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Objective: To find a suitable ecological cultivation measure to solve the problem of root-knot nematode disease of Panax quinquefolium (Panacis Quinquefolii Radix) and the heavy metals accumulating in its roots. Methods: Three-year-old P. quinquefolium was treated with four different combinations of microbial inoculant (MI) and garbage fermentation liquid (GFL) [the joint application of ‘TuXiu’ MI and Fifty potassium MI (TF), the combination use of ‘No. 1′ MI and Fifty potassium MI (NF), ‘Gulefeng’ poly-γ-glutamic acid MI (PGA), GFL], and the untreated control (CK). Here, high-throughput sequencing, ICP-MS and UPLC were employed to systematically characterize changes of microbial diversity and structure composition, heavy metals (As, Cd and Pb) content and ginsenoside content among different treatments. Results: The results revealed that different MIs and GFL could increase the root dry weight of P. quinquefolium, PGA enhanced it by 83.24%, followed by GFL (49.93%), meanwhile, PGA and GFL were able to lessen root-knot nematode disease incidence by 57.25% and 64.35%. The treatment of PGA and GFL can also effectively reduce heavy metals in roots. The As content in GFL and PGA was decreased by 52.17% and 43.48% respectively, while the Cd and Pb contents of GFL and PGA was decreased somewhat. Additionally, the content of total ginsenosides was increased by 42.14% and 42.07%, in response to TF and NF, respectively. Our metagenomic analysis showed that the relative abundance of particular soil microbial community members related to the biocontrol of root-knot nematode disease and plant pathogen (i.e., Chaetomium in NF, Xylari in GFL, and Microascus in PGA), heavy metal bioremediation (Hyphomacrobium in PGA and Xylaria in GFL), and nitrogen fixation (Nordella and Nitrospira in TF) was significantly increased; notably, potential harmful microflora, such as Plectosaphaerella and Rhizobacter, were more abundant in the control group. Conclusion: MI and GFL could improve the quality of P. quinquefolium by modifying its rhizosphere microbial community structure and composition, both of them are beneficial to the development of ecological cultivation of P. quinquefolium.
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Objective: In order to study the application of DNA barcoding in the authentication of Chinese patent medicines, Sanqi Tablets were used as the object to investigate the applicability, specificity and precision of this method. Methods: Fifteen batches of commercially available Sanqi Tablet samples were collected. The conditions of DNA extraction for Sanqi Tablet had been first investigated, and the DNA was used for testing the applicability of the methods such as PCR amplification, sequence acquisition, and species authentication in the principles for molecular identification of traditional Chinese materia medica using DNA barcoding. The specificity and reproducibility of DNA barcoding in identification of Sanqi Tablets and its adulterations from the roots of Panax notoginseng, P. ginseng and P. quinquefolius were also studied. Results: The Sanqi Tablet sample with an amount of sampling to be 100 mg and a water bath at 56 ℃ for 8 h gave an average concentration of 60.7 ng/μL and then the PCR amplification, sequence acquisition and species assignment were all successful. The ITS2 sequences of P. notoginseng, P. ginseng and P. quinquefolius were all 230 bp in length, and there were seven stable SNP loci between P. notoginseng and P. ginseng, P. notoginseng and P. quinquefolius. ITS2 sequences could be successfully obtained from lab-made and the adulterated Sanqi Tablets, and the Sanger sequencing chromatograms of different ratios of P. notoginseng and P. ginseng mixtures, P. notoginseng and P. quinquefolius mixtures had heterozygous peaks with corresponding peak height ratio at SNP positions. The repeatability, intermediate precision and reproducibility were all in line with the requirements of “General Regulation 9101” in the Chinese Pharmacopoeia. Conclusion: The ITS2 sequence can stably and accurately authenticate the raw materials of Sanqi Tablets with substantial specificity and precision. The DNA barcoding identification method of Sanqi Tablets will provide a new technical tool for ensuring the safety of Sanqi Tablets in clinical medications, and provide reference for the identification of other single-herb products documented in the Chinese Pharmacopoeia.
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Medicinal materials in China differ in quality by different ecological types. Our research group found that there were two ecotypes of domestic Panax quinquefolium L. according to the characteristics of ginsenosides, inside versus outside Shanhaiguan. The genetic and ecological mechanisms of quality variation of Panax quinquefolium L. is unknown. Based on the genetic-chemical-ecological strategy, transcriptome and HPLC technology were used for comprehensive correlation analyses of transcriptomic data, ginsenoside content and environmental climate ecological factors. The transcriptomic results showed that key genes of ginsenoside biosynthesis, such as HMGR, AS and FPS, were significantly down-regulated in the inside Shanhaiguan ecotype. HPLC results showed that the quality of outside Shanhaiguan ecotype Panax quinquefolium L. was higher than that of the inside ecotype, with the content of ginsenosides in outside Panax quinquefolium L. was higher than that of inside ecotype except Rb2. Correlation analyses revealed that content of Panax quinquefolium L. ginsenoside is positively related to the expression levels of ginsenoside biosynthesis key genes (MK, HMGS, HMGR, and AS), and negatively related to the expression of glycosyl transferase (GT). The content of ginsenosides is negative related with climate factors, such as temperature, sunshine, and is positively related with moisture in both ecological environments. This study has provided a new mechanistic insight into the quality variations of two ecotypes for Panax quinquefolium L. and established a scientific basis for studying the ecological factors for the quality of traditional Chinese medicine.
