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Objective:This paper aims to clone the cDNA sequence of<italic> limonene</italic>-3-<italic>hydroxylase</italic>（<italic>StL</italic>3<italic>OH</italic>） in <italic>Schizonepeta tenuifolia</italic> and analyze its sequence by bioinformatics. Method:Specific primers were designed based on sequences of<italic> StL</italic>3<italic>OH </italic>gene screened from transcriptome sequencing data of <italic>S. tenuifolia</italic> and the cDNA sequence of <italic>StL</italic>3<italic>OH </italic>gene was cloned by reverse transcription polymerase chain reaction （RT-PCR） and analyzed for its bioinformatics. Result:The <italic>StL3OH</italic> gene cDNA sequence length was 1 598 bp，containing a 1 497 bp long complete open reading frame which encoded 498 amino acids. StL3OH protein had a theoretical relative molecular mass of 56.40 kDa，with a hydrophilic and unstable nature. Bioinformatics analysis showed that StL3OH protein had no signal peptide but had a transmembrane domain which might be located in endoplasmic reticulum. Multiple sequence alignment and cluster analysis showed that the amino acid sequence of MsL3OH protein had a high similarity with StL3OH protein，both of which contained cytochrome P450 heme binding region，belonging to the D subfamily of cytochrome CYP71 family. Codon bias analysis showed that <italic>StL</italic>3<italic>OH</italic> gene preferred guanine/cytosine（G/C） ending codon，with 27 skewed codons， and Nicotiana benthamiana was proven to be the most suitable host for exogenous expression of <italic>StL</italic>3<italic>OH</italic> gene. Conclusion:The cDNA sequence of<italic> StL3OH</italic> gene was cloned from <italic>S. tenuifolia</italic> for the first time，which will provide a basis for further study on the structure and function of StL3OH protein and the regulation mechanism of <italic>StL3OH </italic>gene in the accumulation and biosynthesis of monoterpenes in<italic> S. tenuifolia</italic>.
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Leaves of Euryale ferox are rich in anthocyanins. Anthocyanin synthesis is one of the important branches of the flavonoid synthesis pathway, in which flavonoid 3'-hydroxylase(F3'H) can participate in the formation of important intermediate products of anthocyanin synthesis. According to the data of E. ferox transcriptome, F3'H cDNA sequence was cloned in the leaves of E. ferox and named as EfF3'H. The correlation between EfF3'H gene expression and synthesis of flavonoids was analyzed by a series of bioinforma-tics tools and qRT-PCR. Moreover, the biological function of EfF3'H was verified by the heterologous expression in yeast. Our results showed that EfF3'H comprised a 1 566 bp open reading frame which encoded a hydrophilic transmembrane protein composed of 521 amino acid residues. It was predicted to be located in the plasma membrane. Combined with predictive analysis of conserved domains, this protein belongs to the cytochrome P450(CYP450) superfamily. The qRT-PCR results revealed that the expression level of EfF3'H was significantly different among different cultivars and was highly correlated with the content of related flavonoids in the leaves. Eukaryotic expression studies showed that EfF3'H protein had the biological activity of converting kaempferol to quercetin. In this study, EfF3'H cDNA was cloned from the leaves of E. ferox for the first time, and the biological function of the protein was verified. It provi-ded a scientific basis for further utilizing the leaves of E. ferox and laid a foundation for the further analysis of the biosynthesis pathway of flavonoids in medicinal plants.
