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OBJECTIVE：To esta blish a UPLC fingerprint of Pyrrosia petiolosa from southwest China ，and to determine the contents of 4 kinds of phenolic acids （neochlorogenic acid ，caffeic acid ，chlorogenic acid and cryptochlorogenic acid ）. METHODS：The determination was performed on Waters Cortecs T 3 C18 column（100 mm×2.1 mm，1.6 μm）with mobile phase consisted of methanol- 0.1％ phosphoric acid （gradient elution ）at the flow rate of 0.35 mL/min. The detection wavelength was set at 326 nm. The column temperature was 30 ℃，and injection volume was 1 μL. UPLC method was used to establish the UPLC fingerprint of P. petiolosa in combination with the Similarity Evaluation System of TCM Chromatographic Fingerprints （2012 edition）. Cluster analysis and principle component analysis （PCA）were performed by using SPSS 20.0 software. The contents of 4 kinds of phenolic acids in 20 batches of P. petiolosa were determined by external standard method. RESULTS ：There were 9 common peaks for the UPLC fingerprint of P. petiolosa . Peaks 1，3，4，5 and 9 were identified as neochlorogenic acid ，caffeic acid，chlorogenic acid ，cryptochlorogenic acid and isochlorogenic acid C ，respectively. RSDs of the relative retention time of each peak in different batches of P. petiolosa were 0-0.68％，and the RSDs of the relative peak area were 0-62.35％. The similarities between the fingerprint of 20 batches of medicinal materials and the control chromatogram were not less than 0.990. The result of cluster analysis showed that P. petiolosa from different regions could be sorted into three species. Results of PCA showed the differences among P. petiolosa from different regions. The linear range of neochlorogenic acid ，caffeic acid ，chlorogenic acid and cryptochlorogenic acid were 0.61-61.41，0.18-17.60，2.00-200.11，0.62-61.51 μ g/mL （R2＞0.999 9）. RSDs of precision ， reproducibility and stability tests were all lower than 2.00％. The recoveries were 96.23％-98.17％（RSD＝0.96％-2.28％， n＝6）. Among 20 batches of samples ，the contents of above 4 kinds of phenolic acids were 0.385 3-1.891 9，0.018 0-0.129 5，2.569 5-10.676 0，0.563.5-1.860 5 mg/g. CONCLUSIONS ： The established UPL C fingerprint could reflect the main chemical constituents of P. pedunculata . Phenolic acids could be used as the main evaluation indexes for the quality of P. petiolosa . The quality order of P. petiolosa from southwest China was Chongqing product＞Sichuan product ＞Guizhou product.
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Objective::The complete chloroplast genome of Pyrrosia assimilis was sequenced, its sequence characteristics was analyzed and herbgenomics of P. assimilis was discussed. Method::Its complete chloroplast genome sequence was determined through high-throughput sequencing technology, and its structural characteristics and phylogenetic relationships were analyzed by bioinformatics. Result::The chloroplast genome of P. assimilis was a circular double-chain structure with a total length of 154 964 bp, and the total content of guanine and cytosine (GC) was 41.2%. A total of 131 genes were annotated, including 88 protein-coding genes, 35 transfer RNA (tRNA) genes and 8 ribosomal RNA (rRNA) genes. A total of 43 dispersed repetitive sequences and 56 simple sequence repeats (SSRs) were detected. The frequency of codon encoding leucine was the highest, while the number of codon encoding tryptophan was the lowest. Five highly divergent regions (psbA, rrn16, petA-psbJ, ndhC-trnM, and psbM-petN) were screened, phylogenetic analysis showed that P. assimilis was closely related to P. bonii. Conclusion::Comparative analysis of the complete chloroplast genome of P. assimilis reveals that non-coding regions exhibited a higher divergence than the coding regions, the large single copy region (LSC) and small single copy region (SSC) are more divergent than the reverse repeat region (IR), the selected five highly variable regions can be used as specific DNA barcodes for identification of Pyrrosia species. Study on the chloroplast genome of P. assimilis can provide a reference for the molecular identification, genetic transformation, expression of resistance protein and secondary metabolism pathway analysis of other Pyrrosia medicinal plants.
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Pyrrosia petiolosa, Pyrrosia lingua and Pyrrosia sheareri are recorded as original plants of Pyrrosiae Folium (PF) and commonly used as Chinese herbal medicines. Due to the similar morphological features of PF and its adulterants, common DNA barcodes cannot accurately distinguish PF species. Knowledge of the chloroplast (cp) genome is widely used in species identification, molecular marker and phylogenetic analyses. Herein, we determined the complete cp genomes of three original species of PF via high-throughput sequencing technologies. The three cp genomes exhibited a typical quadripartite structure with sizes ranging from 158 165 to 163 026 bp. The cp genomes of P. petiolosa and P. lingua encoded 130 genes, whilst that of P. sheareri encoded 131 genes. The complete cp genomes were compared, and five highly divergent regions of petA-psbJ, matK-rps16, ndhC-trnM, psbM-petN and psaC-ndhE were screened as potential DNA barcodes for identification of Pyrrosia genus species. The phylogenetic tree we obtained indicated that P. petiolosa and P. lingua are clustered in a single clade and, thus, share a close relationship. This study provides invaluable information for further studies on the species identification, taxonomy and phylogeny of Pyrrosia genus species.
