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An open reading frame (ORF) of isopentenyl-diphosphate delta isomerase gene (FuIPI) was cloned from Fritillaria unibracteata Hsiao et K. C. Hsia. (F. unibracteata). Furthermore, the bioinformatics and functional analyses of FuIPI were performed in this study. The result showed that, the ORF of FuIPI gene was 825 bp, encoding a polypeptide of 274 amino acids in length, with a relative molecular mass of about 31 kD and a theoretical isoelectric point of 5.61. Sequence analysis showed that FuIPI contained conserved structural domains and key residues involved in the catalyzing process. The phylogenetic analysis exhibited that FuIPI was closely related to IPIs of Dendrobium officinale and Musa acuminate. Real-time PCR analysis showed that FuIPI was distributed in different tissues of F. unibracteata, but had the highest transcriptional level in leaves, followed by stems, bulbs, and flowers. Furthermore, the FuIPI protein was successfully expressed in Escherichia coli BL21(DE3). The purified FuIPI protein successfully catalyzed the conversion from isopentenyl diphosphate (IPP) to dimethylallyl pyrophosphate (DMAPP). The above results provided a theoretical basis for further investigation of the molecular role of FuIPI in the biosynthesis of alkaloids.
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Objective:To establish the fingerprint and stoichiometric analysis mode of Ultra Performance Liquid Chromatography Tandem Quadrupole Time-of-Flight Mass Spectrometry (UPLC-Q-TOF-MS) of Fritillaria anhuiensis Bulbus, so as to provide reference for its quality evaluation and standard formulation. Methods:By setting the CORTECS C18 column at 4.6 mm×150 mm, 2.7 μm with the mobile phases of acetonitrile-0.1% formic acid and 10 mmol/L aqueous ammonium formate for gradient elution at a flow rate of 0.4 ml/min and an injection volume of 2.0 μl. The TCM fingerprint similarity evaluation system (2012 version) was used to evaluate 9 batches of Fritillaria anhuiensis Bulbus samples. By using Liquid Chromatography-Mass Spectrometry (LC-MS) technique to make quantity analysis and by combining cluster analysis, principal component analysis and orthogonal least squares-discriminant analysis to make overall quality evaluation. Results:The fingerprint profiles of different batches of Fritillaria anhuiensis Bulbus were established and 21 common peaks were identified, and 12 of them were initially identified. Cluster analysis, principal component analysis and orthogonal least squares-discriminant analysis were used to cluster the nine batches of Fritillaria anhuiensis Bulbus into three categories. Conclusion:The fingerprint established in this study combined with the chemical pattern recognition method are highly sensitive and specific, which could reflect the overall characteristics and differences of Fritillaria anhuiensis Bulbus, providing reference for the quality evaluation of Fritillaria anhuiensis Bulbus and standardization of it.
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Chinese medicinal material from Fritillaria， Beimu in Chinese， is a commonly used antitussive and expectorant traditional Chinese herbal medicine， with the significant functions of clearing heat and moistening lung，resolving phlegm and relieving cough. Five kinds of Fritillaria were recorded in the 2020 edition of Chinese Pharmacopoeia， including Fritillariae Cirrhosae Bulbus， Fritillariae Ussuriensis Bulbus， Fritillariae Thunbergii Bulbus， Fritillariae Hupehensis Bulbus and Fritillariae Pallidiflorae Bulbus. At present， the reports on Fritillaria mainly focus on the pharmacological effect， chemical composition， identification of authenticity and other aspects， while there were few reports on harvesting and primary processing of original medicinal materials. Fritillaria medicines were perennial medicinal plant with various and complex varieties， their quality and curative effect were greatly affected by harvesting and processing in producing area. The processing method differed according to its variety. Fritillariae Cirrhosae Bulbus mainly from western Sichuan plateau， Fritillariae Pallidiflorae Bulbus from Xinjiang and Fritillariae Ussuriensis Bulbus from Northeast China were mostly harvested from June to July and sun dried directly or dried. But Fritillariae Thunbergii Bulbus and Fritillariae Hupehensis Bulbus from Yangtze River basin were harvested when the plants wilted at the beginning of summer， and auxiliary materials such as shell powder and lime must be added during the processing. At present， the drying methods of Fritillaria were still traditional， which is not suitable for large-scale production of cultivated products. Therefore， it is urgent to find a scientific， reasonable and efficient processing methods. Aimed at providing references for standardization production of Fritillaria， this paper made a textual research on the ancient and modern herbal literature， the Chinese Pharmacopoeia and other medicinal standards， combined with modern literature， the harvesting and processing methods of Fritillaria were sorted out and prospected.