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Objective Traditional identification methods of pharmacognosy is difficult to distinguish the seeds of Panax ginseng and Panax quinquefolius. In order to improve the efficacy and accuracy of identification and provide the scientific foundation for the establishment of Chinese herbal medicine seed quality standards, molecular identification methods of the seeds were established by DNA barcoding technology. Methods The pharmacognostical identification method was used to study the morphological identification and microscopic characters of different seeds. DNA barcodes and Chinese Pharmacopoeia species standard barcode database were employed to identify the seeds by ITS2 sequence comparison, genetic distance comparison and systematic NJ tree construction. Results Intraspecific genetic distances of individuals participating were smaller than interspecific genetic distances. Phylogenetic tree map showed that two species were repectively clustered into one. A total of 42 samples of seeds from Panax ginseng and Panax quinquefolius produced by nine areas were all top-quality and easy to distinguish. Conclusion ITS2 DNA barcodes can identify and differentiate the seeds of P. ginseng and P. quinquefolius germplasm resources quickly, accurately and efficiently.
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A new ultrasonic-assisted extraction (UANE) method coupled with solid phase extraction (SPE) using ultrasonic fountain was established for the extraction of eight common ginsenosides from leaves of Panax quinquefolium L. The extraction system has been designed and several experimental parameters,including the type and volume of extraction solvent,pH value and salt concentration of extraction solvent,type and volume of elution solvent,and amount of C18, extraction time were examined and optimized. Under the optimal conditions,the recoveries of ginsenosides were in the range of 96. 3% -110. 6%, the relative standard deviations (RSDs) were in the range of 2.8%-4.3%,indicating that the method has a good performance for the extraction of these ginsenosides. Compared with traditional UANE-SPE method, the modified method simplified the extraction device,shortened the extraction time and improved the extraction efficiency.
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Objective To establish the determination method of four ginsenosides from flower buds of Panax quinquefolium by Waters Acquity UPLC H-Class with Xevo TQD. Methods The chromatographic separation was performed on a Waters Acquity UPLC BEH C18 column (50 mm × 2.1mm, 1.7 μm). The mobile phase was a mixture of acetonitrile and water containing 0.05% ammonium hydroxide with gradient elution; The flow rate was 0.45 mL/min, and the column temperature was 35 ℃; Multiple reaction monitoring (MRM) acquisition under negative ion scan mode by ESI was also performed. Results The method was established with good precision, stability, repeatability, and accuracy. Ginsenoside Re, pseudo-ginsenoside F11, ginsenoside Rb3, and ginsenoside Rd showed a good linearity in the range of corresponding concentrations. Conclusion The UPLC-MS/MS method is rapid, simple, accurate, reliable, which can be used for the analysis of ginsenosides from flower buds of P. quinquefolium.
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Objective: To clone and analyze the coding region of gibberellin 20-oxidase (GA20ox) gene of Panax quinquefolium in seed dormancy and germination process. Methods: According to P. quinquefolium seeds transcriptome annotation, one transcript coding of GA20ox was obtained by comparative analysis of nine related unigenes. The full-length cDNA of PqGA20ox was determined by using RT-PCR method. Then the bioinformatic analysis of this gene and encoded protein was performed. The expression level of PqGA20ox in the roots, stems, leaves, and several seed dormancy periods was detected by real time fluorescent quantitative PCR (RT-qPCR). Results: Bioinformatic analysis showed that PqGA20ox contained 1092 bp and encoded 363 amino acids. PqGA20ox encoded protein had neither transmembrane nor signal peptide. The expression level of PqGA20ox was higher in the inset periods of morphological and physiological dormancy than that in the other periods based on RT-qPCR analysis. Conclusion: The PqGA20ox gene is cloned for the first time, and will provide a foundation for the dormancy release molecular mechanism of P. quinquefolium.
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Objective: To study the resource situation of endophytes in Panax quinquefolium of Jilin province and to select the endophytes with antagonistic effect on pathogenic fungi of P. quinquefolium. Methods: The strains with the antagonistic activity were screened by applying mixed strains method during preliminary screening and adopting the antagonism method of fermentation liquor in re-screening; Besides, the selected endogenous strains were identified via 16 S rDNA and ITS method. Results: Thirty-four endogenous strains were isolated from P. quinquefolium. After preliminary screening and re-screening, six strains with good antagonistic effect on pathogenic fungi of P. Quinquefolium were selected: B3, B11, B17, B23, B24, and F10. Among them, B3 showed an inhibitory effect on seven pathogenic fungi; F10 had a good inhibitory effect on Cylindrocarpon destructans and Alternaria panax, with an inhibitory zone diameter over 35 mm; According to the identification, B3 and F10 were Bacillus methylotrophicus and Penicillium citreonigrum respectively. Conclusion: Endophytes in P. quinquefolium are diversified and there also exist strains with high antagonistic activity, which can be a good source for biocontrol bacterium and fungus of P. quinquefolium diseases.
