ABSTRACT
Objective:To identify 24 <italic>Rana</italic> species such as <italic>Rana dybowskii</italic> by mitochondrial cytochrome C oxidase subunit I （<italic>CO</italic>Ⅰ） gene-based DNA barcoding and build the neighbour-joining （NJ） tree for hierarchical cluster analysis， so as to provide a basis for the identification and classification of <italic>Rana</italic> species as well as the discovery of new species. Method:<italic>R. dybowskii</italic>， <italic>R. chensinensis</italic>， <italic>R. amurensis</italic>， <italic>R. culaiensi</italic>s， and <italic>R. huanrenesis</italic>， ten for each species， were collected for DNA extraction and polymerase chain reaction （PCR） amplification<italic> </italic>and sequencing. A total of 50 <italic>CO</italic>Ⅰ gene sequences were obtained. Then 163 <italic>CO</italic>Ⅰ gene sequences for 24 species of <italic>Rana</italic> and one <italic>CO</italic>Ⅰ gene sequence for <italic>Pelophylax</italic>，<italic> Odorrana</italic>， <italic>Nidirana</italic>， <italic>Hylarana</italic>， and <italic>Amolops</italic> were harvested from GenBank. After sequence alignment by MEGA X， the parsimony-informative sites of <italic>CO</italic>Ⅰ gene sequences were analyzed and the intraspecific and interspecific genetic distances were calculated， followed by the built of NJ tree and hierarchical cluster analysis. Result:The <italic>CO</italic>Ⅰ gene sequences of 24<italic> Rana</italic> species including <italic>R. dybowskii</italic> were 554 bp in length and there were 210 parsimony-informative sites in total. The intraspecific genetic distance of each species was smaller than 2%. Except that the interspecific genetic distance between <italic>R. sangzhiensis</italic> and <italic>R. zhengi</italic> was 0.004， the genetic distances between the other species ranged from 0.024 to 0.228. <italic>R. sangzhiensis</italic> and <italic>R. zhengi</italic> were clustered into one branch and some <italic>R. dybowskii</italic> and <italic>R. uenoi</italic> into one branch. There were two separate branches for <italic>R. chensinensis</italic> and the other species were all clustered independently. Conclusion:<italic>CO</italic>Ⅰ-based DNA barcoding enabled the identification of 24 species of <italic>Rana</italic> including <italic>R.dybowskii</italic>. The findings supported that <italic>R. sangzhiensis</italic>， <italic>R. zhengi</italic>， <italic>R. coreana</italic>， and <italic>R. kunyuensis</italic> were the same species. One branch of <italic>R. chensinensis </italic>might be one of the four undownloaded species in Ranidae or a new species. The results have demonstrated that <italic>CO</italic>Ⅰ-based DNA barcoding allows not only the identification of 24 species of Rana including <italic>R. dybowskii </italic>but also the classification of ranidae species and the discovery of new species or subspecies.
ABSTRACT
We established a quality evaluation method for Shuanghuanglian preparations based on an effect-constituent index (ECI), which is guided by the clinical efficacy of Shuanghuanglian and a dose-efficacy correlation. An HPLC method was used to establish the quantitative fingerprint of Shuanghuanglian from different manufacturers and to determine the content of 10 fingerprint components, including baicalin, chlorogenic acid, forsythin, galuteolin, wogonin, forsythoside A, luteolin, caffeic acid, baicalein, and scutellarin. Using Staphylococcus aureus as biological model, the potency of Shuanghuanglian preparations was determined by antibiotic microbial assay. Using the method of PLC-DA, the efficacious antibacterial components were screened by "dose-efficacy" correlation analysis. According to the antibacterial potency and content of the antibacterial ingredients, combined with the method of the custom weight coefficient, ECI was calculated and verified. The results show that the antibacterial ECI can facilitate evaluation of the efficacy of Shuanghuanglian based on the composition of its contents, providing a new method for the quality control of traditional Chinese medicine.