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ObjectiveTo study the effect of flower removal on the content of three alkaloids in different parts of Fritillaria thunbergii from different regions and at different growth stages. MethodThe content of peiminine， peimine， and peimisine in the bulb， root， stem， and leaf of F. thunbergii after flower removal and with flower un-removed at different growth stages and in different regions were determined simultaneously by ultra-performance liquid chromatography-evaporative light scattering detection （UPLC-ELSD） method. The UPLC was conducted on ACQUITY UPLC BEH C18 column （2.1 mm × 150 mm， 1.7 μm） with the mobile phase of 0.02% triethylamine aqueous solution （A） and methanol （B）elution gradient（0-2 min， 45%A； 2-5 min， 45%-25%A； 5-7 min， 25%A； 7-17 min， 25%-10%A； 17-20 min， 10%A）， flow velocity of 0.20 mL·min-1， column temperature 35 °C， sample room temperature of 20 °C， and injection volume of 3 µL. The ELSD was carried out at drift tube temperature 45 °C and with the sprayer parameter of 40%. ResultThe flower removal significantly increased the yield of F. thunbergii. At the budding stage， the alkaloid content in the bulb of F. thunbergii from Ningbo in Zhejiang， Pan'an in Zhejiang， and Nantong in Jiangsu after flower removal were significantly higher than that of flowering un-removal treatment， while it showed no significant difference between the flower removal and un-removal treatments for the samples from Fengjie in Chongqing. At the flowering stage， the alkaloid content in the bulb of F. thunbergii from Nantong in Jiangsu after flower removal was significantly higher than that of flower un-removal treatment， while it showed an opposite trend for the samples from Pan'an in Zhejiang and Fengjie in Chongqing and had no significant difference between the two treatments for the samples from Ningbo in Zhejiang. At the bulb expansion stage， the alkaloid content in the bulb of F. thunbergii from Ningbo in Zhejiang and Pan’an in Zhejiang after flower removal were significantly higher than that of flower un-removal treatment， which was opposite for the samples from Nantong in Jiangsu and had no significant difference between the treatments for the samples from Fengjie in Chongqing. At the harvest stage， except for the samples from Pan'an in Zhejiang， the samples from the rest 3 regions showed decreased alkaloid content in the bulb after flower removal compared with that of flower un-removal treatment. The alkaloid content in the leaf was higher than that in the bulb of F. thunbergii at all growth stages and from different origins. ConclusionFlower removal can increase the yield of F. thunbergii. The alkaloid content in the bulb of F. thunbergii with flower removed was higher than that with flower un-removed at the budding stage， while this trend was reversed at the harvest stage. Both the yield and the alkaloid content of F. thunbergii from Pan'an in Zhejiang were increased by flower removal. The above-ground part of F. thunbergii has a potential development value.
