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italic>Polygonatum franchetii Hua is a medicinal plant endemic to China from Polygonatum Mill. The chloroplast genomes of two P. franchetii individuals sampled from two different habitats were sequenced by using the DNBSEQ-T7 high-throughput sequencing platform. After assembly and annotation, the two complete chloroplast genomes were characterized, and then comparative and phylogenetic analyses were performed with other published chloroplast genome sequences from Polygonatum. The whole chloroplast genomes of the two P. franchetii individuals were 155 942 and 155 962 bp in length, with a large single copy region (LSC, 84 670 and 84 722 bp), a small single copy region (SSC, 18 564 and 18 566 bp) and a pair of reverse repeats (IRa/IRb, 26 354 and 26 337 bp), respectively. Both of them contained 113 genes, including 79 protein-coding genes (PCGs), 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. Comparative analyses showed that the genome length, the guanine and cytosine (GC) content, genes content and order were highly conserved between the two P. franchetii individuals and among different Polygonatum species. The detected repeat sequences, including dispersed repeats, tandem repeats and simple sequence repeats (SSRs), were also relatively similar in types and positions, though showing a slightly difference in number. No significant expansion or contraction of the inverted repeat regions was found. Sequences variation between the two P. franchetii individuals was lower than that among different Polygonatum species. Besides, coding sequences (CDS) showed less divergence than noncoding sequences, and sequence divergence of IRs regions was lower than that of the LSC and SSC regions, both intraspecifically and interspecifically. Eight sequences with high nucleotide diversity among different species were screened, all of which were found located in the LSC and SSC regions. Phylogenetic inference showed that all Polygonatum species clustered into a monophyletic clade with a 100% bootstrap value, within which, species in section Verticillata formed a distinct group, section Sibirica and section Polygonatum were sister groups. The two P. franchetii individuals grouped together and showed the closest phylogenetic affinity to P. stenophyllum Maxim., belonging to the section Verticillata. The chloroplast genome of P. franchetii and its phylogenetic position in Polygonatum were comprehensively investigated and clearly elucidated in this study, the results may lay a foundation for the resource development and utilization of P. franchetii, as well as further molecular identification and phylogenetic studies of medicinal Polygonatum species.
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In this study, the molecular mechanism of Cinnamomi Cortex-Rehmanniae Radix (CR) in the prevention and treatment of osteoporosis (OP) was investigated by integrating compatibility analysis of compound, bioinformatics and metabolomics. The rat OP models were established, and the Micro-CT indexes and pathological sections were comprehensively evaluated. The results showed that compared with the model group, the indexes such as bone mineral density (BMD) and bone volume/tissue volume (BV/TV) were significantly increased after CR treatment (P < 0.05), and the bone trabeculae were arranged into mesh. The results of UHPLC-Q-TOF/MS mainly involved amino acid metabolism, lipid metabolism and estrogen metabolism pathways. Integrating bioinformatics and metabolomics analysis, it was finally found that: ① cinnamic acid and ethylcinnamate inhibit inflammatory factors such as TNF, IL-1β, and IL-13, thereby preventing and treating OP; ② multiple active ingredients of CR target ESR2, PPARG, and CYP19A1, GABRA1 and other targets, regulate cAMP synthesis, AMPK signaling pathway and lipid metabolism, thereby regulating estrogen levels to prevent and treat OP; ③ oleic acid, arachic acid, etc. act on AR, VDR and other targets, and regulate HIF-1 signaling pathway and AGE-RAGE signaling pathway, thereby regulating osteoblasts and osteoclasts, and affecting calcium and phosphorus absorption to maintain bone homeostasis. This study clarified the molecular mechanism of CR in preventing and treating OP from the perspective of multi-directional regulation of inflammatory factors, estrogen and bone homeostasis, and provided theoretical basis for the clinical application of CR and the development of compound. This experiment complied with the ethical standards of animal experiments and was approved by the Animal Ethics Committee of Shaanxi University of Chinese Medicine (No. SUCMDL20210309002).