Subject(s)
Anthocyanins , Cytochrome P-450 Enzyme System/metabolism , Plant Leaves/metabolism , Plant Proteins/metabolism , TranscriptomeABSTRACT
Based on the characteristics and ISSR molecular marker technology, the study is aimed to compare and perform genetic diversity analysis on Sparganium stoloniferum from 7 regions. Molecular identification method was established for S. stoloniferum from Hunan province. Differences among Sparganii Rhizoma samples from seven habitats were analyzed via measuring weight, length, width and thickness of them. Genetic diversity of S. stoloniferum from 7 regions was analyzed by screening out primers amplifying clear band and showing rich polymorphism, then a cultivars dendrogram was built. The target primer was screened out, and the specific band was sequenced. Nine ISSR primers were selected to amplified clear band, rich polymorphism. A total of 73 bands were amplified by nine ISSR primers selected from 27 ISSR primers. On average, each primer produced 8.0 bands. A total of 38 bands were polymorphic, which occupied 52.8% of all bands. The cultivars dendrogram showed the genetic similarity was 0.54-0.94. Genetic similarity coefficient of S. stoloniferum from Jiangsu province, Anhui province and Jiangxi province was big, indicating the differences among them were slight on genetic level. S. stoloniferum from Hunan province is quite different from samples from the other six habitats on appea-rance and genetic level. A specific band(327 bp) in S. stoloniferum from Hunan province was obtained via ISSR-857 primer, and was sequenced. According BLASTn database, there were few sequences similar to the gene fragment and had little correlation with the growth process of plant. ISSR molecular marker technology provides a new idea for the identification of S. stoloniferum. This result confirmed the particularity of S. stoloniferum from ancient Jingzhou.
Subject(s)
China , Drugs, Chinese Herbal , Genetic Markers/genetics , Genetic Variation , Microsatellite Repeats , Phylogeny , Polymorphism, GeneticABSTRACT
Objective: To elucidate the rationality of integrated processing technology of Leonuri Herba based on comparison of chemical constituents and pharmacological effect of Leonuri Herba between traditional and integrated processing technology. Method:The contents of stachydrine hydrochloride,leonurine hydrochloride,rutin,hyperoside and isoquercetin were used as indexes to compare the differences in the contents of chemical constituents between traditional and integration processing technology of Leonuri Herba.Effect of Leonuri Herba with different processing technology on auricular swelling induced by dimethylbenzene in mice were observed to compared the differences of their anti-inflammatory effect.And rat acute blood stasis model was used to compare the differences of Leonuri Herba with different processing technology on hemorheology and blood coagulation indexes. Result:Contents of stachydrine hydrochloride,leonurine hydrochloride,rutin,hyperoside and isoquercetin in products of integrated processing were 1.558%,0.168%,0.137%,0.113% and 0.078%,they were 1.482%,0.134%,0.125%,0.082% and 0.071% in products of traditional processing,respectively.Both of the processing methods could reduce the degree of swelling and the contents of tumor necrosis factor(TNF)-α,interleukin(IL)-1β,IL-6 in mouse serum.And both of the processing methods could reduce the whole blood viscosity,prolong the thrombin time(TT),prothrombin time(PT),activated partial thromboplastin time(APTT) and reduce the concentration of plasma fibrinogen(FIB) in acute blood stasis model rats. Conclusion:Compared with the traditional processing technology,the integrated processing technology is better in guaranteeing the quality of Leonuri Herba decoction pieces and reducing the production cost,which indicates that the integrated processing of Leonuri Herba is reasonable.
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Farnesyl pyrophosphate synthase of Alisma orientale (Sam.) Juzep. (AoFPPS) is considered as one of the important rate-limiting enzymes in the biosynthetic pathway of protostane triterpenes. In order to investigate the expression and function of AoFPPS, the gene (accession No. HQ724508) was cloned into a bacterial expression vector pCzn1, then the combined plasmid pCzn1-AoFPPS was transformed into Escherichia coli BL21, and a fusion protein was obtained after induction. The fusion protein was purified by Ni resin, and the function was verified through in vitro enzymatic reaction. High performance liquid chromatography (HPLC) analysis revealed that the products were able to catalyze the synthesis of farnesyl pyrophosphate (FPP). High purity recombinant protein was used to immunize New Zealand rabbits to generate a polyclonal antibody. The titer of the antibody was determined by enzyme linked immunosorbent assay (ELISA), and Western blot results demonstrated that the antibody could specifically recognize the AoFPPS protein in A. orientale (Sam.) Juzep. So,the method of rapid immunoassay to detect AoFPPS was established. This study lays the foundation for further study of the AoFPPS gene expression, regulation and mechanism of action in A. orientale (Sam.) Juzep., and it also provides a scientific basis on improving the quality of Alismatis Rhizoma using the plant genetic engineering.