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Objective:To investigate the effect of iron nanoparticles and melatonin on yield and quality of <italic>Fritillaria przewalskii</italic> and provide technical support for its domesticated cultivation. Method:Hundred grain weight was measured by conventional method；alkaloid content was detected according to protocols of the edition of 2020 <italic>Chinese Pharmacopoeia</italic>，chlorophyll，hydrogen peroxide，malondialdehyde，superoxide dismutase （SOD），peroxidase （POD） and catalase （CAT） were detected by spectrophotometric analysis，auxins，cytokinins，gibberellins，salicylic acid，jasmonic acid and abscisic acid were detected by ultra performance liquid chromatography tandem mass spectrometry analysis. Result:Zero-valent iron nanoparticles and melatonin significantly increased the hundred grain weight without affecting the quality. The effect of the two treatments on physiological and biochemical indexes in different stages were quite different，but the effects on content of endogenous hormones were basically the same. Correlation analysis showed that hundred grain weight was negatively correlated with malondialdehyde content，SOD activity and jasmonic acid content，but positively correlated with POD activity，salicylic acid content，gibberellins content，auxin content and abscisic acid content. The two treatments were separated effectively by principal component analysis，indicating that there were some differences in the mechanisms of growth promoting. The treatment of zero-valent iron nanoparticles mainly affected auxins，salicylic acid and abscisic acid. The treatment of melatonin mainly affected SOD，malondialdehyde and gibberellins. Conclusion:Zero-valent iron nanoparticles and melatonin can be used as a simple and practical technology to improve the stress resistance and yields of <italic>F. przewalskii</italic> in domesticated cultivation conditions.
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Objective:To explore the effects of anti-microbial compound （T1） from<italic> Bacillus </italic>（Phylum Firmicutes） and anti-microbial compound （T2） from <italic>Pseudomonas</italic> and <italic>Rhizobium</italic>， two growth-promoting agents， on the physiological characteristics and growth of <italic>Fritillaria przewalskii</italic>， in order to lay a foundation for the development of functional microbial agents and the promotion of ecological planting. Method:The endophytic bacteria of <italic>F. przewalskii</italic> were isolated and identified using conventional methods. The leaves of three-year-old <italic>F. przewalskii</italic> were sprayed with T1 and T2， followed by yield determination. The enzyme activities and physiological and biochemical indexes in the plant and microorganisms were measured using the corresponding assay kits， and the contents of related hormones by liquid chromatography-mass spectrometry （LC-MS）. Result:The isolated endophytic bacteria were classified into Firmicutes，Proteobacteria， and Actinomycetes. The activities of superoxide dismutase（SOD） and peroxidase（POD） and auxin content after T2 treatment were significantly higher than those after T1 treatment， while the contents of siderophore，salicylic acid， and gibberellin were lower. Compared with the blank （CK） group， T1 and T2 increased the contents of endogenous gibberellin，cytokinin， and auxin in <italic>F. przewalskii</italic> leaves，but did not significantly change jasmonic acid and abscisic acid. T1 promoted the accumulation of endogenous salicylic acid in <italic>F. przewalskii</italic> leaves， but there was no significant change after T2 treatment. Compared with CK，T1 and T2 enhanced the activities of SOD， POD， and catalase （CAT） and decreased the content of malondialdehyde. T2 promoted the accumulation of hydrogen peroxide in <italic>F. przewalskii</italic> leaves， but no significant difference was observed after T1 treatment. Compared with CK，both T1 and T2 increased chlorophyll，average iron content in rhizosphere soil， and 100-plant weight. Conclusion:T1 and T2 treatments help to increase the yield，and their specific mechanisms differ from each other. T1 exhibits better effect than T2.
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OBJECTIVE：To establish the fingerprint of Fritillariae thunbergii formula granules and determine the contents of 3 components. METHODS ：HPLC method was used. Using peiminine as reference ，HPLC fingerprints of 13 batches of F. thunbergii formula granules were drawn with Similarity Evaluation System of TCM Chromatogram Fingerprint （2012 edition）. Similarity evaluation and common peak identification were conducted. The contents of peimisine ，peimine and peiminine in F. thunbergii formula granules were determined by the same HPLC method. The quality difference of samples were compared among different manufacturers. RESULTS ：There were 5 common peaks in 13 batches of F. thunbergii formula granules ，and the similarity was 0.669-0.971. Three common peaks of peimisine ，peimine and peiminine were identified. The linear ranges of peimisine ，peimine and peiminine were 30.00-180.00 μg/mL（r＝0.999 9），79.58-477.50 μg/mL（r＝0.999 6）and 97.33-584.00 μg/mL（r＝0.999 4）， respectively. RSDs of precision ，stability（24 h）and reproducibility tests were all lower than 3％. The average recoveries were 95.82％（RSD＝1.17％，n＝6），99.00％（RSD＝1.96％，n＝6）and 95.39％（RSD＝2.00％，n＝6），respectively. In the 13 batches of samples ，the content of peimisine ，peimine and peiminine were 0.17-1.02 mg/g，0.52-2.26 mg/g，and 0.70-3.50 mg/g， respectively. Their average total content was 3.62 mg/g. The average total content of manufacturer C and A was higher （5.02 mg/g and 4.61 mg/g），followed by manufacturer E and B （3.48 mg/g and 3.02 mg/g）；the lowest was manufacturer D（only 1.87 mg/g）. CONCLUSIONS：Established fingerpri nt and content determination method is simple ，feasible and reproducible ，which can be used for the quality evaluation of F. thunbergii formula granules. There are some differences in content among different manufacturers.