ABSTRACT
In this study, we accurately collected the embryonic parenchyma cells and endocarp stone cells of Arctii Fructus at five different growth stages by laser microdissection. Quantitative analyse of caffeic acid, arctiin and arctigenin in these cells were performed using ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS). The results showed that a large amount of arctiin was produced and accumulated in embryonic parenchyma cells from the late flowering stage to mature stage, while much lower content of arctiin was produced and accumulated in endocarp stone cells at these stages. It suggested that the biosynthetic pathways of arctiin were different in embryonic parenchyma cells from endocarp stone cells of Arctii Fructus. Arctigenin was found to be produced and accumulated in both embryonic parenchyma cells and in endocarp stone cells from the late flowering stage to mature stage, but it reached a peak in endocarp stone cells at late flowering stage, then decreased slowly. The concentration of arctigenin was far less than that of arctiin regardless of embryonic parenchyma cells or endocarp stone cells. These results have validated the new method for analysis of dynamic accumulation of arctiin in Arctii Fructus by UFLC-MS/MS with frozen sections and microdissection.
ABSTRACT
Fructus Arctii is a traditional Chinese medicine. The main counterfeit species are the seeds of Arctium tomentosum, Onopordum acanthium, Silybum marianum, Saussurea costus, Amorpha fruticosa. Traditional identification methods or molecular barcoding techniques can identify Fructus Arctii and its counterfeit species. However, the identification of the mixture of it and its spurious species is rarely reported. In this paper, we sequenced the ITS2 sequences of Fructus Arctii and 5 kinds of spurious species mix powder by high-throughput sequencing to identify the mixed powder species and providing new ideas for the identification of Fructus Arctii mix powder. The total DNA in mixed powder was extracted, and the ITS2 sequences in total DNA was amplified. Paired-end sequencing was performed on the DNA fragment of the community using the Illumina MiSeq platform. The sequence was analyzed by the software FLASH, QIIME and GraPhlAn etc. The results showed that the high quality ITS2 sequences of 39910 mix samples were obtained from the mixed samples, of which the total ITS2 sequence of the samples genus was 34 935. Phylogenetic analysis showed that the samples contained Fructus Arctii, A. tomentosum, O. acanthium, S. marianum, S. costus and A. fruticosa. Using ITS2 sequences as DNA barcodes, high-throughput sequencing technology can be used to detect the Fructus Arctii and its spurious specie in mixed powder, which can provide reference for the quality control, safe use of medicinal materials of Fructus Arctii and the identification of mixed powder of traditional Chinese medicine.
Subject(s)
Arctium , Chemistry , Classification , DNA Barcoding, Taxonomic , DNA, Plant , Genetics , DNA, Ribosomal Spacer , Genetics , Drug Contamination , Drugs, Chinese Herbal , Reference Standards , Fabaceae , Fruit , High-Throughput Nucleotide Sequencing , Milk Thistle , Onopordum , Phylogeny , SaussureaABSTRACT
Objective To study the pharmacokinetic features of reactive sulfide in rats after oral administration of Cinnabaris. Methods An HPLC coupled with precolumn derivatization method was developed for the pharmacokinetic features study on reactive sulfide in rats after oral administration of Cinnabaris. Results Good linearity (r>0.99) was found for reactive sulfide in plasma in the concentration range of 0.25–15 μmol/L (r>0.99). The LOQ and LOD of the method were 0.1 μmol/L and 0.02 μmol/L, respectively. The intra- and inter-day precision was less than 4.4% and 3.5% respectively, and the accuracy was -9.9%–6.0%. The average recovery rate was 74.9%. 0.6 g/kg Cinnabaris was given the rats for gavage, and the time-course pharmacokinetics parameters were as follows:Cmax(1.33±0.13) μmol/L, tmax(150±34) min, t1/2(323±62) min, AUC0-∞ (5743±297) ng/mL?h. Conclusion A sensitive, robust and accurate precolumn derivatization-HPLC method for the determination of plasma reactive sulfide is developed and validated. The method is successfully applied in the pharmacokinetic features study on reactive sulfide in plasma of rats after administration of Cinnabaris.
