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Objective: To select the high altitude planting area for improving the quality of Angelica sinensis based on biomass, bioactive compounds accumulation and antioxidant capacity in rhizome. Methods: The experiments were conducted on dry weight (DW), bioactive compounds content and antioxidant capacity in two-year old rhizome of A. sinensis grown at different altitudes (2 100, 2 500, and 2 900 m), under controlling plants reproduced from the seeds of the same plant, the same levels of water and fertilizer in soil. Results: Higher altitude was adverse to rhizome biomass, however, the compounds content (DW and per plant basis) and antioxidant capacity were significantly promoted at higher altitude compared to lower altitude site; On a per plant basis, the content of ferulic acid, soluble sugar, phenolics and flavonoids in rhizome of un-bolted plants grown at 2 900 m respectively was 2.06, 1.13, 1.34 and 1.15 fold greater than that of 2 100 m; The antioxidant capacity also increased with higher altitude. Comprehensive analysis showed that total content of main bioactive compounds in rhizome of un-bolted plants increased with higher altitudes ranging from 2 100 to 2 900 m. Conclusion: Higher-altitude cultivation can significantly enhance bioactive compounds accumulation in rhizome. The biosynthesis and accumulation of bioactive compounds were regulated by low temperature and light. These findings will provide theoretical references for improving production and quality of rhizome as well as large-scale cultivation.
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Objective: To establish a biological potency assay for Xiaojin Pills against platelet aggregation in vitro, evaluate the quality consistency of Xiaojin Pills, and screen traditioanal Chinese medicines which play the role of promoting blood circulation in Xiaojin Pills. Methods: Xiaojin Pills and ten Chinese medicines [artificial musk, Momordica cochinchinensis, Aconitum kusnezoffii, Liquidambar formosana, Boswellia carterii, Commiphora myrrha, Faeces Trogopterori, Angelica sinensis, Pheretima aspergillum, Fragrant Ink] in its formula were extracted by ultrasound in 40% methanol. The antiplatelet aggregation rate of the extract was measured by platelet aggregation meter. The platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared from abdominal aorta of rats. The platelet aggregation was induced by adenosine diphosphate (ADP). With sodium ferulate as a standard reference material, the biological potency of antiplatelet aggregation of Xiaojin Pills was calculated by the simplified probit principle. Results: The results showed that the biological potency of Xiaojin Pills was between 0.598 and 1.338 U/mg among different manufacturers and batches. In Xiaojin Pills group, Pheretima, Faeces Trogopterori, and Momordicae Semen had stronger inhibitory effects on platelet aggregation with inhibition rates of 70.87%, 31.83% and 67.52%, respectively. Conclusion: The quality consistency of Xiaojin Pills from different manufacturers and batches is poor, and Pheretima, Faeces Trogopterori, and Momordicae Semen may be the key drugs for Xiaojin Pills to play the role of promoting blood circulation.
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Objective: To study the ecological suitability of Angelica sinensis growing in Gansu Province and guide the rational cultivation. Methods: Through visiting and field investigation, 1 545 batches of Chinese angelica samples were collected from the county areas in Gansu Province. The information about the longitude, latitude, altitude of each sampling point was collected by using the GPS, combining with national environmental factor data, and using Maxent model and spatial analysis function of ArcGIS software. Results: The areas with high suitability of A. sinensis distribution are in the southeast of Gansu Province. The main ecological factors affecting the suitability distribution of A. sinensis were altitude, rainfall in March, May and December, wettest month precipitation, soil pH and other ecological factors. Conclusion: The research findings are basically consistent with the living habits of A. sinensis that cultivated in high-cold mountain areas and plateau flat pasture areas with a cool climate, moderate soil, moisture, slightly acidic to neutral fertile and loose brown sandy loam soil. This result can provide scientific basis for the reasonable distribution of A. sinensis cultivation area in Gansu Province.