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A gas chromatography-mass spectrometry(GC-MS)method was established for the analysis of volatile components in Mentha haplocalyx, and seven principal components were quantified by gas chromatography(GC). Based on these analyses, the differences of volatile components in M. haplocalyx from Jiangsu, Anhui and other regions were compared. The results showed that the volatile oil of M. haplocalyx was divided into four chemical types：menthol-menthone type, pulegone-menthone type, piperitone-menthol type, piperitone epoxide type, and menthol-menthone type was the principal type. Menthol was the highest and pulegone was the lowest. The differences of M. haplocalyx from Anhui and other regions were obvious. The major volatile components and the differences of M. haplocalyx from different regions were confirmed and a quantitative method was established for the determination of volatile components, which provided the basis for improving the quality standard of M. haplocalyx.
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The global antimicrobial resistance has been a big challenge to the human health for years. It has to make balance between the safety of animal products and the use of antimicrobials in animal husbandry. Any methods that can minimize or even phase out the use of antimicrobials in animal husbandry should be encouraged. We herein describe the research strategies for feed additives and veterinary medicines from the side products of Chinese medicine resources industrialization. Killing two birds with one stone-besides the major purposes, the rational utilization of non-medicinal parts and wastes of industrialization of Chinese herbal medicines is also achieved under the proposed strategies.
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Squalene synthase of Alisma orientale catalyzes farnesyl diphosphate (FPP) to form squalene, which is the key regulatory enzyme of the carbon source flow to protostane triterpenes biosynthesis. For further research on the function and expression of AoSS gene, the open reading frame (ORF) of squalene synthase gene (accession no. JX866770) from A. orientale was subcloned into a prokaryotic expression vector pCzn1 and induced the expression of AoSS gene in Escherichia coli BL21(Roseta). The fusion protein was mainly in the form of inclusion bodies and purified to obtain high purity protein. By verifying its functionality through vitro enzymatic reaction, the results showed that the catalytic protein had the catalytic activity of FPP into squalene. In order to research the expression of AoSS in A. orientale, the purified protein was used to immunized rabbits to prepare polyclonal antibody which was then purified, the titer of the antibody was greater than 1∶51 200 by ELISA detection, and displayed good specificity by Western blotting. The prepared antibody was used for immunoassay of AoSS in different organs of A. orientale, and the results showed that the AoSS expression level was the highest in tubers, followed by leaves, and lowest in root. Successful construction of prokaryotic expression vector, validation of gene functions and establishment of rapid immunoassay lay the foundation for further researches on the function and regulation of AoSS gene, and also provide scientific basis on the application of the protostane triterpenes of A. orientale in the field of synthetic biology.
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OBJECTIVE@#To explore the effect of high glucose and lipid intervention on islet cell apoptosis through the inhibition of prostate apoptosis response factor-4 (Par-4) expression and the underlying mechanisms.@*METHODS@#The mice islet β cells (NIT-1 cells) were randomly divided into a control group, a Par-4 inhibited group, a glucose and fatty acid intervented group and a glucose and fatty acid intervented+Par-4 inhibited group. Cell apoptosis was detected by terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), and the protein expression levels of Par-4 and glucose regulated protein 78 (GRP78) were detected by Western blot.@*RESULTS@#Compared with the control group [(3.14 ± 1.08)%], the apoptosis rate of islet beta cell [(33.82 ± 3.15)%] in the glucose and fatty acid intervented group was significantly increased accompanied by the dramatically elevated Par-4 and GRP78 expression (both P<0.05). Compared with the glucose and fatty acid intervented group, the apoptosis rate in glucose and fatty acid intervented+Par-4 inhibit group [(18.3 4 ± 2.11)%] was significantly decreased concomitant with the significantly decreased Par-4 and GRP78 expression (both P<0.05).@*CONCLUSION@#The glucose and fatty acid-induced apoptosis of mice islet β cells could be improved through the inhibition of Par-4 expression, which might be related to reduction of endoplasmic reticulum stress.