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MYB transcription factors play many important regulatory roles in plant growth and development, secondary metabolism, and stress adaptation processes. In this work, an MYB gene containing a complete open reading frame (ORF) was selected from the transcriptome database of R. palmatum L. RpMYB4 ORF and cloned, encoding a polypeptide of 245 amino acids with a molecular weight of 26.99 kDa. RpMYB4 lacks a signal peptide or transmembrane domain but contains two conserved DNA binding domains (HTH-MYB) of the R2R3-MYB subfamily at the N-terminus. Multiple-sequence alignment demonstrated that RpMYB4 shared as high as 61% identity with many MYB proteins from other species. Phylogenetic analysis showed that RpMYB4 had the closest relationship with FtMYB8 and was clustered in the S4 subfamily. Subcellular localization by confocal microscopy showed that an RpMYB4-GFP-fusion protein localized to the nucleus in tobacco. Real-time fluorescence quantitative PCR analyses revealed that RpMYB4 was differentially expressed in various tissues, with the highest expression in leaves, followed by petioles, rhizome, and roots, and with the lowest level in mature seeds. After treatment of R. palmatum L. seedlings with 200 μmol·L-1 MeJA, the expression of RpMYB4 in leaves was down-regulated within 24 h, and significantly up-regulated after 200 μmol·L-1 SA treatment at 12 h and 24 h. However, gene expression did not change with 200 μmol·L-1 ABA treatment. The transcripts of RpMYB4 under drought, high temperature, and mechanical injury stresses reached a peak at 24 h, 24 h, and at 3 h, respectively, while RpMYB4 expression was inhibited by low temperature stress, reaching its lowest value at 6 h. The gene showed no significant response to salt stress. Overall, RpMYB4 was cloned from R. palmatum L. for the first time, showed high expression in leaves, and was responsive to SA and various abiotic stress treatments including drought, high temperature, and mechanical injury. The results will be useful for further analysis of secondary metabolism and stress adaptations in R. palmatum L.
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italic>Bupleurum L. (Apiaceae) is an economically important genus, in which many species are of medicinal value. In this study, the complete plastid genomes (plastomes) of B. chinense DC. and B. boissieuanum H. Wolff were sequenced and their characteristics were investigated. Comparative and phylogenetic analyses were conducted with other published Bupleurum plastomes. The complete plastomes of B. chinense and B. boissieuanum were 155 458 and 155 800 bp in length, and both exhibited the typical quadripartite circular structure consisting of a large single copy region (LSC, 85 343 and 85 804 bp), a small single copy region (SSC, 17 495 and 17 410 bp), and a pair of inverted repeat regions (IRa/b, 26 310 and 26 293 bp), respectively. A total of 129 genes, including 84 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes were identified from each of the two plastomes. Repeat sequences detected were similar in types and distribution patterns, but the numbers were slightly different. Comparative analyses revealed that the Bupleurum plastomes were highly conserved in length, structure, the guanine and cytosine (GC) content, and gene content and order, both intraspecifically and interspecifically, and no obvious expansion or contraction of the inverted repeat regions occurred. Sequence variation was lower within the same species than among different species, noncoding sequences (including intergenic regions and introns) showed a higher divergence than the protein-coding sequences, and sequences in the LSC and SSC regions were more divergent than those in the IR regions. In addition, 11 sequences with higher nucleotide diversity among species were detected in the LSC and SSC regions. All studied Bupleurum species were inferred forming a monophyletic group with a 100% bootstrap value. Bupleurum chinense and B. boissieuanum were phylogenetically closest to B. commelynoideum and B. falcatum, separately, with all three B. chinense accessions clustered into a distinct clade. These results provide genetic information for further species identification, phylogenetic resolution, and will assist in exploration and utilization of medicinal Bupleurum species.