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The dried roots of Angelica sinensis has been widely applied in clinical care due to its efficiencies in nourishing and activating blood, regulating female menstrual disorders and relieving pains, relaxing bowels, etc. The cultivated two-year-old plants normally harvested roots for medicinal uses emerge over 30% early bolting rate, which leads to the lignified roots that are useless in medicinal agents. The early bolting and flowering that are leading to serious yield reduction has been one of the most serious problems in the production of A. sinensis for many years. Here, based on previously published research articles, monographs, patents as well as practice experiences, the research progresses on the internal and external factors affecting bolting and flowering of A. sinensis, the pathways regulating bolting and flowering, the mechanism revealing bolting and flowering by biotechnological interventions were summarized, with the aim of providing effective pathways jointly internal and external factors for regulating bolting and flowering, as well as references for further revealing the mechanism of the bolting and flowering of A. sinensis.
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Objective: To established a rapid nondestructive determination method for the multi-marker constituents in Angelica sinensis by near infrared spectroscopy (NIRS) combined with the partial least squares (PLS) method in order to improve the quality control for A. sinensis. Methods: A total of 108 batches of A. sinensis from different origins were collected for the research. An Ultra performance liquid chromatography (UPLC) method was established to measure the content of the seven components in A. sinensis, which were adopted as the reference value. And the integrating sphere diffuse reflection mode was employed to collect the NIR spectrum. The quantitative calibration model between the near infrared spectrum and the reference content of each component to be measured was established by PLS and other chemometrics methods. Each part of the modeling process was optimized respectively, including the selection of calibration set and validation set, different pretreatment methods and different spectral section. Results: The correlation coefficient for calibration set of chlorogenic acid, ferulic acid, isochlorogenic acid A, ligustilide, butylidenephthalide, senkyunolide I and levistilide A were 0.937 6, 0.970 2, 0.963 4, 0.991 1, 0.962 4, 0.966 6 and 0.947 6, respectively; The root mean square error of prediction (RMSEP) were 0.072 1, 0.038 9, 0.011 3, 0.483 0, 0.017 5, 0.178 0 and 0.097 0, respectively. The predicted values of NIRS models and the measured values of UPLC showed a good linear relation, which presented a great prediction ability of the models. Conclusion: The methods of NIRS combined with PLS can be applied for the rapid content determination of seven components in A. sinensis including chlorogenic acid, ferulic acid, isochlorogenic acid A, ligustilide, butylidenephthalide, senkyunolide I and levistilide A, which is proved to be simple and reliable.
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Objective: By screening the quality markers of Ca2+ antagonistic ingredients, a rapid evaluation system about the vasodilatory effect of Angelica sinensis (AS) by near infrared spectroscopy (NIR) was established. Methods: The Ca2+ antagonists in AS were screened by UPLC/Q-TOF combined with Ca2+ double luciferase reporter gene system. To establish the quality markers, the antagonistic effects were further evaluated in cells and in vitro. The quality markers in multi batches of AS were quantitatively analyzed, and the corresponding NIR was obtained, and then the NIR fitting algorithm was established. Meanwhile, the relationship between Ca2+ antagonistic holistic activity of AS extract and the content of quality markers was investigated, and the prediction model for vasodilation efficacy of AS was constructed based on quality markers check analysis via NIR technique. Results: The screening result showed that ligustilide (X1) and levistilide A (X2) in AS had significant Ca2+ antagonistic effects, and the change of the content was consistent with the capacity of Ca2+ antagonistic action of AS extracts, so they were confirmed as quality markers. According to nonlinear regression analysis, the relationship between Ca2+ antagonistic effect (Y) of AS and the content of two quality markers satisfied the following functions: Y = 31.257 9 X1 + 381.352 0 X2 - 248.979 0 X1X2 + 18.482 2. In addition, the measured values from NIR simulation method for ligustilide and levistilide A detection showed a good correlation with the predicted values. Conclusion: In this study, it was found that ligustilide and levistilide A were quality markers responsible for vasodilation efficacy in AS, and the quantitative correlation between quality markers and the function of vasodilation effect was established. With the help of NIR technology, a novel solution for the rapid monitoring of the quality of traditional Chinese medicine was demonstrated.