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Objective: To clone and analyze the gibberellin 2-oxidase (GA2ox) gene of Panax quinquefolium in seed germination. Methods: Gene sequences about gibberellins synthesis and catabolism were found out from annotation information of 78 207 unigenes obtained by high-throughput sequencing in the early study. Then one transcript coding GA2ox was obtained from 11 unigenes related to GA2ox. Primers were designed according to selected sequence to get the full-length cDNA of P. quinquefolium using PCR method. Predictive analysis and expression analysis of PqGA2ox were obtained by bioinformatics and real-time PCR. Results: A GA2ox gene containing 987 bp encoding 328 amino acids was cloned and named as PqGA2ox. Bioinformatics analysis showed that PqGA2ox had no transmembrane domain or signal peptide, but had the 20G-Fell_Oxy conserved domains. The expression level of PqGA2ox was lower in the metaphase of morphological dormancy and physiological dormancy than that in the intitial period and release period based on real-time PCR analysis. Conclusion: The PqGA2ox gene from the seeds of P. quinquefolium is cloned for the first time, which will provide a foundation for the molecular mechanism of dormancy release of P. quinquefolium.
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Objective To study the chemical constituents of the acid hydrolytic products from the stems and leaves of Panax quinquefolium. Methods The stems and leaves of P. quinquefolium were hydrolyzed with acid;Isolation and purification were carried out by various chromatographic techniques,such as silica gel and so on. Compounds were identified and elucidated by spectral and chemical methods. Results Eight compounds were obtained from the acid hydrolytic products in the stems and leaves of P. quinquefolium and identified as 20 (S)-panaxadiol (Ⅰ),20 (S)-protopanaxadiol (Ⅱ),20 (R)-protopanaxadiol (Ⅲ),20 (S)-panaxatriol (Ⅳ),24 (R)-ocotillol (Ⅴ),20 (R)-protopanaxatriol (Ⅵ),20 (R)-dammarane-3?,12?,20,25-tetrol (Ⅶ),and 20 (R)-dammarane-3?,6?,12?,20,25-pentol (Ⅷ). Conclusion Compounds Ⅰ-Ⅷ are the derivates of the aglycones of ginsenosides,isolated from the acid hydrolytic products from the stems and leaves of P. quinquefolium for the first time. Among these compounds,Ⅳ,Ⅶ,and Ⅷ are discoverd from the roots,stems,leaves,fruits,and buds of P. quinquefolium for the first time. Compound Ⅶ is discovered and reported that it is the derivate of the aglycone of protopanoxadiol ginsenoside,with the conspicuous anticancer activity from the fruits of P. ginseng for the first time.
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Objective To investigate the polysaccharides constituents from the transgenic crown gall cultures of Panax quinquefolium. Methods The crude polysaccharides PPQ50 and PPQ70 were extracted by water-ethanol method from the crown gall of P. quinquefolium. Four polysaccharides, named PPQ50-1, PPQ50-2, PPQ70-1, and PQ70-2, were isolated and purified by DEAE Sepharose and Sephacry S-100 column chromatography. Results The homogeneity of four polysaccharides was examined by gel chromatography and polarimetry. Their molecular weights were determined by gel filtration chromatography. They were 4.7?104, 2.9?105, 7.5?104, and 1.3?105, respectively. The sugar composition was analyzed by HPLC. The polysaccharides are mainly composed of neutral sugar particularly of D-glucose. The structure elucidation of PPQ50-2 was also supported by 1H-NMR and 13C-NMR spectroscopy. Conclusion All of the four polysaccharides are first isolated and purified from the transgenic crown gall cultures of P. quinquefolium.The result shows the PPQ50-2 and PPQ70-2 are both glucans.
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Objective To investigate the biosynthesis of arbutin by suspension culture of crown gall of transgenic Panax quinquefolium. Methods Hydroquinone in methanol(60 mg/mL) was added to the medium of the crown gall of P.quinquefolium after precultured for 20 d,then they were co-cultured for another 60 h.The product was isolated and purified by column chromatography and its structure was identified by physicochemical properties and spectroscopic data.The dynamic curve of biotransformation was investigated by quantative analysis of arbutin with HPLC.Results The product could be obtained from both of the culture and medium,which was isolated and elucidated as 4-hydroxyphenyl-?-D-glucopyranoside(arbutin).After co-cultured for 60 h,the mole conversion ratio of hydroquinone is 88.4%,the product contents in culture and medium are 63.69 mg/flask and 1.86 mg/flask,respectively,and the excretion ratio of arbutin reaches the highest(2.92%).Conclusion It's the first time around the world that the crown gall of transgenic P.quinquefolium is used as a biotransformation system and arbutin which shows varied pharmacological activites have been got successfully.