Subject(s)
Animals , Mice , Apoptosis , Apoptosis Regulatory Proteins , Metabolism , Cell Line , Fatty Acids , Gene Expression Regulation , Glucose , Heat-Shock Proteins , Metabolism , In Situ Nick-End Labeling , Islets of Langerhans , Cell BiologyABSTRACT
The objects of research on the resources chemistry of Chinese medicinal materials (RCCMM) are promotion of efficient production, rational utilization and improving quality of CMM and natural products. The development of TCM cause depends on the efficient utilization and sustainable development of CMM, hinges on the technologies and methods for using and discovering medicinal biological resources, stand or fall on the extension of industy chains, detailed utilizaion of resource chemical components by multi-way, multi-level. All of these may help to the recycling utilization and sound development of RCMM. In this article, five respects were discussed to the RCCMM researches and resources recycling utilization ways and goals and tasks. First, based on the principle of resource scarcity, discovering or replacing CMM resources, protecting the rare or endangered species or resources. Second, based on the multifunctionality of CMM, realizing the value-added and value compensation, and promoting the utilization efficiency through systermatic and detailed exploitation and utilization. Third, based on the resource conservation and environment-friendly, reducing raw material consumption, lowering cost, promoting recycling utilization and elevating utilization efficiency. Fourth, based on the stratege of turning harm into good, using the invasive alien biological resources by multi-ways and enriching the medicial resources. Fifth, based on the method of structure modification of chemical components, exploring and enhancing the utility value of resouces chemical substances. These data should provide references and attention for improving the utilization efficiency, promoting the development of recycling economy, and changing the mode of economic growth of agriculture and industry of CMM fundamentally.
Subject(s)
Agriculture , Economics , China , Conservation of Natural Resources , Economics , Drugs, Chinese Herbal , Chemistry , Economics , Materia Medica , Chemistry , Economics , Medicine, Chinese Traditional , Economics , Plants, Medicinal , ChemistryABSTRACT
Based on the infrared spectra of Lophatheri Herba and Commelinae Herba, one-dimensional infrared spectra, second derivative spectra and two-dimensional correlated spectra were used to find out the differences between Lophatheri Herba and its imitations, respectively. The common peak ratio and variant peak ratio dual-indexes sequential were calculated and established according to infrared spectra of eleven batches of herbs. Infrared spectral data of Lophatheri Herba cluster analysis was applied to explore the similarity between each sample. The grouping results trend of sequential analysis of dual-indexes and cluster analysis was accordant. The results showed that the differences could be found by multi-level identification, and the source and the quality of the herbs could be effectively distinguished by the two analysis methods. Infrared spectroscopy, used in the present work exhibited some advantages on quick procedures, less sample required, and reliable results, which could provide a new method for the identification of traditional Chinese medicine with the imitations and adulterants, and the control of quality and origin.
Subject(s)
Drugs, Chinese Herbal , Chemistry , Plant Leaves , Chemistry , Plants, Medicinal , Chemistry , Reproducibility of Results , Spectrophotometry, Infrared , Methods , Spectroscopy, Fourier Transform Infrared , MethodsABSTRACT
The industrialization chains and their products, which were formed from the process of the production of medicinal materials-prepared drug in pieces and deep processed product of Chinese material medica (CMM) resources, have generated large benefits of social and economic. However, The large of herb-medicine castoff of "non-medicinal parts" and "rejected materials" produced inevitably during the process of Chinese medicinal resources produce and process, and the residues, waste water and waste gas were produced during the manufactured and deep processed product of CMM. These lead to the waste of resources and environmental pollution. Our previous researches had proposed the "three utilization strategies" and "three types of resources models" of herb-medicine castoff according to the different physicochemical property of resources constitutes, resources potential and utility value of herb-medicine castoff. This article focus on the conversion efficiency of resources model and analysis the ways, technologies, practices, and application in herb-medicine cast off of the conversion efficiency of resources model based on the recycling economy theory of resources and thoughts of resources chemistry of CMM. These data may be promote and resolve the key problems limited the industrialization of Chinese material medica for long time and promote the realization of herb-medicine castoff resources utilization.