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Objective: To establish an HPLC method for the fingerprint analysis and content determination of standard decoction of Angelicae Sinensis Radix (ASR), and to provide an effective method to ensure the quality of standard decoction of ASR. Methods: Fingerprint of standard decoction of ASR was established by HPLC. The similarity evaluation combined with cluster analysis (CA), principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA) were applied to explore chromatographic peak of main affecting the quality of standard decoction of ASR, and to determine the contents of ferulic acid, ligustilide H, ligustilide I, ligustilide and tryptophan. All samples were analyzed by Kromasil C18 column (250 mm × 4.6 mm, 5 μm) maintained at 35 ℃ and eluted with acetonitrile-0.1% formic acid at the flow rate of 1.2 mL/min, and the detection wavelength was 280 nm. Results: There were 17 common peaks in the HPLC fingerprint of established standard decoction of ASR, and the similarity of 15 batches of standard decoction of ASR was between 0.788 and 0.983. The samples were broadly divided into three categories by CA and PCA. Seven markers were verified by PLS-DA, and peaks 8, 17, 10, and 12 were identified as ferulic acid, ligustilide, ligustilide I, and ligustilide H, respectively. In quantitative analysis, the five components showed good regression (r2 > 0. 999 0) with linear range, the content respectively was 0.041%-5.596% in ferulic acid, 0.026%-1.583% in ligustilide H, 0.201%-6.461% in ligustilide I, 0.126%-4.942% in ligustilide, and 0.481%-2.753% in tryptophan, and the average recoveries were in the range of 99.43%-104.35%. Conclusion: The analysis method established in this experiment is stable, reliable, and repeatable, which can provide reference for the quality evaluation of standard decoction of ASR.
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Objective To compare the populations genetic diversity between wild and cultivated populations of Angelica sinensis. Methods ISSR markers were used to analyze the genetic diversity of 1 038 samples, which included 48 cultivated populations and seven wild populations. Shannon’s information index (I) and other parameters of genetic information were calculated by Popgen 1.32. Using independent sample t-test in SPSS19.0 to examine whether there was significant difference. The AMOVA analysis of the genetic variation of two groups was carried out by using GenAlEx 6.5; UPGMA dendrogram relationship between wild and cultivated populations were clustered by Ntsys, and the correlation of genetic distance and geographic distance was analyzed by using the mental test in GenAlEx 6.5. Results The average percentages of polymorphie bands (PPB) of wild and cultivated populations were 83.77% and 96.10%; Shannon’s information indexes (I) were 0.221 7 and 0.282 8; The average values of Nei’s genetic diversity index (He) were 0.344 4 and 0.434 30, and there had extremely significant differences (P < 0.001). The genetic differentiation coefficient (Gst) and gene flow (Nm) of wild populations were 0.299 1 and 1.171 8, which of cultivated populations were 0.733 4 and 0.181 7. The variation percentage was 15.85% between groups of wild and cultivated. The variation percentage among populations was 54.3%, and the variation percentage within populations was 54.3%; The genetic distance varied from 0.014 4 to 0.730 7. Conclusion The genetic diversity of wild populations was lower than the cultivated populations; The level of genetic differentiation among wild populations was obviously lower than the cultivated populations; The total genetic variation of A. sinensis mainly existed among populations; It has a distant relationship between wild and cultivated populations, and has correlation between genetic distance and geographic distance.