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Stroke has been harmful to human health for a long time, and there is no satisfactory treatment strategy because of its complex pathogenesis. Taohechengqi decoction has been effective in the treatment of stroke. In this study, the components were collected by TCMSP, TCMIP, BATMAN-TCM and TCMID databases, the targets were predicted and screened by PharmMapper and BATMAN-TCM databases, and the functional enrichment analysis of the targets was carried out by using R language package clusterProfiler. Finally, the key targets are verified by GEO database and molecular docking. The results showed that 51 active components of Taohechengqi decoction may regulate 15 key targets such as nitric oxide synthase, endothelial (NOS3), prostaglandin G/H synthase 2 (PTGS2), matrix metalloproteinase-9 (MMP9), affecting vascular endothelial growth factor signaling pathway and other pathways to play a role in the prevention of stroke, affecting tumor necrosis factor signaling pathway and other pathways to play a role in the treatment of stroke. GEO data analysis showed that androgen receptor (AR), caspase-8 (CASP8), intercellular adhesion molecule 1 (ICAM1), interleukin-1 beta (IL1B), mitogen-activated protein kinase 14 (MAPK14), MMP9, myeloperoxidase (MPO), peroxisome proliferator-activated receptor gamma (PPARG), PTGS2 and cellular tumor antigen p53 (TP53) were up-regulated genes, while serum albumin (ALB), estrogen receptor 1 (ESR1), NOS3, transcription factor p65 (RELA) and proto-oncogene tyrosine-protein kinase Src (SRC) were down-regulated genes. GEO analysis explained that Taohechengqi decoction may prevent stroke by down-regulating ESR1, NOS3, and treat stroke by up-regulating ICAM1, IL1B, MAPK14, MMP9, PPARG, PTGS2, TP53, and down-regulating RELA and SRC. The study found that in the process of prevention and treatment of stroke, Taohechengqi decoction played a two-way regulation role through multi-genes and multiple ways, which provided a new strategy for the treatment of stroke.
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Objective:To investigate the allelopathic effects of water extracts from rhizosphere soil of three medicinal plants Rehmannia glutinosa，Pinellia ternata and Isatis indigotica on seed germination and seedling growth of Polygala tenuifolia， screen the stubble varieties suitable for crop rotation with P. tenuifolia， and provide some scientific basis for continuous cropping obstacles of P. tenuifolia. Method:The bioassay method was used to study the effects of rhizosphere soil water extracts from three medicinal plants Rehmannia glutinosa，Pinellia ternata and Isatis indigotica at concentrations of 0.3，0.6，0.9 g·mL-1 on the germination of P. tenuifolia seed and seedling growth. Result:The rhizosphere soil water extracts of Rehmannia glutinosa and Pinellia ternata showed basically low-promotion and high-inhibition concentration effects on the final germination rate，germination potential，and germination index of P. tenuifolia seeds，while the water extract of Isatis indigotica showed significant allelopathic inhibition effect. All three rhizosphere soil water extracts showed significant allelopathic inhibition effects on the growth index of P. tenuifolia seedlings. Among them，the rhizosphere soil water extract of Rehmannia glutinosa showed lower inhibitory effect on the plant height and root length of P. tenuifolia seedlings than the other two water extracts. The photosynthetic pigment content，proline（Pro） content，and soluble sugar content of P. tenuifolia chinensis seedlings were the highest under 0.3 g·mL-1 soil water extract of Rehmannia glutinosa， with relatively higher content of soluble protein， and relatively lower content of hydrogen oxide（H2O2）. Under the treatment of 0.9 g·mL-1 soil water extract of Rehmannia glutinosa，P. tenuifolia seedlings had the highest peroxidase（POD） and superoxide dismutase（SOD） activities，low catalase（CAT） activity，and lowest content of malondialdehyde（MDA）. Conclusion:Based on the comprehensive analysis of the above experimental data and allelopathic effects，the water extract of rhizosphere of Rehmannia glutinosa can promote the germination of P. tenuifolia seeds to a certain extent，and lay the foundation for seedling resistance to biochemical stress. Therefore， Rehmannia glutinosa is more suitable for crop rotation with P. tenuifolia.