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Traditional Chinese medicine Baitouweng have a long history of application. The pharmacopoeia included dry roots of Pulsatilla chinensis (Bge.) Regel of Ranunculaceae. There are easily confused species in the market circulation, such as P. cernua (Thunb.) Bercht. et Opiz., P. dahurica (Fisch.) Spreng., P. turczaninovii Kryl. et Serg., and P. chinensis (Bge.) Regel var. kissii (Mandl) S. H. Li et Y. H. Huang, etc. In this study, using the method of metagenomics, based on high-throughput sequencing technology, the ITS2 sequence of mixed samples of five species of Baitouweng medicinal materials was sequenced. First, the total DNA extraction of medicinal materials mixing powder, and the ITS2 fragment of total DNA was amplified by PCR. Second, the Illumina MiSeq platform was used to carry out Paired-end sequencing for DNA fragments. Last, using FLASH, QⅡME and GraPhlAn software to arrange and analyze, and clustering analysis with the sequences of uploaded to GenBank by our group in the early stage. The results showed that a total of 53 024 sequences of ITS2 were obtained from the mixed samples, there are 52 295 effective sequences, there are a total of 49 079 of five species of medicinal materials of P. Miller. After the representative sequences and the sequence of uploaded to GenBank by our group in the early stage were clustering analysis, 5 species of Baitouweng medicinal materials were clustered into one branch separately, presenting monophyletic. The results showed that using the high-throughput sequencing technology, using ITS2 sequence as DNA barcode, the mix powder of 5 species of Baitouweng medicinal materials could be effectively identified. It provides a new method and thought for the origin identification of mixed Chinese medicinal materials.
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A rapid and accurate method was established by psbA-trnH sequence to distinguish Dioscorea nipponica from other species belonging to the same genus.In this study, we collected 144 samples of D. nipponica from 17 different producing areas in China, the psbA-trnH sequenced of 23 nucleotide sequences were downloaded from GenBank, the sequences from Dioscorea genus. DNAMAN 8.0 software was used to show splicing, MEGA 6.0 software was applied for sequence analysis and comparison. The results showed that the genetic relationship between D. nipponica and D. subcalva was the closest, and the genetic relationship with D. zingiberensis was the furthest. It is indicated that the psbA-trnH sequence as a DNA barcode can effectively distinguish between D. nipponica and other plants in the genus of Dioscorea.
ABSTRACT
By using the drug metabolizing enzyme inhibitors, the effects of metabolic factors on potential liver injury induced by the main component, trans-2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside(trans-SG), in Polygonum multiflorum was investigated. The main metabolic enzyme isoforms involved in trans-SG metabolism were also screened. The results showed that trans-SG at the dosage 31 mg·kg-1 did not cause liver injury; and the combination of trans-SG with the phase I metabolic enzyme inhibitor, 1-benzylimidazole (10 mg·kg-1), did not change the degree of liver injury(compared with LPS + trans-SG group, P > 0.05). However, the combination of trans-SG with phase II metabolic enzyme inhibitor, ketoconazole(35 mg·kg-1), significantly increased the degree of liver injury(compared with LPS + trans-SG group, P < 0.05). The phase I metabolites of trans-SG were not detected in human liver microsomes phase I metabolism system, while the phase II trans-SG metabolites were detected in recombinant human UGT isozymes phase II metabolism system. Six isoforms of uridine diphosphate glucuronate transferase(UGT)exhibited abilities to metabolize trans-SG and the order of metabolic ability was: UGT1A1 > UGT1A9 > UGT1A7 > UGT1A10 > UGT2B7 > UGT1A8. The results showed that trans-SG was mainly metabolized by UGT in phase II metabolism. The inhibition of drug metabolizing enzymes of phase II can increase the liver injury susceptibility of trans-SG, which provides a reference to the evaluation of susceptible factors and drug incompatibility research of Polygonum multiflorum.