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Objective: To improve the screening efficiency of the active ingredients in natural products by building up a kind of novel and efficient magnetic nanoparticle-assisted cell membranes (MN-CMs) fishing assay employing specific affinity interactions between active ingredients and receptors on cell membranes (CMs). Methods: The magnetic nanoparticles (MNPs) were combined with erythrocyte membrane of rabbits to fish active ingredients from water extracts of Angelica sinensis, and the fishing results were analyzed by GC-MS. Results: The particle size of the self-made magnetic beads was about (250.6 ± 3.3) nm (n = 3) with PDI index at 0.010 ± 0.003 (n = 3), and the beads were monodisperse, strongly magnetic and superparamagnetic, the saturation magnetization was 83.4 emu/g. The combination of MNPs and CMs was stable, the maximum combined amount was 1.02 mg CMs/10 mg MNPs, and the combination was able to keep better enzyme activity of CMs in the fishing assay. GC-MS results showed that ligustilide was fished out as the active compound from water extracts of A. sinensis by MN-CMs assay with the retention time at 20 min, and the new established fishing assay could effectively avoid the interference of inactive components. Conclusion: Ligustilide, one of the active ingredients in A. sinensis, can be screened out by the established fishing assay of MN-CMs. The developed fishing method in this workmakes up for some deficiencies of traditional screening method and provides a novel and efficient way to screen ingredients from natural products.
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Objective To evaluate the safety of new Angelica sinensis species with ionizing radiation. Methods GC was adopted to determine the organochlorine pesticide residues; ICP-MS was used to identify the contents of lead (Pb), cadmium (Cd), and copper (Cu); Mercury vapourmeter was used to determine the content of mercury (Hg); Atomic fluorescence spectrometry was used to identify the content of asenic (As). Adopting gray correlation as experiment method and taking the total ash, acid-insoluble ash, heavy metals, and harmful elements content as evaluation indexes, combined with acute toxicity test on mice, the study is to evaluate the safety of the new A. sinensis varieties cultivated with ionizing radiation. Results The results show that the organochlorine pesticide residues, heavy metals, and harmful elements contents in Mingui No.3 and Mingui No.4 comply with regulations in "Green Industry Standards for the Import and Export of Medicinal Plants and Preparations of the People's Republic of China". Comprehensive evaluation of the safety of the samples with gray correlation degree showed that the safety of Mingui No.3 and Mingui No.4 was superior to other samples. The enrichment degrees of different parts of A. sinensis on heavy metals and harmful elements were different: The head of A. sinensis presented higher enrichment degree on the heavy metals lead (Pb), cadmium (Cd), and copper (Cu), the tail shows higher enrichment degree on such heavy metals as arsenic (As) and mercury (Hg), and the body presented lower enrichment degree on heavy metals. In the acute toxicity experiment of mice, LD50 was not measured in the mice and the maximum tolerance of mice was 120 g/kg, equivalent to 480 times the clinically maximum amount of adults (60 kg). Conclusion There is no obvious toxicity in new A. sinensis species with ionizing radiation, and the clinical usual dosage is safe and feasible.
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Objective: To investigate the active ingredients and molecular mechanism of Astragalus membranaceus var. mongholicus, Angelica sinensis, Angelicae dahurica, and Gleditsia sinensis in Tuoli Xiaodu Powder in promoting of diabetic wound healing. Methods: UPLC-Q-TOF/MS in positive and negative ion modes was applied to analyze the components in the ethanol extract from Tuoli Xiaodu Powder. Molecular docking technology was used to predict the targets proteins of these components. The function and pathway annotations of target proteins were performed through relevant databases such as Uniprot and KEGG. The drug components-target-function diagram was constructed using Cytoscape software. Results: Twenty-eight compounds containing flavonoids, saponins, coumarins, alkaloids, and triterpenoids were identified in positive and negative ion modes. Among these compounds, 17 compounds could interact with 17 target proteins, and there were 210 pairs of component-target relationships by analyzing the results of molecular docking. Among them, five targets were related to immune regulation, six targets were related to antibacterial and anti-inflammatory effects, six targets were related to cell differentiation, 10 targets were related to cell migration, six targets were related to angiogenesis, two targets were related to stimulation of epithelial growth factor, six targets were related to vasodilation, and two targets were related to estrogen. Conclusion: The flavonoids, saponins, coumarins, steroids, and triterpenoids contained in the simplified formula possess many biological effects such as antibacterial, anti-inflammatory, immune regulation, and angiogenesis. These functions may be related to its modulation of NF-κB, PI3K/Akt/eNOS, and MAPK pathway through regulating NF-κB, MAPK, PI3K, and ERK2 targets.