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The whole chloroplast genome ofthe medicinal plant Paeonia mairei H. Lév. was sequenced using the Illumina HiSeq X Ten platform and then assembled, annotated, and characterized by bioinformatic methods in this study. The complete chloroplast genome of P. mairei is 152 731 bp in length with the typical quadripartite structure, which consists of a large single copy-region (LSC, 84 402 bp), a small single copy-region (SSC, 16 969 bp), and a pair of inverted repeat regions (IRa and IRb, 25 680 bp), with an overall GC content of 38.4%. A total of 136 predicted genes, including 90 protein-coding genes, 38 tRNA genes and eight rRNA genes were identified. Among these, seven protein-coding genes, seven tRNA genes and four rRNA genes were found duplicated in the IR regions. In addition, 28 dispersed repeats, 10 tandem repeats, and 64 simple sequence repeats were detected within the whole chloroplast genome of P. mairei. Comparative analyses between 12 Peaonia species showed that the chloroplast genomes are highly conserved in length, gene content, gene order, and GC content. Meanwhile, the noncoding sequences (intergenic regions and introns) show a higher variation than the protein coding sequences, and sequences from the LSC region and SSC region are more variable than those from the IR regions. P. mairei was inferred forming in a distinct clade with P. lactiflora, P. obovate, and P. anomala subsp. veitchii with a 100% bootstrap value and is phylogenetically closest to P. lactiflora. These results may provide a basis for further genetic studies and the development and utilization of medicinal P. mairei.
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Objective: To establish the HPLC fingerprint of wild and cultivated Notholirion bulbuliferum,and recognize them according to the chemical pattern, in the expectation of providing the basis for the quality control and domestication cultivation of N. bulbuliferum of origins. Method: Twenty samples of wild and cultivated N. bulbuliferum collected from different origins were detected by HPLC, and a common mode of fingerprint was established. The Similarity Evaluation System for Chromatographic Fingerprint of TCM (2012A edition) was used to evaluate the similarity of the samples. The differences among the samples were identified by chemical pattern recognition methods, including principal component analysis (PCA),cluster analysis (HCA) and partial least squares discriminate analysis (PLS-DA). Result: The HPLC fingerprint of N. bulbuliferum was obtained,and 26 common peaks were found in the chromatograph. Similarities of all samples were over 0.9,PCA,and HCA and PLS-DA results demonstrated obvious distinctions between wild and cultivated N. bulbuliferum. Eight constituents,such as pcoumaric acid were identified as biomarkers,representing major differences between the two varieties. Conclusion: The HPLC chromatogram of N. bulbuliferum developed in this paper has strong characteristics and repeatability. After being combined with the pattern recognition mode, it can be used as an effective method for evaluating the quality of N. bulbuliferum and distinguishing wild and cultivated N. bulbuliferum,and provide a reference for the quality control and domestication introduction of N. bulbuliferum.
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Anthraquinones are not only the main active constituents but also the index components for the quality control of Rhei Radix et Rhizoma. To study the anthraquinone biosynthesis, Rheum palmatum L. seedlings were subjected to a high-throughput transcriptomic sequencing analysis by Illumina HiSeqTM 2000 150PE. The Illumina sequencing generated a total of 11.04 G clean data resulting in 736 309 74 clean reads, deposited in the sequence read archive (SRA accession SRP160030). Trinity do novo assembly yielded 93 646 unigenes, with an average of 1 108 nt. Functional annotation revealed that all unigenes were successfully annotated in the NR, NT, Swiss-port, PFAM, and KOG databases. GO enrichments showed that 57 subgroups were involved in biological process, cellular component, and molecular function. KEGG analysis indicated that 1 107 unigenes were implicated in 19 standard secondary metabolic pathways. 172 unigenes were analyzed to encode 28 key enzymes during the MVA, MEP, shikimic acid, and polyketide pathways related to anthraquinone biosynthesis. 125 CYP450 and 73 UGTs unigenes were related the modification of secondary metabolites in R. palmatum L. Furthermore, seven unigenes with full length cDNAs were successfully verified by RT-PCR and sequencing analyses. Then, MISA prediction produced a number of 18 885 simple sequence repeats (SSRs). Herein, the transcriptomic gene expression profiles of R. palmatum L. and candidate genes during the anthraquinone biosynthesis pathway were obtained for the first time. The results provided basic information for subsequent gene function characterization, secondary metabolic pathway analysis, and anthraquinone biosynthesis and regulation elucidation in R. palmatum L.
