively thicker with more leaves, and the fresh weight and dry weight of stems and leaves were significantly higher than other 6 treatments. Leaves in all treatments contained dendrobine. Under the light intensity treatments of 75.02% with 50%, 65%, 80% water content of medium, dendrobine content of leaves was lower while dendrobine contents of other treatments were more than 0.60%. After comprehensive assessment through the principal component analysis and total dendrobine accumulation, the results showed that 3 treatments with relative light intensity of 75.02% ranked the top three. Conclusions In brief, the moderately strong light intensity and water content of medium from low to medium can facilitate the growth and yield of D. nobile plants, while light intensity from moderately weak to weak can enhance the dendrobine content.
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OBJECTIVE: To study the effect of mycorrhizal fungi on the growth and the main chemical composition accumulation of Dendrobium nobile seedlings. METHODS: Seedlings which inoculated mycorrhizal fungus MF23 grew for 18 months, and were sampled regularly. The stem height and the dry weight of plant organs were measured and the content of dendrobine and polysaccharide were determined to investigate the effect of MF23 on the plant growth and the main chemical composition accumulation of stem. RESULTS: After 18 months growth, the stem height and the dry weight of root, stem and leaf of inoculated seedlings were significantly higher than those of the control, increased by 25.34%, 29.28%, 34.64% and 47.12%, respectively. MF23 increased the highest amount of dendrobine and polysaccharide in stem and also prolonged the accumulation process of polysaccharide. After 12 months growth, the content of dendrobine in both the treatment and the control group reached the highest levels, which were 0.59% and 0.51%, respectively. The content of polysaccharide in the treatment and the control group reached the highest levels after 15 (20.05%) and 12 (17.89%) months growth, respectively. CONCLUSION: Mycorrhizal fungus MF23 significantly promotes the growth of D. nobile seedlings, and had a good application prospects on the cultivation and production of D. nobile.
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To determine the content of dendrobine in Dendrobium nobile from different harvest times and plant parts, to research the inherent rule about it. GC with internal standard was used to determine. The content of dendrobine had significant differences in different periods and parts. The dendrobine content is higher in four-year root than in three-year root. The dendrobine content in the upper segment of stem is the highest, secondly is in the middle seg-ment, and in the low segment is the lowest. This offered evidence to determine the most appropriate harvest time and fair use of different parts for D.nobil.
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[Objective] cDNA expression libraries from the leaves of Dendrobium candidum Wall. ex lindl. were constructed to supply evidence for the clones of the full-length genes involved in the biosynthesis of bioactive compounds. [Methods] Total RNA was isolated and purified from tender leaves of 4-year-growing Dendrobium candidum by using Trizol single-step method. cDNA was synthesized by long distance polymerase chain reaction (LD-PCR) and then was connected to ?TripIEX2. After that, the recombinant bacteriophages were packaged and cDNA library of Dendrobium candidum was constructed. The titer of cDNA library was expressed as the number of phage formation unit (pfu) per milliliter and the inserted fragment was identified by PCR amphfication. [Results] cDNA expression libraries from the leaves of Dendrobium candidum were constructed successfully. The titer of the original library was 4.3?105 pfu/mL and 3.1?105 clones in total, with a recombinant rate of 97.6%. The amplified titer was 6.8?109pfu/mL. PCR amplification suggested that the inserted cDNA fragments ranged from 0.5 to 2.0 kb, mostly from 1.2 to 1.7 kb. [Conclusion] The constructed Dendrobium candidum cDNA library has a higher titer, a higher recombinant percentage and larger inserted fragments.
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[Objective] Subtractive cDNA libraries from the leaves of D. candidum were constructed to supply evidence for the clone of dendrobine-biosynthesis-associated genes. [ Methods ] Suppression subtractive hybridization ( SSH) technology was used. With the cDNA of perennial leaf as the tester and that of annual leaf as the driver, forward and reverse hybridization was performed. The two obtained subtracted cDNA fragments were cloned into PointTMXa-1 plasmid vectors, and then the vectors were transformed into E. Coli JM109. The inserted fragments were amplified by PCR and then identified. [Results] The subtractive cDNA libraries related with dendrobine-biosynthesis-associated genes were successfully constructed. The forward and reverse subtractive libraries included 560 and 220 clones respectively. Detected by PCR, the length of the inserted fragments was 550 bp on average. [Conclusion] The constructed subtractive libraries are suitable for further study on the functional genes associated with dendrobine biosynthesis of D. candidum.
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AIM: Determination of total alkaloids in Dendrobium nobile Lindl.METHODS: Dried materials were subjected to exhaustive extraction with ethanol: water (9:1,v/v) under reflux.Following filtration and condensation,the residue was dissolved in 3% hydrochloric acid and filtered.The solution was refined using a strongly acidic cation exchange resin column through sequential elution.After elution of deionized H2O,the eluent with 50% of alcoholic plus 5% of ammonia was collected until alkaloids were completely desorbed and evaporated under reduced pressure.The test sample was obtained.The test sample was dissolved in glacial acetic acid and titrated with perchloric acid titrand(0.1 mol/L) with automatic titrator.The herb should contain alkaloids,calculated as dendrobium(per 1ml of perchloric acid titrand is equivalent to 26.3 mg dendrobium).RESULTS: The results of three batches of material were 0.85% ,0.63% ,1.03% respectively.CONCLUSION: The method is convenient,fast,effective and reliable.Automatic potentiometric titration can be used for determination of alkaloids in Dendrobium nobile Lindl.