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Objective To analyze the material basis and molecular mechanisms of Danggui Beimu Kushen (DBK) Pills in treating prostatic diseases based on the method of integrated pharmacology. Method The platform of Integrative Pharmacology of Traditional Chinese Medicine (TCM-IP, www.tcmip.cn) was utilized to predict the main active ingredients and functional targets of DBK Pills in treating prostatic disease, key targets were screened for enrichment analysis of pathways, and the network of “herb-core component-key target-main pathway” was constructed, and the possible mechanisms of DBK Pills in treating prostatic diseases were explored. Results A total of 532 candidate key targets for the treatment of prostatic diseases by DBK Pills were predicted, and 1 840 terms of gene function and 194 signal pathways were analyzed by gene ontology (GO) and KEGG, respectively. The network analysis of “herb-core component-key target-main pathway” showed that 65 core components were predicted, including 29 ingredients from Angelica sinensis, 11 from Fritillaria thunbergii and 26 from Sophora flavescens. Those predicted components acted on the key targets of prostatic diseases, such as transcription factor binding, negative regulation of apoptosis, et al, through the estrogen, apoptosis, chemokines and other signal pathways, and thus played a role in the regulation of cell cycle, apoptosis and proliferation imbalance, which might be the molecular mechanisms of DBK Pills for the treatment of prostatic disease. Conclusion DBK Pills regulate the development of BPH, prostate cancer and other diseases through multiple pathways with multi-component interacting with multiple targets.
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Objective In this study, Angelica sinensis and its adulterants, close relatives were analyzed by ITS2 sequences to investigate the genetic structure from Min County, Gansu Province. Methods In this study, the internal transcribed spacer 2 (ITS2) regions of samples were amplified by PCR and sequenced bi-directionally. Obtained sequences were assembled using Codon Code Aligner. The genetic distances were computed by MEGA 6.0 in accordance with the kimura 2-parameter (K2P) model and the phylogenetic tree was constructed by Neighbor-joining (NJ) method. Moreover, the secondary structure of ITS2 was predicted using ITS2 database websites. Results The ITS2 region was 230 bp and base composition was GC content of 55.46% of A. sinensis in Min County. In its adulterants and close relatives of A. sinensis, the ITS2 region ranged in size from 228-233 bp and base composition was with G + C content ranged from 51.53%-65.65%. A total of 220 polymorphic sires were detected from 53 sequences, in which parsimony informative sires were up to 213. By K2P model, the intra-specific genetic distances of A. sinensis in Min County were smaller than inter-specific ones of A. sinensis and its adulterants in ITS2 regions. NJ tree and secondary structure results could distinctly differentiate quality product and adulterants. Conclusion ITS2 sequence can accurately identify the authenticity of A. sinensis, which provide a effective technology for the healthy and sustainable development in A. sinensis market.
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Objective: To study the effect of volatile from isolated garlic sprouts on the germination characteristics of Angelica sinensis seeds under simulated continuous cropping stress. Methods: Treating the obstacle of A. sinensis seeds under simulate continuous cropping stress with the extract from Angelica Sinensis Radix and using isolated garlic sprouts to simulate garlic volatile allelopathy environment, so as to study the germination characteristics of A. sinensis seeds. Results: The extracts from Angelica Sinensis Radix inhibited the germination of A. sinensis seeds, and the higher the concentration was, the stronger the inhibition effects will be. The volatile from the isolated garlic sprouts promoted the germination of A. sinensis seeds, which was not treated with the extracts from Angelica Sinensis Radix at low concentration (50 g isolated garlic sprouts) and inhibited the germination at high concentration (100-200 g isolated garlic sprouts). The isolated garlic sprouts volatile had certain promoting allelopathy on the germination properties of A. sinensis seeds which were treated with the extracts from Angelica Sinensis Radix. When the fresh weight of the donor garlic sprouts was 50-100 g, the promoting effects on A. sinensis seeds germination reached extremely significant level (P<0.01). When the fresh weight of garlic sprouts was 200 g, the promoting effects were not significant (P<0.05). Conclusion: The volatile from the isolated garlic sprouts could alleviate the germination inhibition by extracts from Angelica Sinensis Radix.
