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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.
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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.
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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.
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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.
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Objective: To clone and characterize a 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) gene DnHMGS in Dendrobium nobile. Methods: RT-PCR and RACE technologies were used for gene cloning. Characteristics including the physicochemical properties and conserved domain of the deduced DnHMGS protein were determined by a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed by DNASTAR and MEGA softwares, respectively. qRT-PCR was employed to examine the tissue specific expression pattern of DnHMGS. Results: The full length cDNA of DnHMGS was 1 816 bp (GenBank accession No. KX789180) and encoded a 474-amino-acid protein with a molecular weight of 52 458.47 and an isoelectric point (pI) of 5.98. The deduced DnHMGS protein, like other HMGS proteins, constituted typical domain and active site. Multiple sequence alignment and phylogenetic analyses demonstrated that DnHMGS was closely related to Ananas comosus, rice, and maize monocots. Proteins analysis revealed that DnHMGS was expressed in the three included organs. The transcripts were the most abundant in the leaves with more than twice that in the roots and stems. However, the expression of DnHMGS changed to stems > leaves > roots when D. nobile infected by Mycena sp. Conclusion: The full length cDNA of DnHMGS is indentified from D. nobile for the first time. Molecular characterization of DnHMGS will be useful for further functional elucidation of the gene involving in isoprenoid biosynthesis pathway in D. nobile, and help us understand the molecular mechanism of Mycena sp. which better encourages the biosynthesis of dendrobine.
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OBJECTIVE: To clone and characterize a 3-hydroxy-3-methylglutaryl-CoA reductase(HMGR) gene DnHMGR2 in Dendrobium nobile. METHODS: RT-PCR and RACE technologies were used for gene cloning. Characteristics including the physicochemical properties and conserved domain of the deduced DnHMGR2 protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR and MEGA softwares, respectively. qRT-PCR was employed to examine the specific expression pattern of DnHMGR2 before and after inoculation. RESULTS: The full length cDNA of DnHMGR2 was 2 095 bp (GenBank accession No. KX825920) and encoded a 562-amino-acid protein with a molecular weight of 59.68×103 and an isoelectric point (pI) of 6.18. The deduced DnHMGR2 protein, like other HMGR proteins, constituted typical domain and active site. Multiple sequence alignment and phylogenetic analyses demonstrated that DnHMGR2 had highly similar identity to a number of HMGR genes from various plants and was closely related to Dendrobium candidum. Real time quantitative PCR (qRT-PCR) analysis revealed that DnHMGR2 was expressed in the three included organs. The transcripts were abundant in the leaves and stems with more than 2.59 and 1.60 fold over that in the roots, respectively. However, the expression levels of DnHMGR2 changed to the sequence of stems > leaves > roots when D. nobile was infected by Mycena sp.. CONCLUSION: The full length cDNA of DnHMGR2 from D. nobile is indentified. Molecular characterization of DnHMGR2 will be useful for further functional elucidation of this gene involving in isoprenoid biosynthesis pathway in D. nobile, and help us understand the molecular mechanism of Mycena sp., which encourages the biosynthesis of dendrobine.
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Objective: To investigate the chemical constituents of Dendrobium nobile from Guizhou province. Methods: The constituents were isolated and purified by silica gel, MCI column chromatography, and preparative HPLC technology. The structure of the isolated compound was elucidated by MS and NMR spectra. Results: A new sesquiterpene, identified as δ-cadinen-12,14-diol (1), was isolated from the stem of D. nobile. Conclusion: Compound 1 is a new compound.