ABSTRACT
By determination the section color and lustre indexes as well as the content of baicalin in 30 batches of Scutellariae Radix slices, calculate the correlation of these two, screen the color and lustre indexes which could represent their intrinsic quality, and establish a new grade classification method based on the results. The results showed that samples met the conditions of △L≥－37, △b≥45 simultaneously were picked grade and the content of baicalin was of ≥200 mg•g⁻¹ definitely; Samples inconsistent with any one of above conditions were general grade. This research indicated that indexes of △L and △b could characterize both the color and luster of slice and intrinsic quality, so that could be used as the indexes to classify the grades of Scutellariae Radix slices accurately, easily and objectively. The research results would provide new ideas and references for grade classification of traditional Chinese medicine slices.
ABSTRACT
Ranae Oviductus has a high economic and social value, but its adulterants are more numerous, which causes a great confusion to the market. Using DNA bar code technology based on COI sequence for PCR amplification and sequencing of the identified Rana dybowskii, R. chensinensis, R. huanrensis and R. amurensiss, the COI gene database of four species of Rana was established, and comparing the measured sequence with the sequence of GenBank, four kinds of Rana were identified. The MEGA (molecular evolutionary genetics analysis) 7 .0 software was used to calculate the genetic distance of K2P and construct the NJ (neighbor-joining) system cluster tree. The sequence of the four species of Rana measured were clustered into one group with the sequence of the four kinds of Rana downloaded from GenBank, but separated from the two outer groups downloaded from GenBank. The COI gene of the R. dybowskii was likely to have regional differences, however this technique failed to distinguish male and female Rana. The results showed that DNA bar code technology could accurately identify the base of original animal of R. oviductus. It indicates that DNA bar code COI provides a new method for the identification of R. oviductus.
ABSTRACT
To identify origin of the medicinal materials Dryopteridis Crassirhizomatis Rhizoma by using the psbA-trnH sequence, the polymerase chain reaction (PCR) amplification and product sequencing of the experimental samples were performed. In order to expand the scope of the study, the psbA-trnH sequences of 8 genera and 3 species were downloaded from GenBank for analysis. DNAMAN 8.0 software was used to show splicing and comparison results of the peak diagrams with analysis of them, and MEGA 6.0 software was to calculate K2P genetic distances and establish clustering tree adjacent genus. The results showed that by using the psbA-trnH sequence, Dryopteridis Crassirhizomatis Rhizoma, its original plant and other easy-confused medicinal materials and plants can be distinguished with each other obviously, with the psbA-trnH sequence of Dryopteridis Crassirhizomatis Rhizoma completely consistent with that of its original plant. Consequently, it is revealed that it's feasible to identify Dryopteridis Crassirhizomatis Rhizoma and its original plant, and separate from its adulterants by means of the psbA-trnH sequence, which can provide more scientific bases for the further study of the identification of the ferny medicinal herbs.
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A method coupling ultra-performance liquid chromatography (UPLC) with quadrupole time-of-flight mass spectrometer (Qtof MS) using the electrospray ionization (ESI) source was developed for the identification of the major saponins from Panax notoginseng powder (PNP). Ten different PNP samples were analyzed and evaluated for their quality by similarity evaluation and principle component analysis (PCA). Based on the accurate mass, summarized characteristic fragmentation behaviors, retention times of different types of saponins, related botanical biogenesis, and reported chromatographic behavior of saponins, fifty-one common peaks were effectively separated and identified, including 28 protopanaxadiol saponins and 18 protopanaxatriol saponins. Simultaneously, 15 significant discrepancy compounds were identified from the disqualified PNP samples. The established UPLC/Qtof MS fingerprint method was successfully applied for profiling and identifying the major saponins of PNP, providing a fast quality evaluation tool for distinguishing the authentic PNP and the adulterated products.