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Objective: To clone the full-length cDNA of caffeic acid O-methyltransferase (COMT) encoding gene from Angelica sinensis and to perform bioinformatic analysis for the cDNA sequence. Methods: Extracting the total RNA from the leaves of A. sinensis as cDNA synthesis template, the full length COMT cDNA of A. sinensis was cloned through homology-based cloning and rapid amplification of cDNA ends (RACE) technique. The bioinformatics of the cDNA sequence was analyzed by Blast N, Blast P, ORF Finder, Compute PI/Mw, ProtScale, PROSITE, and SWISS-MODEL sequences online analysis tools on NCBI, ExPASy, DNAMAN, and MEGA softwares. Results: The full-length of COMT cDNA (1 436 bp) was obtained (GenBank accession number: KP188587). It included 5'-UTR (76 bp) and 3'-UTR (362 bp), with an open reading frame (ORF) of 1 098 bp, encoding 365 amino acid polypeptides. The relative molecular mass of COMT calculated was 40 230, theoretical isoelectric point (PI) was 5.43, and there was no signal peptide in COMT. The protein sequence contained typical class II O-methyltransferases domain: SAM_OMT_II. Conclusion: A novol cDNA encoding COMT from A. sinensis is cloned. This work might establish an experimental basis for exploring COMT gene function and the biosynthetic and regulation of ferulic acid (FA) in A. sinensis.
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Objective: To investigate the effects of different proportions of Astragalus and Angelica on the proliferation ability and cell senescence of hematopoietic progenitor cells (HPC) in the mice model of bone marrow hematopoiesis suppression, and to probe the mechanism of Astragalus-Angelica compatibility on promoting hematopoiesis. Methods: ICR male mice were randomly divided into normal group, model group, positive control group of recombinant human granulocyte colony stimulating factor (rhG-CSF), Astragalus group, Angelica group, and different proportion combination groups of Astragalus and Angelica, and the animals in Chinese medicinal herb groups were ig administered, once a day, for 8 d. In the positive control group, rhG-CSF was sc injected on days 6, 7, and 8 of administration. Except for the normal group, the others were received cyclophosphamide (CTX) by ip injection on days 4, 5, and 6 of administration to establish the model of bone marrow hemopoiesis suppression. The mice were killed on day 9 to obtain bone marrow cells for the culture of HPC, and the senescence rate of bone marrow nucleated cell (BMNC) was detected by SA-β-galactosidase staining method. Results: Compared with the model group, Angelica and Astragalus-Angelica with proportions of 5:1, 1:1, and 1:5 could significantly raise colony forming unit-granulocyte (CFU-GM), colony forming unit-megakaryocyte (CFU-MK), colony forming unit-erythroid (CFU-E), and burst forming unit-erythroid (BFU-E) (P < 0.01). Astragalus-Angelica with 10:1 made CFU-MK, CFU-E, BFU-E remarkably increase (P < 0.05, 0.01), and had no effect on CFU-GM. Furthermore, Astragalus-Angelica with 1:1 promoting the formation of HPC was evidently stronger than that of single Astragalus, single Angelica, and other combinations (P < 0.05). Compared with the model group, the senescence positive rate of BMNC was markedly decreased in Astragalus group, Angelica group, and Astragalus-Angelica combination groups (P < 0.01), while being lowest in Astragalus-Angelica with 1:1 proportion (P < 0.05, 0.01). Conclusion: Astragalus, Angelica, Astragalus-Angelica with 10:1, 5:1, 1:1, and 1:5 proportions can prompt the proliferation and differentiation of HPC that bone marrow hematopoiesis is suppressed in mice, inhibit the senescence of bone marrow hematopoietic cell, moreover, the effect of Astragalus-Angelica with 1:1 proportion is the best. It suggests that promoting the proliferation and differentiation of HPC is one of the mechanisms that Astragalus-Angelica prompt hematopoiesis.