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To screen the active fractions with lithagogue effects of Pyrrosia lingua from Guizhou province and preliminarily investigate its mechanism. The rats were fed with 1% ethylene glycol and 2% ammonium chloride to establish the nephrolithiasis models, which were used to evaluate thelithagogue effects of different polar fractions of P. lingua from Guizhou province. The level of urinary calcium and oxalic acid in urine, renal calcium, oxalic acid, superoxide dismutase (SOD), catalase(CAT) and malondialdehyde (MDA) in renal tissues,as well as crystalline deposit and lithogenesis in renal tissues and the levels of creatinine(Cr) and blood urea nitrogen (BUN) in the serum were detected. The effective compounds were inferred from the analysis of active fractions extract based on LC-MS technology. Petroleum ether fraction and dichloromethane fraction of P. lingua from Guizhou province can reduce renal oxalic acid and renal calcium concentration, increase urinary oxalic acid and urine calcium, with significant inhibitory effect on the formation of renal calculus in rats, significantly increase SOD and CAT activities in renal tissues, and significantly reduce MDA levels. LC-MS analysis showed that the caffeine, citric acid and tartaric acid among the compounds from petroleum ether fraction and dichloromethane fraction had lithagogue effects. Both the petroleum ether fraction and dichloromethane fraction of P. lingua from Guizhou province showed good effect on prevention and treatment of calculus in middle dose groups, and the mechanism may be associated with antioxidation, reducing calcium oxalate crystal deposition, and promoting calcium oxalatecrystal release, in addition, caffeine, citric acid and tartaric acid had lithagogue effects.
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OBJECTIVE:To establish a method for the contents determination of 4 amino acids in Pyrrosia lingua. METHODS:The samples were analyzed by RP-HPLC. It was performed on a column of Welchrom C18 with the mobile phase A of 0.1 mol/L So-dium acetate buffer(pH6.5)- acetonitrile(93∶7,V/V)and mobile phase B of acetonitrile-water(4∶1,V/V)(gradient elution)at the flow rate of 1.0 ml/min,detection wavelength was 254 nm,column temperature was 30 ℃ and the sample size was 5 μl. RE-SULTS:The linear range was 4.92-49.2 μg/ml for glutamate(r=0.999 1),3.168-31.68 μg/ml for glycine(r=0.999 9),2.6-26 μg/ml for alanine(r=0.999 4)and 2.88-28.8 μg/ml for leucine(r=0.999 9);RSDs of precision,stability and reproducibility tests were no more than 2.2%;average recoveries were 98.4%(RSD=2.2%,n=9),99.6%(RSD=2.5%,n=9),98.3%(RSD=2.2%,n=9)and 100.1%(RSD=2.3%,n=9),respectively. CONCLUSIONS:The method is simple,accurate and reproducible,and can be used for the contents determination of 4 amino acids in P. lingua.
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Pyrrosia petiolosa (Christ) Ching, Polypodiaceae, is an important medicinal pteridophyte used for the treatment of nephritis and bronchitis, while P. davidii (Giesenhagen. ex Diels) Ching, Polypodiaceae, often substitutes medicinal Pyrrosia in clinic. The present study was aimed to compare the pharmacognosy of P. petiolosa and P. davidii, including plant morphology, microscopic characteristics, physico-chemical parameters, UV and IR spectrum, and HPLC fingerprint. It was revealed that the two herbs had basically similar pharmacognostical characteristics but with certain differences. The present study contributes to the standardization and verification of these medicinal materials.
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Objective To establish an HPLC method for simultaneous separation and determination of chlorogenic acid, quercetin and kaempferol inPyrrosia lingua (Thumb.) Farwell. Methods The separation was performed on Phenomenex C18 column (250 mm×4.6 mm, 5μm) with the mobile phase of methanol-acetic acid (pH=3.0) solution and gradient elution. Flow rate was 1.0 mL/min;the UV detection wavelength was 254 nm;column temperature was 35℃.Results The calibration curves for chlorogenic acid, quercetin, and kaempferol were in good linearity in the range of 0.000 24-3.00μg (r=0.999 9), 0.000 16-2.00μg (r=0.999 9), and 0.000 16-2.00μg (r=0.999 9), respectively. The limits of detection (S/N=3) were 3.29, 0.43 and 0.33 ng/mL, respectively. The average recovery rates were 97.73%, 98.07% and 96.92%, respectively. ConclusionThe method is simple, precise and sensitive. It provides scientific proof for separation and determination of chlorogenic acid, quercetin and kaempferol inPyrrosia lingua (Thumb.) Farwell.
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OBJECTIVE: To analyze the essential oils from the leaves of Pyrrosia petiolosa or Pyrrosia subfurfuracea.METHODS: The essential oils from the leaves of P.petiolosa or P.subfurfuracea were analyzed by head-space solid microextraction coupled with GC-MS(HS-SPME-GC-MS) on capillary column HP-5 MS (30.0 m?250 ?m?0.25 ?m).The carrier gas was helium(with a high purity of 99.999%) at a flow rate of 1.0 mL?min-1.The ionization mode: EI ionization source with electron energy of 70 eV.RESULTS: For the first time,39 constituents were identified from the leaves of P.petiolosa and 29 constituents were identified from the leaves of P.subfurfuracea.Of which,12 constituents were mutual in both samples.CONCLUSION: The components of the essential oil in leaves of the two Pyrrosia species bear some similarities,but the essential components of the two Pyrrosia species are different from each other fundamentally.Butylated hydroxytoluene in the leaves of P.petiolosa and caryophyllene in the leaves of P.subfurfuracea have medicinal value.