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The molecular identification of Fritillaria taibaiensis and its relatives was studied by real-time PCR with a TaqMan-MGB probe. DNA was extracted from F. taibaiensis and its relatives. According to the sequence of ITS1 region, the mutation sites of F. taipaiensis and its related species were identified by MEGA7.0 software. The specific primers (a pair) and a TaqMan-MGB probe were designed by Primer Premier 6.0 software. In the Roche LightCycler 96 system, the lowest limit of detection for F. taipaiensis DNA template was 0.002 39 ng·μL-1, and the optimal Tm value range was 60 and 61 ℃. Specificity identification showed that the method had good specificity for F. taipaiensis, as it could be distinguished from other 13 different Fritillaria species including F. unibracteata. Since this method could accurately identify F. taipaiensis and its related species, it provides technical support for rational development of F. taipaiensis resources, management of Chinese medicinal market and supervision of raw materials in Chinese medicine manufacturing enterprises.
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Squalene is a key metabolic intermediate for sterols and various other triterpenoids. Its biosynthesis is catalyzed by squalene synthase (SQS), which converts two molecules of farnesyl pyrophosphate to squalene. The biosynthetic pathway of Fritillaria thunbergii Miq isosteroid alkaloids is similar to that of triterpenoids. In this study, a full-length cDNA of squalene synthase from Fritillaria thunbergii Mig (FtSQS) was cloned using rapid amplification from cDNA ends (RACE) technology. GenBank accession number was KF551097. 2. Bioinformatics methods were used to characterize the FtSQS in detail, including the detection of conserved regions, sequence homology analysis, secondary and tertiary structure prediction, and phylogenetic tree analysis. The results showed that its open reading frame (ORF) was 1 230 bp and encoded 409 amino acids. Protein-Blast alignment found that amino acid homology with SQS of Indian pine, Truncate alfalfa, Purple shirt, Potato, Bupleurum, Golden iron lock and Arabidopsis reached 73. 84%, 73. 23%, 72. 24%, 70. 66%, 70. 66%, 69. 44%, 68. 14%. Promoter analysis indicated that the 5' upstream region of FtSQS possessed various potential elements associated with physiological and environmental factors. To obtain a soluble recombinant protein, 24 hydrophobic amino acids were deleted from the carboxyl terminus, and the C-terminal truncated mutant FtSQS (FtSQSATM) was expressed in E. coli BL21 (DE3). SDS-PAGE analysis suggested that approximately 66 kD recombinant protein was checked. The in vitro enzymatic reaction proved that FtSQS could catalyze farnesyl pyrophosphate to generate squalene. Expression level of FtSQS mRNA in leaves was the highest, followed by stem and root, but in bulb was much lower than that in other tissues. It suggests that leaves are active organ for biosynthesis of peimine. The identification and function of FtSQS provides an important basis for the study of secondary metabolites of Fritillaria thunbergii Miq.