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Forsythia suspensa is a herbal medicine that widely used for heat-clearing and detoxification in clinical practice. However, the molecular mechanism of its heat-clearing and detoxifying effect is still unclear. Based on the theory and methods of network pharmacology, the efficacy of the heat-clearing and detoxification of Forsythia suspensa was analyzed in this study. A total of 114 of compounds in Forsythia suspensa were collected, and 15 of effective compounds were obtained by analyzing the bioavailability (OB) and drug-like properties (DL). Then 26 corresponding targets were obtained using reverse pharmacophore-docking method. Using the BioGPS database, the organ location of the target initially was revealed. The compound-targetdisease network model of Forsythia suspensa was constructed by using the Cytoscape, which showed that the material basis of the heat-clearing and detoxification of Forsythia suspensa was to synthesize and synergize the effects by combining various active ingredients of multiple targets, simultaneously. This study explains the scientific mechanism of the heat-clearing and detoxification of Forsythia suspensa, and provides a theoretical foundation for clinical rational usage of Forsythia suspensa.
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Objective To investigate the medicinal plant group and resource of Sect. Euthya in Paris L. in Shaanxi Province. Methods Through literature analysis and interview survey, combined with line transect method, the medicinal plant group, the natural distribution and the status of medicinal plant group and resources about Sect. Euthya in the specific county territory in Shaanxi Province were investigated.Results According to related records about the medicinal plants of Sect. Euthya in Paris L. included P. polyphylla, var. stenophylla, var. apetala, and Paris fargesii var. petiolatain in Shaanxi Province. Based on field investigation, it was found that, the medicinal plants of Sect.Euthya in Paris L.also included five variations of P.polyphylla var.latifolia,var.apperdiculata,var. thibetica, var. chinensis, and var. yunnanensis, which were new distribution records. No var. apetala was found under field investigation. Most of the rhizomes of the Sect. Euthya plants were used as Chinese materia medica Paridis Rhizoma, with wide distribution and good growth condition. The natural resources of these plants are endangered. Conclusion In this study, two species and six variations in the Sect. Euthya are identified as new distribution records. Consequently, the medicinal plant distribution record of Paris L. in Shaanxi Province is complete. The natural resources are investigated, which have laid the foundation for further research, development and protection.
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Objective: To study the effects of growth factors, altitude, and illumination (shady slope and sunny slope) on anthraquinones and tannins in medicinal rhubarb, and to find the theoretical basis for choosing the optimum growth conditions for rhubarb. Methods: The contents of aloe-emodin, rhein, emodin, chrysophanol, emodin, chrysophanol-1-O-glucoside, emodin-8-O-glucoside and (+)-catechin of different growth years at different altitudes and with different illumination were analyzed by variance analysis. Results: The mean contents of free anthraquinones in 1-, 2-, and 3-year rhubarb were 4.26, 5.18, and 8.10 mg/g, respectively. The mean contents of conjugated anthraquinones were 4.67, 5.62, and 6.76 mg/g, and the contents of (+)-catechin were 3.12, 4.18, and 4.72 mg/g, respectively. There were significant differences among the three altitude ranges of (1 300 ± 50), (1 500 ± 50), and (1 700 ± 50) m. There were significant differences between (1 300 ± 50) m and (1 500 ± 50) m and (1 700 ± 50) m in three different growth years (P 0.05). Conclusion: The contents of anthraquinones and tannins in rhubarb increase significantly with the increase of altitude with the same growth range and in the same growth year. There is significant difference between the growth years. When the illumination (shady slope and sunny slope) was different, there is no significant difference in the anthraquinone and tannin contents, but the average contents of anthraquinone and tannin in the sunny slope are higher than those in the shady slope. Artificial cultivation of medicinal rhubarb should be selected at an altitude of 1 400 m above the growth year of not less than 3 years, direct sunshine environment, which is conducive to improve the total contents of anthraquinone and tannin in medicinal rhubarb.