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Objective To exPlore the ProtectiVe mechanism of Dendrobium nobile lindl ( DNL ) decoction on the exPression of Peroxisome Proliferator actiVated recePtor gamma ( PPARγ) in the renal cortex of diabetic nePhroPathy ( DN) rats. Methods Sixty SD rats were randomly diVided into six grouPs (n=10 Per grouP) as follows: normal control grouP (NC), diabetic nePhroPathy control grouP ( DN) ,rosiglitazone grouP ( RGZ) ,the Dendrobium nobile lindl low ( LD) ,medium ( MD) and high ( HD) dose grouPs. After DN rat model was established, the rats were administrated with resPectiVe medications for 12 weeks,resPectiVely. The renal Pathology of rats was obserVed. The mRNA and Protein exPression of PPARγ in the renal cortex were detected via Real_time PCR and Western blotting,resPectiVely. Results High dose DNL decoction significantly alleViated thickening of kidney tissue basement membrane and fusion of foot Process in model rats. The leVels of PPARγmRNA and Protein exPression in the MD and HD grouPs were significantly increased as comPared with the DN grouP (P<0. 05,P<0. 01). Conclusion DNL decoction can effectiVely reduce kidney injury by uP_regulating the PPARγ mRNA and Protein exPression in diabetic nePhroPathy rats.
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Objective To study the chemical constituents from the stems of Dendrobium nobile. Methods Compounds were isolated through various chromatographic techniques and identified by spectral data. Results Twelve phenolic compounds were obtained. Their structures were characterized as dihydroconiferyl dihydro-p-coumarate (Ⅰ), vanillin (Ⅱ), apocynin (Ⅲ), p-hydroxybenzaldehyde (Ⅳ), syringaldehyde (Ⅴ), syringic acid (Ⅵ), syringylethanone (Ⅶ), α-hydroxypropiosyringone (Ⅷ), coniferyl aldehyde (Ⅸ), dihydroconiferyl alcohol (Ⅹ), 2-hydroxyphenylpropanol (Ⅺ), and 3-hydroxy-4-methoxyphenylethanol (Ⅻ), respectively. Conclusion All above compounds are reported from this plant for the first time. Compounds Ⅰ and Ⅲ -Ⅻ are reported for the first time from the plants of Dendrobium Sw.
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Object To speed up the artificial propagation of the costly medicinal plant Dendrobium nobile Lindl. Methods The tender stems with lateral buds were tried as the explants and cultured on various culture media with different portions of hormon. Results The best medium for bud induction was the MS basic medium with addition of 6 BA (0.5 mg/L), NAA (0.2 mg/L), while MS with the addition of 6 BA (3.0 mg/L) NAA (0.5 mg/L), was suitable for propagation, MS with addition of IBA (0.3 mg/L), NAA (0.1 mg/L) was better for root growing, the striking growth rate was up to 95%. The young sprout was cultured fine on the base material mixed with coco crumbs, volcanic rocks and brick fragments (1∶1∶ 1) and covered by dried pteridophyte. Conclusion To provide the effective ways to reserve the resources of D. nobile and last its utlization.
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Object To study the effect of plant growth regulator on medicinal constituents of Dendrobrium nobile Lindl Methods The root of D nobile was treated by immersing in solutions of plant growth regulator 6 BA and GA 3 Results and Conclusion Both plant growth regulator at certain concentration, resulted in an increase of total soluble sugar content, but without effect on the content of dendrobine GA 3 can significantly elevate the content of total alkaloid in D nobile
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primer.Conclusion The present reaction system could provide clear bands,abundant polymorphisms,and reliable reaction.It is proved to be suitable for molecular biology research of D.nobile.
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Objective To develop a convenient and effective molecular authentication method for identifying Dendrobium nobile from other species of Dendrobium Sw.Methods Based on rDNA ITS sequences of D.nobile and other species of Dendrobium Sw.of Huangcaos and Fengdous,unusual sequence sites were confirmed and an allele-specific diagnostic primer pair was designed for D.nobile. Diagnostic PCR was performed using the primers with the total DNA of D.nobile and other species of Dendrobium Sw.as templates to establish the positive PCR condition for D.nobile.Results An about 300 bp DNA positive fragment was amplified from D.nobile with anneal temperature at 66 ℃ and anneal time at 40 s,whereas no any DNA fragment was amplified from the rest 39 species of Dendrobium Sw.Conclusion D.nobile could be effectively distinguished from other species of Dendrobium Sw.by diagnostic PCR with allele-specific diagnostic PCR,which is not only effective and specific,but also simple and timesaving,and has a comprehensive application prospects.
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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.