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Objective:To investigate the effect of different nitrogen forms and concentrations on yield and quality in Fritillaria thunbergii，and provide basis for improving scientific utilization of nitrogenous fertilizer and its introduction to Chongqing area. Method:The pot culture experiment was conducted to investigate the changes in growth，physiological and biochemical characteristics，soil factors，alkaloid content and yield of Fritillaria thunbergii under the ratio of nitrate nitrogen（NO3--N） to ammonium nitrogen（NH4+-N）of 15∶0（N1），12∶3（N2），7.5∶7.5（N3），3∶12（N4） and 0∶15（N5）. Result:As compared with no-nitrogen（CK） treatment group，the growth and quality of F. thunbergii were significantly improved by different nitrogen nutrition treatments，with differences among them.With the increase of ammonium nitrogen concentration：①plant height and the activity of superoxide dismutase（SOD） reached the maximum when the ratio of NO3--N to NH4+-N was 3∶12，increased by 9.27% and 206.62% respectively compared with the CK group，② the length and width of leaf，stem diameter，chlorophyll a，chlorophyll b，total chlorophyll content，the content of available P and organic matter，total alkaloid content and yield reach the maximum when the ratio of NO3--N to NH4+-N was 0∶15，increased by 14.02%，16.44%，13.68%，40.75%，45.31%，41.72%，77.70%，14.70%，24.61%/47.39% respectively compared with the CK group，with the increase of nitrate nitrogen concentration，③the leaf index，soluble protein content，peimisine content/yield，yield of peiminine and dry weight of bulbs reached the maximum when the ratio of NO3--N to NH4+-N was 7.5∶7.5，increased by 2.54%，5.92%，21.76%/54.55%，60.61% and 26.93%，respectively compared with the CK group，④the content of carotenoids，pigment and peiminine，the activity of peroxidase（POD） and catalase（CAT），the content/yield of peimine，both peimine and peiminine，both peimine，peiminine and peimisine，dry weight of bulbs reached the maximum when the ratio of NO3--N to NH4+-N was 12∶3，increased by 45.39%，45.31%，36.01%，271.38%，67.45%，39.82%/64.87%，38.90%/63.80%，37.03%/61.57%，20.29% respectively compared with the CK group. Conclusion:All the results indicated that a higher proportion of NH4+-N is beneficial to the growth of F. thunbergii，while NO3--N is beneficial to the accumulation of alkaloids and the growth of bulbs.Therefore，the combined application of ammonium and nitrate（NO3--N to NH4+-N ratio of 12∶3） is more effective than pure nitrate or pure ammonium applications to improve the yield and quality of F. thunbergii．
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Objective::To explore the correlation between bulb quality and rhizosphere soil factors of Fritillaria taibaiensis of different origins and years, in order to provide theoretical basis for the high quality and safe production of F. taibaiensis. Method::Totally 11 samples of bulb and rhizospheric soil of F. taipaiensis of different origins and years were taken as the research objects. Available N, available P, available K, organic matter, pH and six soil enzyme activities in rhizosphere soils were determined by soil agrochemical analysis method. Peimisine and nine nucleosides in F. taibaiensis bulbs were determined by HPLC, and total alkaloid content was determined by UV. SPSS 22.0 software was used to analyze the correlation of the measured data. Result::There were significant differences in rhizosphere soil factors and bulb quality between F. taibaiensis of different origins and years (P<0.05). In terms of soil factors, the contents of available N, available K, organic matter and six soil enzyme activities in rhizosphere soil of wild varieties were higher than those of cultivated varieties, while the contents of available P and pH were lower than those of cultivated varieties. With the increase of growth years, the soil nutrient index of cultivated varieties showed different change trends, while that of wild varieties did not change significantly. However, most of the soil enzymes in both groups decreased in varying degrees. In terms of bulb quality, the contents of nine nucleosides and alkaloids in F. taibaiensis bulbs decreased with the increase of growth years, with larger change trends of cultivated varieties, while that of wild varieties was not significant. The contents of nucleosides and alkaloids in most cultivated varieties were higher than those in wild varieties. The correlation analysis showed certain correlations between soil factors in rhizosphere as well as soil factors and bulb quality. In general, soil nutrient status and bulb quality decreased with the increase of years. Conclusion::The quality of F. taibaiensis is mainly affected by its rhizosphere soil factors. In the process of field conservation and artificial cultivation, attention shall be paid to increase or decrease of the content of soil nutrients and their proportional relationship according to actual situations, so as to ensure the quality of F. taibaiensis.