Subject(s)
Biotransformation , Conservation of Natural Resources , Methods , Drug Industry , Drugs, Chinese Herbal , Chemistry , Metabolism , Materia Medica , Chemistry , Metabolism , Research DesignABSTRACT
<p><b>OBJECTIVE</b>To establish a method for determining anticoagulation potency of Sparganii Rhizoma, and evaluate the effect of Sparganii Rhizoma herbs from different producing areas on promoting blood circulation and removing blood stasis; and study the material basis of Sparganii Rhizoma through the correlation analysis on its anticoagulation potency, ferulic acid and total flavonoid content.</p><p><b>METHOD</b>The anticoagulation time of Sparganii Rhizoma from different producing areas with activeated partial thromboplastin time for their active extracts. Their biopotency was calculated by using the method of "parallel lines of dose effect" (3, 3). The degree of correlation between their anticoagulation potency and chemical constituents were calculated by using Pearson correlational analysis method.</p><p><b>RESULT</b>Sparganii Rhizoma and is control drugs had a good linear relationship between dose and effect (Y = 172.76X - 193.39, R2 = 0.9955). The method had better accuracy (RSD 4.7%), repeatability (RSD 2.3%) and intermediate precision (RSD 5.4%), finding that the biopotency of Sparganii Rhizoma from different producing areas ranged between 52.33-238.58 U x g(-1), and all of them passed the test on reliability. The results of correlation analysis showed no remarkable relationship between the anticoagulation potency of Sparganii Rhizoma and the contents of the two chemical constituents.</p><p><b>CONCLUSION</b>This biopotency determination method established in the experiment can be used as one of approaches for qulaity evaluation on Sparganii Rhizoma.</p>
Subject(s)
Animals , Rabbits , Anticoagulants , Chemistry , Pharmacology , Blood Coagulation , Drugs, Chinese Herbal , Chemistry , Pharmacology , Rhizome , Chemistry , Typhaceae , ChemistryABSTRACT
Triterpenes, which have large application potential in the treatment of cancer, are the main active components of genuine medicinal material Alisma orientale (Sam.) Juzep. Farnesyl pyrophosphate synthase (FPPS) is one of the important rate-limiting enzymes in the synthetic pathway of triterpenes. In this study the FPPS full length cDNA of the A. orientale, was cloned via homology-based cloning approach and rapid amplification of cDNA ends (RACE). The full length of the FPPS cDNA was 1 531 bp (accession no. HQ724508), which contained a full 1 032 bp ORF that encoded 343 amino acids. The deduced protein sequence exhibited five conserved motifs, two of which is riched of Asp (DDXXD). The result of real-time quantitative PCR (QRT-PCR) showed that FPPS gene was expressed in different organs of A. orientale. The expression increased from October to the first ten-day period of December, and then decreased. The FPPS gene expression was higher in leaves but lower in leafstalk, tuber and root. HPLC analysis of active components 23-acetyl-alismol B of A. orientale. during different periods indicated that its change trend should be consistent with FPPS gene expression. It can be primarily deduced that FPPS gene should be an important control point in the synthetic pathway of Alisma terpenes. This study may facilitate the quality of medicinal plants through gene engineering in the future.
Subject(s)
Alisma , Genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Computational Biology , Conserved Sequence , DNA, Complementary , Genetics , DNA, Plant , Genetics , Gene Amplification , Geranyltranstransferase , Genetics , Metabolism , Molecular Sequence Data , Phylogeny , Plant Leaves , Genetics , Plant Roots , Genetics , Plants, Medicinal , Genetics , RNA, Messenger , Metabolism , Real-Time Polymerase Chain Reaction , MethodsABSTRACT
<p><b>OBJECTIVE</b>To optimize the conditions of supercritical fluid extraction (SFE) for curcumin in Curcuma longa.</p><p><b>METHOD</b>Optimum extraction conditions were studied by orthogonal tests. The extracts were analyzed by HPLC.</p><p><b>RESULT</b>The optimal extraction conditions were pressure 25 MPa, temperature 55 degrees C, static time 4 h, dynamic time 5 h, flow rate of CO2 3.5 L x min(-1), co-solvent ethanol 30% (mL x g(-1)).</p><p><b>CONCLUSION</b>It is feasible to extract curcumin by SFE.</p>