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Objective To evaluate Persicae Semen-Rhei Radix et Rhizoma (PS-RRR) of different compatibility of acute blood stasis rats blood rheology and blood coagulation function, and to reveale the effect of PS-RRR for promoting blood circulation to remove blood stasis effect scope, nature, and degree of interaction. Methods With ice water bath and injected adrenaline hydrochloride copy of acute blood stasis rats model, different ratio (0:1, 1:5, 2:5, 2:3, 1:1, 3:2, 5:2, 5:1, and 1:0) of different concentration of PS-RRR was given for later. Through the determination of whole blood viscosity (WBV), blood sedimentation (ESR), erythrocyte aggregation index (EAI), prothrombin time (PT), partial thrombin (APTT), fibrinogen (FIB) content of coagulation, thrombin time (TT), and the blood rheology of blood stasis rats was observed, the influence of the blood coagulation indexes. Then response surface analysis and multi-index comprehensive index method of PS-RRR different compatibility of promoting blood circulation was used to remove blood stasis effect comprehensive comparative analysis. Results The ratio of PS-RRR between 2:3 to 3:2 showed obvious synergy (strength of synergy: -0.8); In PS dose from 5.5-10 g and RRR dose from 2.1-5.8 g area showed the antagonism function (antagonism effect strength maximum: 0.6); While other percentage did not show obvious synergy or antagonism. Conclusion The results of reveal that scope, nature, and degree of PS-RRR for promoting blood circulation to remove blood stasis interaction effect, the and its prescriptions of traditional Chinese medicine (TCM) and clinical use of PS-RRR 1:1 is consistent with the highest frequency of conclusion. PS-RRR provides scientific basis for clinical applications.
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Objective: To establish an HPLC method for determing nine components from the leaves of Rheum officinale, such as gallic acid, senna glycosides B, rhubarb phenol-1-O-glucoside, emodin-8-O-glucoside, aloe-emodin, rhein, emodin, rhubarb, and emodin methyl ether, as well as to explore the scientific evidence for reasonable exploitation and utilization of medicinal rhubarb leaf. Methods: Takeing C18 chromatographic column (250 mm × 4.6 mm, 5 microns), methanol-0.2% acetic acid water as mobile phase, gradient elution and flow rate of 1 mL/min, column temperature 30℃, and detection wavelength of 260 nm. Results: Gallic acid, senna glycosides B, rhubarb phenol-1-O-glucoside, emodin-8-O-glucoside, aloe-emodin, rhein, emodin, rhubarb, and emodin methyl ether had the good linear relationship, respectively in the range of 20.2-606.0, 79.3-2 379.0, 10.1-301.8, 14.8-443.7, 0.7-2.2, 0.13-3.9, 12.4-372.0, 38.8-1 164.0, 6.2-185.4 ng (r > 0.999 6); The nine kinds of the ingredients of the average recovery were 95.76% - 95.76%, RSD was 1.46% - 2.43%. Conclusion: This method is simple and accurate, and with good effect and reliable results for the separation and determination of the components from the leaves of R. officinale, which can provide the reference for its rational development and utilization.