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Genuine medicinal materials-Fritillariae Cirrhosae Bulbus and its active components are widely used in clinical medicine in China and even in many countries in the world because of their good medicinal effect. However, with the increase of the demand for the resources of Fritillariae Cirrhosae Bulbus, and the low yield of itself, the small number of seeds under natural conditions, the extremely low germination rate and survival rate and so on, the wild resources of Fritillariae Cirrhosae Bulbus are endangered. This paper summarized researches of several aspects like herbal research of Fritillariae Cirrhosae Bulbus, distribution of wild source plant resources and its influencing factors, conservation measures, and using endophyte to extract Fritillaria alkaloids, based on the articles published by China National Knowledge Internet and Web of Science. Hoping to provide some development ideas for the protection of wild source plant resources of Fritillariae Cirrhosae Bulbus from the perspectives of traditional conservation measures and active products extracted by microbial vigorously developed at this stage, so as to realize the balance of supply and demand of Fritillariae Cirrhosae Bulbus as soon as possible.
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Objective: To establish a method for simultaneous determination of 53 kinds of pesticides residual in different category of Fritillaria by using QuEChERS and gas chromatography tandem mass spectrometry, and applying to 193 batches sample screening. Methods: The forbidden, restricted and commonly used pesticides were selected as detecting indexes according to the result of this investigation. The samples were prepared by QuEChERS, and quantitative analysis was carried out by GC-MS/MS multiple-reaction monitoring (MRM) model. There were three supplemental levels for determining recoveries and RSD. Results: The results showed that all the 53 pesticides had good linearity in certain ranges with the correlation coefficients (R) higher than 0.997 8. The recoveries of more than 86.8% pesticides were ranged from 60% to 140% at three supplemental levels (1×LOD, 2×LOD, and 10×LOD), with the RSDs less than 15%. The LOD ranged of all pesticides were below 0.01 mg/kg. The result of 193 batches sample screening showed that 91 batches sample were detected 14 pesticides, and the detection rate was 47.2%. Conclusion: The detecting indexes is meaningful and the developed method is simple, rapid, sensitive, and reliable for screening multiple pesticide residues in different category of Fritillaria. The result has certain reference value for the cultivation and circulation supervision of different category of Fritillaria.
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OBJECTIVE： To establish UPLC-Q- TOF-MS/MS fingerprint of Fritillaria thunbergii ， and to define its anti-inflammatory quality markers. METHODS ：The determination was performed on Eclipse Plus C 18 column with mobile phase consisted of methanol- 0.1％ formic acid solution （gradient elution ）at the flow rate of 0.4 mL/min. The column temperature was set at 30 ℃，and injection volume was 2 µL. The electrospray ion source was used to scan in the range of m/z 50-1 200 with positive and negative ion detection mode. UPLC-Q-TOF-MS/MS fingerprints of 10 batches of F. thunbergii from different habitats were established by using the Similarity Evaluation System of TCM Chromatographic Fingerprints （2004A edition ）. With ear swelling degree，the serum levels of MDA and NO as anti-inflammatory indexes ，the correlation between the relative area of common peaks in fingerprint and the anti-inflammatory indexes was analyzed by using bivariate correlation analysis and grey correlation analysis ， and the quality markers were screened and identified. RESULTS ：In positive and negative ion mode ，10 batches of F. thunbergii had 26 peaks and 10 peaks. Based on bivariate correlation analysis and grey correlation analysis ，nine quality markers related to anti-inflammatory effect were found ，which were identified as peiminine ，peimine，cyclobalamine，daucidin，polyphyllin Ⅴ， bitumen podophyllotoxin ，phytosterol，ent-kaur-15-en-17-ol，ent-17-norkauran-16-one. CONCLUSIONS ：Established UPLC-Q- TOF-MS/MS fingerprint can be used to evaluate the quality of F. thunbergii ；peiminine，peimine and cyclobalamine and so on may be the quality markers of anti-inflammatory effect of F. thunbergii .