Subject(s)
Chromatography, High Pressure Liquid , Methods , Drugs, Chinese Herbal , Chemistry , Panax notoginseng , Chemistry , Powders , Chemistry , Spectrometry, Mass, Electrospray Ionization , MethodsABSTRACT
AIM@#To investigate the chemical constituents from the leaves of Broussonetia papyrifera.@*METHODS@#The chemical constituents were isolated and purified by macroporous adsorptive resin D101, silica gel, and ODS column chromatography and preparative HPLC. Their structures were elucidated on the basis of 1D and 2D NMR analyses. In addition, their cytotoxic activity against human hepatoma carcinoma cells (HepG-2) were evaluated by the MTT method. Furthermore, RP-HPLC and colorimetric methods were used for the analysis of cosmosiin and total flavonoids.@*RESULTS@#A new lignan, together with five known compounds were obtained, and their structures were characterized as (+)-pinoresinol-4'-O-β-D-glucopyranosyl-4″-O-β-D-apiofuranoside (1), cosmosiin (2), luteolin-7-O-β-D-glucopyranoside (3), liriodendrin (4), 3, 5, 4'-trihydroxy-bibenzyl-3-O-β-D-glucoside (5), and apigenin-6-C-β-D-glucopyranside (6). Furthermore, RP-HPLC and colorimetric methods were established for the analysis of cosmosiin and total flavonoids.@*CONCLUSION@#Compound 1 was a new lignan, and compounds 5 and 6 were isolated for the first time from the title plant. Compounds 1, 4 and 6 showed definite activities against HepG-2, while the other compounds didn't show inhibitory effects. The optimal harvest time of B. papyrifera (L.) Vent. is September.
Subject(s)
Humans , Broussonetia , Chemistry , Cell Proliferation , Cytotoxins , Chemistry , Toxicity , Hep G2 Cells , Lignans , Chemistry , Toxicity , Molecular Structure , Plant Extracts , Chemistry , Toxicity , Plant Leaves , ChemistryABSTRACT
To investigate the chemical constituents of the processed rhizomes of Panax notoginseng, their 70% ethanol extract was chromatographed on macroporous resin (SP825), silica gel, RP-C18 and semi-preparative HPLC to afford compounds 1-23. On the basis of physicochemical properties and spectral data analysis, their structures were identified to be 6'-O-Acetylginsenoside Rh1 (1), ginsenoside RK3 (2), ginsenoside Rh4 (3), 20S-ginsenoside Rg3 (4), ginsenoside Rk1 (5), 20R-ginsenoside Rg3 (6), ginsenoside Rg5 (7), ginsenoside F2 (8), 20S-ginsenoside Rh1 (9), 20R-ginsenoside Rh1 (10), gypenoside X VII (11), notoginsenoside Fa, (12), ginsenoside Ra3 (13), ginsenoside Rg1 (14), ginsenoside Re (15), notoginsenoside R2 (16), ginsenoside Rg2 (17), notoginsenoside R1 (18), ginsenoside Rd (19), ginsenoside Rb1 (20), notoginsenoside D (21), notoginsenoside R4 (22) and ginsenoside Rb2 (23), respectively. Among them, compound 1 was isolated from P. notoginseng for the first time, and compounds 4, 6, 8 and 11 were isolated from the processed P. notoginseng for the first time. According to the fingerprint profiles of raw and processed P. notoginseng, the putative chemical conversion pathways of panoxatriol and panoxadiol compounds in the processing procedure was deduced, and the results revealed the main reactions to be dehydration and glycosyl hydrolysis.
Subject(s)
Chromatography, High Pressure Liquid , Drug Compounding , Drugs, Chinese Herbal , Chemistry , Molecular Structure , Panax , Chemistry , Rhizome , Chemistry , Spectrometry, Mass, Electrospray IonizationABSTRACT
<p><b>OBJECTIVE</b>To find out the optimum extract process for Ligusticum chuanxiong in Gan-ning Granule, and studyed the methods of concentration and dry for the extract.</p><p><b>METHOD</b>With the yield of ferulic acid as the assessment index, to optimize the 80% alcohol totalling, extracting times and circumfluence time for extract process by the orthogonal design, to optimize the inlet-air temperature, feed speed and density of feed for spry drying by the orthogonal design.</p><p><b>RESULT</b>The optimum procedure was the ferulic acid were extracted for 1 hour with 3 times of 80% alcohol. While extracting times effected it most porminently. The optimal processing conditions of spry drying were inlet-air temperature 120 degrees C, feed speed 8.5 mL x min(-1) and density of feed 1.15, While feed speed effected it most porminently.</p><p><b>CONCLUSION</b>The experimental results provide the basis for the extraction process and drying process of the ferulic acid in ligusticum chuanxiong.</p>