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Objective: To study the chemical constituents from the roots of Angelica sinensis. Methods: The chemical constituents in the roots of A. sinensis were separated and purified through various kinds of chromatographic methods, including silica gel, MCI-Gel, Sephadex LH-20, and RP-HPLC. Their structures were elucidated by spectral data and physical-chemical properties. Results: 6-Hydroxy-3-(4-hydroxyphenyl)-7-methyl-coumarin (1) was isolated from the 95% ethanol extracts of this plant. Conclusion: Compound 1 is a new compound, named Angelica coumarin A. And it displays the cytotoxicity against A549 and MCF7 cells with IC50 values of 3.2 and 2.8 μmol/L, respectively.
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Objective: To investigate the genetic diversity of Angelica sinensis from different habitats in Gansu province. Methods: ISSR markers were used to analyze the genetic diversity of 41 populations of A. sinensis. Nei's genetic diversity index (H) and other parameters of genetic information were calculated by Popgene 32. UPGMA dendrogram relationship was clustered by Ntsys. Results: Eight primers produced 154 bands, among which 119 were polymorphic bands, the average percentage of polymorphie bands (PPB) was 77.27%. H and Shannon's information index (I) were 0.222 9 and 0.337 4, the genetic differentiation coefficient (Gst) and gene flow (Nm) were 0.6839 and 0.231 1 within the population levels. The genetic distance varied from 0.042 9 to 0.327 8. Conclusion: The genetic diversity among species of A. sinensis is at higher level, but the level of genetic diversity between populations is higher than that within populations. There is a great degree of genetic differentiation among populations, and gene flow does not almost exist between populations.
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Objective: To clone the conserved cDNA fragment of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) gene from Angelica sinensis and to analyze its tissue-specific expression in roots, stems, and leaves. Methods: A pair of degenerate primers were designed according to the conservative sequences of the cloned DXR from other plant species. The total RNA from the leaves of A. sinensis was as template, DXR fragment was obtained by reverse transcription polymerase chain reaction (RT-PCR), connected to pGEM-T Easy vector then transformed into Escherichia coli DH5α. The positive clone identified by PCR was sequenced. Taking Actin of A. sinensis as a reference gene, SYBR Green quantitative RT-PCR was used to detect the relative expression levels of DXR different tissues of A. sinensis. Results: The DXR fragment of A. sinensis containing 564 bp encoding 187 amino acids was registered in Genbank (No. KJ000259). The sequence identity analysis suggested that both the obtained nucleotide sequence and its corresponding amino acid sequence shared more than 80% of homology with GenBank DXRs from Gossypium barbadense and other five higher plant species. DXR was expressed at the highest level in the leaves, and the relative expression levels in the leaves were 2.5 and 3.2 times relative to the stems and roots, respectively (P<0.01). Conclusion: A novel DXR fragment is cloned from A. sinensis and its tissue-specific expression in A. sinensis is investigated. This work might establish an experimental basis for the effective application of DXR in the future.
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OBJECTIVE: To identify Angelica sinensis and its adulterants by ISSR markers. METHODS: Genome polymorphism of 26 A. sinensis and its adulterants were analyzed by ISSR-PCR. The genetic similarity coefficients and genetic relationships were calculated by NTSYS-pc version 2.10. The dendrogram was constructed based on UPGMA clustering. RESULTS: Ten out of 100 ISSR primers are screened out and 96 bands were amplified, among which 85 were polymorphic fragments (percentage of polymorphic bands was 88.54%). The length of bands ranged from 200 to 2100 bp. One specific band was amplified by the primer UBC848.A. sinensis and its adulterants could be discriminated by primer UBC848 and UBC834. Results of UPGMA dendrogram indicated that A. sinensis from nine different places clustered together and related to A. acutiloba. The other species were clustered far away from A. sinensis. Three commercially crude materials purchased in the market were adulterants. CONCLUSION: The specific primers were found to identify A. sinensis by ISSR method. There is genetic diversity among A. sinensis from different production areas. The results provide molecular evidence for identification of A. sinensis and its adulterants and technical support of building up the stable quality evaluation system of A. sinensis.