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OBJECTIVE：To optimize the extractio n technology of Fritillaria pallidiflora polysaccharides（FPSP），and to study its structure. METHODS ：Using the yield of FPSP as response value ，Box-Behnken design-response surface methodology was adopted to optimize solid-liquid ratio ，extraction temperature and extraction time of FPSP extraction technology. Structural properties of FPSP was characterized by UV spectrum ，FTIR，GC-MS，Congo red staining ，SEM，XRD and thermogravimetric analysis. RESULTS：The optimal technology parameters of FPSP were solid-liquid ratio of 1∶28（g/mL），extraction temperature of 94 ℃， extraction time of 2.5 h；the yield of FPSP was 16.25％（n＝3），the error of which to theoretical yield （16.58％）was 0.33％. FPSP contained xylose ，glucose and galactose with a molar ratio of 1∶58.02∶0.73，and trace amount of mannose ；there was a weak absorption peak near the wavelength of 280 nm；belonged to α-configuration pyranopolysaccharide. FPSP was in triple-helical structure. The surface of FPSP was a network structure formed by irregular particles. FPSP had both crystalline and amorphous structures. FPSP had good thermostability. CONCLUSIONS ：The optimized extraction technology of FPSP is reasonable ，and has high extraction yield. The research might provide reference for the further development and utilization of F. pallidiflora .
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Fritillaria thunbergii is a commonly used traditional Chinese medicine, which has the effects of clearing heat and resolving stagnation, eliminating phlegm and relieving cough. At present, it is mostly produced by cultivation, and the cultivation process requires application of base fertilizer, winter fertilizer, seedling fertilizer and late top dressing. Now farmyard manure or organic fertilizer can be used to replace the base fertilizer and winter fertilizer, but the research on the replacement of organic fertilizer has not been completed for the late top dressing. Potassium fulvate is a kind of fulvate fertilizer, which can not only regulate the growth of crops but also supplement potassium necessary for the growth of crops. In this paper, using F. thunbergii as a model plant with mature cultivation techniques, the effect of potassium fulvate on the quality and yield of rhizome traditional Chinese medicine F. thunbergii was systematically studied for the first time. HPLC-ELSD was used to determine the contents of peimine A and peimine B, hot dip method was used to determine the content of alcohol extract, and the SPAD-502 Plus chlorophyll meter was used to detect SPAD value. The results showed that applying 1.5 to 2.25 kg·hm~(-2) of potassium fulvic acid per hectare could effectively improve the yield of F. thunbergii and there was significantly difference between potassium phosphate monobasic and potassium fulvic acid in terms of quality. After the application of range 1.5 to 2.25 kg·hm~(-2) of potassium fulvic acid per hectare, the content of alcohol soluble extract of F. thunbergii was ranged 21.61% to 22.27%, the total amount of peimine A and peimine B were ranged 0.09% to 0.10%. Applying 1.5 to 2.25 kg·hm~(-2) of potassium fulvic acid per hectare could replace the conventional pure chemical fertilizer potassium phosphate monobasic, which could be used as top dressing fertilizer for the cultivation of F. thunbergii.
Subject(s)
Benzopyrans/administration & dosage , Fertilizers , Fritillaria/chemistry , Phytochemicals/analysis , Potassium/administration & dosageABSTRACT
Objective: To evaluate antitumor activities of Fritillaria imperialis and Eryngium caucasicum methanolic extracts on human hepatoma (HepG2) and colon cancer (HCT116) cell lines in comparison to human foreskin fibroblasts as the normal cells. Methods: Methanolic extracts of Fritillaria imperialis and Eryngium caucasicum were prepared by the maceration method. The effect of the extracts at various concentrations (100, 200, 400, 600, and 800 μg/mL) on cell survival was evaluated using the MTT method. Besides, fluorescence staining was used to evaluate death patterns of the cells. Results: MTT assay showed that Fritillaria imperialis significantly decreased the viability of all cell lines after 24 and 48 hours of treatments. However, Eryngium caucasicum extract did not show any significant cytotoxicity effect on the cell lines. Fluorescence staining revealed that Fritillaria imperialis induced apoptosis of HCT116 cells at 550 μg/mL. Conclusions: Fritillaria imperialis extract has antiproliferative and cytotoxic effects on HCT116 and HepG2 cancer cells and therefore, may serve as an anticancer agent.
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OBJECTIVE: To establish an effective high performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD) method to simultaneously detect multi alkaloids in Fritillaria wabuensis, which can be used to control the quality of Fritillaria wabuensis. METHODS: The method was conducted on an InertSustain C18 column (4.6 mm×250 mm, 5 μm) with 0.1% TFA (V/V) aqueous solution-acetonitrile as mobile phase eluted in a gradient program. RESULTS: The method had good linearity (r2≥0.999 0).The accuracy was between 90% and 105%. This method was simple, sensitive, accurate and specific, which can be applied to determine imperialine-3-β-D-glucoside, imperialine, peimisine, verticine, verticinone, isoverticine and chuanbeinone in Fritillaria wabuensis. CONCLUSION: Nearly all fritillariae alkaloids can be detected under this condition, therefore, the chromatogram can be used as the characteristic chromatogram to testify different kinds of Fritillaria.The method can also be combined with mass spectrometric detector. It′s the first time for isoverticine and chuanbeinone to be detected in Fritillaria wabuensis by HPLC and also the first time for seven isosteroidal alkaloids in Fritillaria wabuensis to be simultaneously determined.
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Objective: To evaluate antitumor activities of Fritillaria imperialis and Eryngium caucasicum methanolic extracts on human hepatoma (HepG2) and colon cancer (HCT116) cell lines in comparison to human foreskin fibroblasts as the normal cells. Methods: Methanolic extracts of Fritillaria imperialis and Eryngium caucasicum were prepared by the maceration method. The effect of the extracts at various concentrations (100, 200, 400, 600, and 800 μg/mL) on cell survival was evaluated using the MTT method. Besides, fluorescence staining was used to evaluate death patterns of the cells. Results: MTT assay showed that Fritillaria imperialis significantly decreased the viability of all cell lines after 24 and 48 hours of treatments. However, Eryngium caucasicum extract did not show any significant cytotoxicity effect on the cell lines. Fluorescence staining revealed that Fritillaria imperialis induced apoptosis of HCT116 cells at 550 μg/mL. Conclusions: Fritillaria imperialis extract has antiproliferative and cytotoxic effects on HCT116 and HepG2 cancer cells and therefore, may serve as an anticancer agent.
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Objective: To study the variation patterns of rhizospheric soil nutrient and the content of nutrient elements for Fritillaria taipaiensis,in order to provide the theoretical base for the soil improvement and balanced fertilization during the artificial cultivation. Method: Totally 14 samples of rhizospheric soil of Fritillaria taipaiensis from different origins and years were taken as the research objects. Total N,available N,total P,available P,total K,available K,organic matter,pH and 7 nutrient element contents (Ca,Mg,Na,Mn,Zn,Cu and Ni) were analyzed by the soil agrochemical analysis method combined with the atomic absorption spectrophotometry. SPSS 22.0 software was applied for data multiple comparison and correlation analysis. Result: The all results showed significant differences (PF. taipaiensis. The content of total N,available N,total P,available P and organic matter of rhizospheric soil collected from cultivated varieties decreased with the increase of years,and the content of total K,available K and pH decreased first and then increased. However,the soil physical and chemical properties of wild varieties had no obvious change with the increase of years. The content of Ca,Mg,Na and Cu of soil from cultivated varieties decreased with the increase of years,while the content of Mn decreased first and then increased. And Zn and Ni showed no significant change with the increase of years. Compared with cultivated varieties,the content of Ca,Mg,Na,Mn and Cu increased first and then decreased. The content of Zn and Ni showed no obvious change. In general,the rhizospheric soil nutrient and the content of nutrient elements for wild F. taipaiensis were superior to those of cultivated varieties. Conclusion: The third year is the turning point of F. taipaiensis growth. The rhizospheric soil nutrient and the content of nutrient elements decreased obviously after three years. Attention shall be given to the balanced fertilization,the improvement of soil quality and the prevention of the cropping during cultivation of F. taipaiensis.